Metabolic immunomodulators that potentiate Anti-tumor immunity in cancer

Abstract

Described herein are methods of modulating the immune response to a cancer cell in a patient comprising administering to the patient a therapeutically effective amount of an inhibitor of acetyl-CoA synthetase short-chain (ACSS). In some aspects, the ACSS inhibitor may be described by the formula: (I)

Description

[0001] METABOLIC IMMUNOMODULATORS THAT POTENTIATE ANTI-TUMOR IMMUNITY IN CANCER This application claims the benefit of priority to United States Provisional Application Nos. 63 / 583,183, filed on September 15, 2023, 63 / 597,999, filed on November 10, 2023, and 63 / 651,227, filed on May 23, 2024, the entire contents of which are hereby incorporated by reference. BACKGROUND This invention was made with government support under CA010815 and CA249950 awarded by the National Institutes of Health. The government has certain rights in the invention. I. Field This disclosure relates to the fields of biology, pharmacology, medicine, and chemistry. In particular, new uses of compounds, compositions, and methods related to the treatment of breast cancer are disclosed. II. Description of Related Art Triple-negative breast cancer (TNBC) often presents as high-grade and has the poorest survival rate among the different subtypes of breast cancer. TNBC is a heterogenous disease typically diagnosed immunohistochemically by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. The lack of growth factor receptor expression has hampered the development of targeted therapies for the treatment of TNBC and therefore chemotherapy remains the standard of care (Bianchini et al, 2016). Immunotherapy was first approved in 2019 for metastatic TNBC patients with PD-L1 positive tumors, and there are currently numerous clinical trials investigating the efficacy of immunotherapy in TNBC (Atezolizumab Combo Approved for PD-L1-positive TNBC. Cancer Discov 9, OF2 (2019); Emens et al., 2021; Emens et al., 2019; Nanda et al., 2016; Adams et al., 2019a; Adams et al., 2019b; Loi et al., 2017; Winer et al., 2021; Dirix et al., 2018; Vonderheide et al., 2017). Given the success of immunotherapies in the clinic, it would be beneficial to gain a more thorough understanding of the tumor-immune axis in TNBC to better inform immunotherapeutic and combinatorial treatment modalities. There are three acyl-CoA synthetase short-chain family members (ACSS) that convert short-chain fatty acids to short-chain acyl-CoA’s in mammalian cells. ACSS’s mainly differ in their subcellular localization and substrate selectivity. Acetyl-CoA synthetase 1 (ACSS1) and ACSS3 are found in the mitochondria (Fujino et al., 2001; Yoshimura et al., 2017). It was previously showed that ACSS1 has a high specific activity for both acetate and propionate, while ACSS3 greatly prefers propionate and has almost no activity towards acetate (Miller et al., 2021). ACSS2 is a nucleo-cytosolic enzyme that strongly prefers acetate (Miller et al., 2021; Luong et al., 2000). Given that acetyl-CoA does not freely cross the mitochondrial membrane, ACSS1 and ACSS2 control distinct subcellular pools of acetate-derived acetyl- CoA. Acetyl-CoA is a versatile metabolite that is oxidized in the tricarboxylic acid (TCA) cycle or used for fatty acid biosynthesis and protein (histone) acetylation (Schug et al., 2016). Many cancer cells with expression of ACSS2 can use acetate as an alternative source of carbon and energy, competing in significance with glucose (Bulusu et al., 2017; Schug et al., 2015; Kamphorst et al., 2014). Production of acetyl-CoA from acetate by ACSS2 is a common metabolic adaptation in many cancers, including breast, prostate, glioblastoma, melanoma, fibrosarcoma, multiple myeloma, and hepatocellular carcinoma (Miller et al., 2021; Schug et al., 2015; Comerford et al., Chen et al., 2015; Chen et al., 2017; Gao et al., 2016; Li et al., 2017; Li et al., 2021; Lakhter et al., 2016). Moreover, ACSS2 expression has been shown to be higher in breast cancer, ovarian cancer, brain cancer, and prostate cancer compared to normal adjacent tissue (Schug et al., 2015; Comerford et al., 2014; Li et al., 2017).11C-acetate positron emission tomography (PET) imaging has shown that many tumors consume higher amounts of acetate compared to the surrounding normal tissue (Oyama et al., 2009; Ho et al., 2014; Delbeke & Pinson 2003). ACSS1 and ACSS2 have been shown to be upregulated by 4-hydroxytamoxifen treatment and long-term estrogen deprivation suggesting that acetate metabolism may be activated in ER+ breast cancer with acquired resistance to selective estrogen receptor modulator or in TNBC (Calhoun et al., 2022). Many recent studies suggest that metabolic inhibitors can also act as immunomodulators (Triplett et al., 2018; Leone et al., 2019; Scharping et al., 2021; Zhang et al., 2019; Zhao et al., 2018; Guerra et al., 2020). This class of metabo-immunomodulators dually act by impairing tumor metabolism while also modulating immune cell function in the tumor microenvironment. A potent, specific ACSS2 inhibitor called VY-3-135 was previously characterized that causes significant reductions in tumor growth in immune-deficient mice, suggesting a cancer cell intrinsic role for acetate metabolism in supporting tumor growth (Miller et al., 2021). With an ACSS2 inhibitor now in first-in-human clinical trials (NCT04990739) for cancer treatment, gaining a better understanding of the metabolic consequences of blocking tumor acetate metabolism on cancer cells and the tumor microenvironment will help guide the use of ACSS2 inhibitors in the clinic. SUMMARY In some aspects, the present disclosure provides methods of inducing an immune response to a cancer cell using ACSS inhibitors. In some embodiments, the induced immune response may increase the activity of one or more chemotherapeutic agents. In some aspects, the present disclosure provides a method of modulating the immune response to a cancer cell in a patient comprising administering to the patient a therapeutically effective amount of an inhibitor of acetyl-CoA synthetase short-chain (ACSS). In some embodiments, the ACSS is ACSS2. In some embodiments, the immune response is the increased production of one or more immune cells. In some embodiments, the immune cell is a T cell, such as a CD4+T cell or a CD8+T cell. In some embodiments, the immune cell is a natural killer cell. In some embodiments, the immune response is a change in the amount of an immune cell of at least 10% over the amount before administration of the ACSS inhibitor. In some embodiments, the change is at least 25%. In certain embodiments, the change is at least 50%. In some embodiments, the patient exhibits a normal immune response. In some embodiments, the cancer cell is a part of a solid tumor. In some embodiments, the cancer cell is a cancer cell that shows increased expression of one or more ACSS enzymes. In some embodiments, the cancer cell is a breast cancer cell, an ovarian cancer cell, a brain cancer cell, a prostate cancer cell, a pancreatic cancer cell, a sarcoma cancer cell, a lung cancer cell, a colorectal cancer cell, a skin cancer cell, a bone or connective tissue cancer cell, a blood cancer cell, or a liver cancer cell. In some embodiments, the cancer cell comprises one or more genetic markers that impair the cancer cell’s effectiveness to one or more treatments. In some embodiments, the cancer cell is a breast cancer cell, a glioblastoma cell, an ovarian cancer cell, a prostate cancer cell, a melanoma cancer cell, a fibrosarcoma cancer cell, a multiple myeloma cancer cell, or a hepatocellular carcinoma cancer cell. In some embodiments, the patient is a mammal. In certain embodiments, the mammal is a human. In some embodiments, the ACSS inhibitor is further defined as: ); wherein: R1 and R1′ are each i ndependently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2 is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3is alkyl(C≤12)or substituted alkyl(C≤12); X1 is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3is N, O, or C; and R4is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), substituted alkyl(C≤12), cycloalkyl(C≤12), substituted cycloalkyl(C≤12), alkenyl(C≤12), substituted alkenyl(C≤12), alkynyl(C≤12), substituted alkynyl(C≤12), aryl(C≤12), substituted aryl(C≤12), heteroaryl(C≤12), substituted heteroaryl(C≤12), heterocycloalkyl(C≤12), substituted heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: wherein: R1 and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R2 is amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3is alkyl(C≤12)or substituted alkyl(C≤12); Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is further defined as: II) wherein: R1and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R3is alkyl(C≤12)or substituted alkyl(C≤12); Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is further defined as: wherein: R1and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R3is alkyl(C≤12)or substituted alkyl(C≤12); and R4is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is further defined as: V) wherein: R1and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, R1is aryl(C≤12)or substituted aryl(C≤12). In some embodiments, R1 is aryl(C≤12), such as phenyl. In some embodiments, R1′ is aryl(C≤12) or substituted aryl(C≤12). In some embodiments, R1′ is aryl(C≤12), such as phenyl. In some embodiments, R2is hydroxy. In some embodiments, R3 is alkyl(C≤12) or substituted alkyl(C≤12). In some embodiments, R3 is alkyl(C≤12), such as ethyl. In some embodiments, Rais hydrogen. In some embodiments, R4is alkyl(C≤12) or substituted alkyl(C≤12). In some embodiments, R4 is substituted alkyl(C≤12), such as 2-hydroxypropyl. In some embodiments, the ACSS inhibitor is further defined as: or In some embodiments, the present disclosure provides a method of treating cancer comprising administering to the patient in need thereof a therapeutically effective amount of an ACSS inhibitor and a therapeutically effective amount of a therapeutic modality, wherein the therapeutic modality is a treatment for the cancer. In some embodiments, the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. In some embodiments, the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid. In some embodiments, the therapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. In certain embodiments, the therapeutic modality is a chemotherapeutic agent. In some embodiments, the therapeutic modality is an immunotherapy. In some embodiments, the patient is a mammal. In certain embodiments, the mammal is a human. In some embodiments, the ACSS inhibitor is administered no more than 12 hours before the therapeutic modality. In certain embodiments, the ACSS inhibitor is administered no more than 24 hours before the therapeutic modality. In some embodiments, the ACSS inhibitor is administered no more than 3 days before the therapeutic modality. In some embodiments, the patient is immunocompromised. In other embodiments, the patient has a healthy immune system. In some embodiments, the ACSS inhibitor is further defined as: wherein: R1 and R1′ are each independently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2 is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); X1is N or CRb, wherein Rbis selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), substituted alkyl(C≤12), cycloalkyl(C≤12), substituted cycloalkyl(C≤12), alkenyl(C≤12), substituted alkenyl(C≤12), alkynyl(C≤12), substituted alkynyl(C≤12), aryl(C≤12), substituted aryl(C≤12), heteroaryl(C≤12), substituted heteroaryl(C≤12), heterocycloalkyl(C≤12), substituted heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: II) wherein: R1and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R2is amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: or In some embodiments, the present disclosure provides a method of treating cancer in a patient who has been administered an ACSS inhibitor comprising administering to the patient a therapeutically effective amount of a therapeutic modality that is a treatment for the cancer, such as a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. In some embodiments, the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid. In some embodiments, the therapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. In certain embodiments, the therapeutic modality is a chemotherapeutic agent. In some embodiments, the therapeutic modality is an immunotherapy. In some embodiments, the patient is a mammal. In certain embodiments, the mammal is a human. In some embodiments, the ACSS inhibitor has been administered no more than 12 hours before the therapeutic modality. In certain embodiments, the ACSS inhibitor has been administered no more than 24 hours before the therapeutic modality. In some embodiments, the ACSS inhibitor has been administered no more than 3 days before the therapeutic modality. In some embodiments, the patient is immunocompromised. In other embodiments, the patient has a healthy immune system. In some embodiments, the ACSS inhibitor is further defined as: I); wherein: R1and R1′ are each independently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); X1 is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), substituted alkyl(C≤12), cycloalkyl(C≤12), substituted cycloalkyl(C≤12), alkenyl(C≤12), substituted alkenyl(C≤12), alkynyl(C≤12), substituted alkynyl(C≤12), aryl(C≤12), substituted aryl(C≤12), heteroaryl(C≤12), substituted heteroaryl(C≤12), heterocycloalkyl(C≤12), substituted heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: wherein: R1 and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R2 is amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3is alkyl(C≤12)or substituted alkyl(C≤12); Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C ≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: or In some aspects, the present disclosure provides a method of modulating the effectiveness of a chemotherapeutic modality comprising administering to a patient an ACSS inhibitor and the chemotherapeutic modality. In some embodiments, the method results in a 10% increase in the effectiveness of the chemotherapeutic modality as measured in tumor size. In some embodiments, the effectiveness is a 25% increase. In some embodiments, the ACSS inhibitor is administered before the chemotherapeutic modality. In some embodiments, the ACSS inhibitor is administered concurrently with the chemotherapeutic modality. In some embodiments, the ACSS inhibitor is administered within 3 hours after with the chemotherapeutic modality. In some embodiments, the chemotherapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. In certain embodiments, the chemotherapeutic modality is a chemotherapeutic agent. In some embodiments, the chemotherapeutic modality is an immunotherapy. In some embodiments, the patient is a mammal. In certain embodiments, the mammal is a human. In some embodiments, the ACSS inhibitor has been administered no more than 12 hours before the chemotherapeutic modality. In some embodiments, the ACSS inhibitor has been administered no more than 24 hours before the chemotherapeutic modality. In some embodiments, the ACSS inhibitor has been administered no more than 3 days before the chemotherapeutic modality. In some embodiments, the patient is immunocompromised. In other embodiments, the patient has a healthy immune system. In some embodiments, the ACSS inhibitor is further defined as: I); wherein: R1and R1′ are each independently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); X1is N or CRb, wherein Rbis selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), substituted alkyl(C≤12), cycloalkyl(C≤12), substituted cycloalkyl(C≤12), alkenyl(C≤12), substituted alkenyl(C≤12), alkynyl(C≤12), substituted alkynyl(C≤12), aryl(C≤12), substituted aryl(C≤12), heteroaryl(C≤12), substituted heteroaryl(C≤12), heterocycloalkyl(C≤12), substituted heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: I) wherein: R1 and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R2 is amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3is alkyl(C≤12)or substituted alkyl(C≤12); Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: or In some embodiments, the patient is a mammal. In some embodiments, the mammal is a human. In some aspects, the present disclosure provides a method of modulating the effectiveness of a chemotherapeutic modality comprising administering to a patient who has been administered an ACSS inhibitor the chemotherapeutic modality. In some embodiments, the method results in a 10% increase in the effectiveness of the chemotherapeutic modality as measured in tumor size. In some embodiments, the effectiveness is a 25% increase. In some embodiments, the ACSS inhibitor is administered before the chemotherapeutic modality. In other embodiments, the ACSS inhibitor is administered concurrently with the chemotherapeutic modality. In some embodiments, the ACSS inhibitor is administered within 3 hours after with the chemotherapeutic modality. In some embodiments, the chemotherapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. In some embodiments, the chemotherapeutic modality is a chemotherapeutic agent. In certain embodiments, the chemotherapeutic modality is an immunotherapy. In some embodiments, the patient is a mammal. In some embodiments, the mammal is a human. In some embodiments, the ACSS inhibitor has been administered no more than 12 hours before the chemotherapeutic modality. In some embodiments, the ACSS inhibitor has been administered no more than 24 hours before the chemotherapeutic modality. In certain embodiments, the ACSS inhibitor has been administered no more than 3 days before the chemotherapeutic modality. In some embodiments, the patient is immunocompromised. In other embodiments, the patient has a healthy immune system. In some embodiments, the ACSS inhibitor is further defined as: wherein: R1 and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2 is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3is alkyl(C≤12)or substituted alkyl(C≤12); X1is N or CRb, wherein Rbis selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), substituted alkyl(C≤12), cycloalkyl(C≤12), substituted cycloalkyl(C≤12), alkenyl(C≤12), substituted alkenyl(C≤12), alkynyl(C≤12), substituted alkynyl(C≤12), aryl(C≤12), substituted aryl(C≤12), heteroaryl(C≤12), substituted heteroaryl(C≤12), heterocycloalkyl(C≤12), substituted heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: wherein: R1and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R2is amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: or In some embodiments, the patient is a mammal. In certain embodiments, the mammal is a human. In some aspects the present disclosure provides a pharmaceutical composition comprising: (A) an ACSS inhibitor; and (B) a chemotherapeutic agent or an immunotherapy. In some embodiments, the ACSS inhibitor and the chemotherapeutic agent or the immunotherapy are formulated into a single unit dose. In some embodiments, the pharmaceutical composition comprises a chemotherapeutic agent. In some embodiments, the pharmaceutical composition comprises an immunotherapy. In some embodiments, the ACSS inhibitor is further defined as: I); wherein: R1 and R1′ are each independently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2 is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); X1 is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), substituted alkyl(C≤12), cycloalkyl(C≤12), substituted cycloalkyl(C≤12), alkenyl(C≤12), substituted alkenyl(C≤12), alkynyl(C≤12), substituted alkynyl(C≤12), aryl(C≤12), substituted aryl(C≤12), heteroaryl(C≤12), substituted heteroaryl(C≤12), heterocycloalkyl(C≤12), substituted heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: wherein: R1 and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R2 is amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3is alkyl(C≤12)or substituted alkyl(C≤12); Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: or In some embodiments, the immunotherapy is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD1 checkpoint inhibitor such as an anti-PD1 antibody. In yet another aspect, the present disclosure provides methods of treating cancer in a patient who has been treated with a checkpoint inhibitor with a therapeutically effective amount of an ACSS inhibitor of the formula: I); wherein: R1 and R1′ are each independently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); X1is N or CRb, wherein Rbis selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), substituted alkyl(C≤12), cycloalkyl(C≤12), substituted cycloalkyl(C≤12), alkenyl(C≤12), substituted alkenyl(C≤12), alkynyl(C≤12), substituted alkynyl(C≤12), aryl(C≤12), substituted aryl(C≤12), heteroaryl(C≤12), substituted heteroaryl(C≤12), heterocycloalkyl(C≤12), substituted heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: II) wherein: R1and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R2 is amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In still yet another aspect, the present disclosure provides methods of treating cancer in a patient in need thereof with a therapeutically effective amount of a checkpoint inhibitor and a therapeutically effective amount of an ACSS inhibitor of the formula: wherein: R1and R1′ are each independently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); X1 is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), substituted alkyl(C≤12), cycloalkyl(C≤12), substituted cycloalkyl(C≤12), alkenyl(C≤12), substituted alkenyl(C≤12), alkynyl(C≤12), substituted alkynyl(C≤12), aryl(C≤12), substituted aryl(C≤12), heteroaryl(C≤12), substituted heteroaryl(C≤12), heterocycloalkyl(C≤12), substituted heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy (C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: II) wherein: R1 and R1′ are each alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; R2 is amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); and R4is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12), or a pharmaceutically acceptable salt thereof. In some embodiments, the ACSS inhibitor is further defined as: or or a ph In some embodiments, the patient is a mammal such as a human. In some embodiments, the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. In some embodiments, the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid. In some embodiments, the patient is a mammal such as a human. In some embodiments, the ACSS inhibitor is administered no more than 12 hours before the checkpoint inhibitor. In some embodiments, the ACSS inhibitor is administered no more than 24 hours before the checkpoint inhibitor. In some embodiments, the ACSS inhibitor is administered no more than 3 days before the checkpoint inhibitor. In one aspect, the present disclosure provides compounds of Formula (I): I); wherein: R1and R1′ are each independently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); X1 is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicy cloalkylamino(C≤12); or a tautomer, solvate, and / or salt thereof. In some embodiments, Y3 is achiral. In some embodiments,Y1 is C(O)NRa. In some embodiments, Rais hydrogen. In some embodiments, R4is −Y2−R4′; wherein Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version thereof; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version thereof; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12). In some embodiments, Y2 is arenediyl(C≤12) or substituted arenediyl(C≤12). In further embodiments, Y2is arenediyl(C≤12), such as benzenediyl. In some embodiments, R4′ is −S(O)2R4′′′, wherein R4′′′ is amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), or substituted dicycloalkylamino(C≤12). In some embodiments, R4′′′ is alkyl(C≤12), such as methyl. In some embodiments, Y3is C or N. In some embodiments, Y3 is C. In other embodiments, Y3 is N. In some embodiments, R1 or R1′ is aryl(C≤12) or substituted aryl(C≤12). In some embodiments, R1and R1′ are both aryl(C≤12)or substituted aryl(C≤12). In further embodiments, R1 and R1′ are both aryl(C≤12), such as phenyl. In other embodiments, R1 and R1′ are not both aryl(C≤12)or substituted aryl(C≤12). In further embodiments, R1and R1′ are not both aryl(C≤12). In some embodiments, R1 and R1′ are not both phenyl. In some embodiments, R1 is aryl(C≤12) or substituted aryl(C≤12). In furtjer embodiments, R1 is aryl(C≤12), such as phenyl. In some embodiments, R1′ is aryl(C≤12) or substituted aryl(C≤12). In some embodiments, R1′ is aryl(C≤12), such as phenyl. In some embodiments, R1 or R1′ is substituted aryl(C≤12), such as 4-fluorophenyl. In some embodiments, R1 is aryl(C≤12) and R4 is 3-methylsulfonylphenyl. In some embodiments, the compound is further defined as: or or a tau In another aspect, the present disclosure provides pharmaceutical compositions comprising: (a) a compound as described above; and (b) an excipient. In another aspect, the present disclosure provides methods of treating a disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of a compound or a pharmaceutical composition as disclosed above. In some embodiments, the disease or disorder is cancer. In further embodiments, the disease or disorder is a breast cancer, an ovarian cancer, a brain cancer, a prostate cancer, a pancreatic cancer, a sarcoma cancer, a lung cancer, a colorectal cancer, a skin cancer, a bone or connective tissue cancer, a blood cancer, or a liver cancer. In another aspect, the present disclosure provides methods of modulating the immune response to a cancer cell in a patient comprising administering to the patient a therapeutically effective amount of a compound or a pharmaceutical composition disclosed herein. In some embodiments, the cancer cell is a part of a solid tumor. In some embodiments, the cancer cell is a cancer cell that shows increased expression of one or more ACSS enzymes. In some embodiments, the cancer cell is a breast cancer cell, an ovarian cancer cell, a brain cancer cell, a prostate cancer cell, a pancreatic cancer cell, a sarcoma cancer cell, a lung cancer cell, a colorectal cancer cell, a skin cancer cell, a bone or connective tissue cancer cell, a blood cancer cell, or a liver cancer cell. In some embodiments, the cancer cell comprises one or more genetic markers that impair the cancer cell’s effectiveness to one or more treatments. In some embodiments, the cancer cell is a breast cancer cell, a glioblastoma cell, an ovarian cancer cell, a prostate cancer cell, a melanoma cancer cell, a fibrosarcoma cancer cell, a multiple myeloma cancer cell, or a hepatocellular carcinoma cancer cell. In some embodiments, the patient is a mammal. In further embodiments, a human. In another aspect, the present disclosure provides methods of treating cancer comprising administering to the patient in need thereof a therapeutically effective amount of a compound or a pharmaceutical composition disclosed herein and a therapeutically effective amount of a therapeutic modality, wherein the therapeutic modality is a treatment for the cancer. In some embodiments, the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. In other embodiments, the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid. In some embodiments, the therapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. In further embodiments, is a chemotherapeutic agent. In other embodiments, the therapeutic modality is an immunotherapy. In some embodiments, the patient is a mammal. In some embodiments, the mammal is a human. In some embodiments, the compound or composition is administered no more than 3 days before the therapeutic modality. In further embodiments, the compound or composition is administered no more than 24 hours before the therapeutic modality. In still further embodiments, the compound or composition is administered no more than 12 hours before the therapeutic modality. In another aspect, the present disclosure provides methods of treating cancer in a patient who has been administered a compound or a pharmaceutical composition disclosed herein comprising administering to the patient a therapeutically effective amount of a therapeutic modality that is a treatment for the cancer. In some embodiments, the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. In other embodiments, the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid. In some embodiments, the therapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. In further embodiments, the therapeutic modality is a chemotherapeutic agent. In other embodiments, the therapeutic modality is an immunotherapy. In some embodiments, the patient is a mammal. In further embodiments, the mammal is a human. In some embodiments, the compound or composition is administered no more than 3 days before the therapeutic modality. In further embodiments, the compound or composition is administered no more than 24 hours before the therapeutic modality. In still further embodiments, the compound or composition is administered no more than 12 hours before the therapeutic modality. In another aspect, the present disclosure provides ACSS PROTAC conjugates comprising an acetyl-CoA synthetase short chain (ACSS) targeting ligand covalently linked to an E3 ligase recruiting ligand. In some embodiments, the ACSS targeting ligand is of formula (I): I); wherein: R1 and R1′ are each independently absent, halo; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2 is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3is alkyl(C≤12)or substituted alkyl(C≤12); X1is N or CRb, wherein Rbis selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Rais hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; or −Y2−R4′, wherein: Y2is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), alkoxy(C≤12), substituted alkoxy(C≤12), cycloalkoxy(C≤12), substituted cycloalkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), cycloalkylamino(C≤12), substituted cycloalkylamino(C≤12), dicycloalkylamino(C≤12), substituted dicycloalkylamino(C≤12); or a tautomer, solvate, and / or salt thereof. In some embodiments, the ACSS targeting ligand is further defined as:

[0002] or a tautomer, solvate, and / or salt of any of these formulae.

[0003] In some embodiments, the E3 iigase recruiting ligand is selected from among a CRBN recruiting ligand, a VHL recruiting ligand, an MDM2 recruiting hgand, or a cIAP recruiting ligand. In some embodiments, the E3 ligase recruiting ligand is selected from among

[0004] In some embodiments, the linker is a polymer comprising 2 to 20 repealing units. In some embodiments, the linker is a polyethylene glycol linker.

[0005] In another aspect, the present disclosure provides ACSS binding probes comprising an acelyl-CoA synthetase short chain (ACSS) targeting ligand covalently linked to a fluorescent moiety. In some embodiments, the ACSS targeting ligand is of formula (I): ); wherein: R1and R1′ are eac epe e y a se , a o; or alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; R2is hydrogen, halo, amino, hydroxy, alkoxy(C≤12), substituted alkoxy(C≤12), alkylamino(C≤12), substituted alkylamino(C≤12), dialkylamino(C≤12), substituted dialkylamino(C≤12), acyloxy(C≤12), substituted acyloxy(C≤12), amido(C≤12), or substituted amido(C≤12); R3 is alkyl(C≤12) or substituted alkyl(C≤12); X1 is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(C≤12), cycloalkyl(C≤12), alkoxy(C≤12), alkylamino(C≤12), dialkylamino(C≤12), or a substituted version of any of these groups; Y1 is a covalent bond, O, C(O), C(O)NRa, or NRa; wherein Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is N, O, or C; and R4 is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; or −Y2−R4′, wherein: Y2 is alkanediyl(C≤12), arenediyl(C≤12), heteroarenediyl(C≤12), or heterocycloalkanediyl(C≤12), or a substituted version of any of these groups; and R4′ is alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(C≤12), heterocycloalkyl(C≤12), or a substituted version of any of these groups; −C(O)R4′′, or −S(O)2R4′′′, wherein R4′′ and R4′′′ are each independently amino, hydroxy, alkyl(C≤12), cycloalkyl(C≤12), alkenyl(C≤12), alkynyl(C≤12), aryl(C≤12), heteroaryl(c<:i2), heterocycloalkytywu), alkoxy (c-ui), substituted alkoxy(C, cycloalkoxy(c<i2), substituted cycloalkoxy (c-zi ?). alkylamino(c<i 20, substituted alkylamino(c<i2). dialkylainino(c: ,12), substituted dialkylamino(csi2), cycloalkylaminoywu), substituted cycloalkylarnino(c<i2), dicycloalkylaminoicsw), substituted dicycloalkylaniino(c<i2)t or a tautomer, solvate, and / or salt thereof.

[0006] In some embodiments, wherein the ACSS targeting ligand is further defined as: or a tautomer, solvate, and / or salt of any of these formulae.

[0007] In some embodiments, the fluorescent moiety is FITC. In other embodiments, the fluorescent moiety is a BODIPY dye, such as BODIPY 630 / 650. In some embodiments, the fluorescent moiety is a pyrylium dye, such as Py-1. In some embodiments, the probe binds to ACSS with a KD of about 500 nM. In some embodiments, the probe binds to ACSS with a KD less than 500 nM. In some embodiments, the probe binds to ACSS2 with a KD of about 500 nM. In some embodiments, the probe binds to ACSS2 with a KD less than 500 nM.

[0008] In another aspect, the present disclosure provides methods for determining the binding affinity of a compound to an ACSS enzyme comprising:

[0009] (A) contacting an ACSS enzyme with a probe disclosed herein; and

[0010] (B) determining the amount of luminescence generated from the probe.

[0011] In some embodiments, the binding affinity is determined in vivo. In some embodiments, n the ACSS enzyme is ACSS2, In some embodiments, the ACSS enzyme is a modified ACSS enzyme. In further embodiments, the modified ACSS enzyme is modified with luciferase subunit, hi some embodiments, the modified ACSS enzy me is an ACSS enzyme with a 19 kDA luciferase subunit. In further embodiments, the modified ACSS enzy me is an ACSS enzyme tagged with a 19 kDa subunit derived from Oplophorus gracilirostris.

[0012] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description, it should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Note that simply because a particular compound is ascribed to one particular generic formula doesn’t mean that it cannot also belong to another generic formula.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] FIGS. 1A-J - Inhibition of ACSS2 triggers stronger tumor growth inhibition in immune-competent mice compared to immune-deficient mice, a. Diagram outlining the approach to testing inhibition of ACSS2 in immune-competent and immune-deficient tumorbearing mice. VY-3-135 = ACSS2 inhibitor. sgAcss2 = single guide RNA targeting Acss2. NSG= NOD.Cg-Prkdcscid 112rgtmlWjl / SzJ. b-g, Allograft tumor growth in NSG, C57B1 / 6, or BABL / c mice treated with VY-3-135. n:=:8 mice per group, h, Orthotopic allograft tumor growth of Brpkpl lO cells in C57BI / 6 mice treated with VY-3-135. n ~ 5 mice per group, i and j. Orthotopic allograft tumor growth of Cas9 (WT) or two different sgAcss2 (Acss2-KO) Brpkpl 10 cell lines in NSG or C57B1 / 6. n = 5 to 10 mice per group. P values are displayed on the graph and were generated using 2-way ANOV A with Sidak’s multiple testing. Values represent the mean tumor volume ± standard error of the means (SEM).

[0016] FIGS.2A-K- Knockout of Acss2 in mouse breast cancer cell lines often leads to tumor clearance, a. LC-MS analysis of13C2-acetate labeling of metabolites and fatty acids in T12 tumor tissue taken from vehicle versus VY-3-135 treated BALB / c mice. Lines represent the mean with individual data points displayed. P values were generated using Mann-Whitney U testing, n = 5 tumors per group. AU = arbitrary units b, Allograft tumor growth of Cas9 (WT) A7C11 cells in C57BI / 6 mice. P values are displayed on the graph and were generated using 2-way ANOV A with Sidak’s multiple testing. V alues represent the mean tumor volume ± SEM. n = 5 mice per group, c, Representative western blots for ACSS2 expression in Ti l and 4T1 sgAcss2 cell lines, d. Allograft tumor growth of Cas9 (WT) or sgAcss2 (Acss2~KO) T11 cells in B ALB / c mice, n = 8 mice per group, e, Kaplan-Meier survival plot of C57B1 / 6 tumor-bearing mice injected with either wild type (Cas9) or Acss2-KO (sgAcss2) T1 1 ceils. P values are displayed on the graph and were generated using a log-rank (Mantel-Cox) test, n::::16 mice per group, f, Allograft tumor growth of Cas9 (WT) or sgAcss2 (Acss2-KO) 4T1 cells in BALB / c mice. P value is displayed on the graph and was generated using 2-way ANOVA with mixed effects analysis of time x tumor volume. Values represent the mean tumor volume ± SEM. n ~ 8 mice per group g, Syngeneic allograft tumor growth of 4T1 sgAcss2 revertant (sgAcss2 rev) tumors in BALB / c mice, n = 7 to 8 mice per group. Take rates indicate the number of mice with tumors in each group at the end of the study. h,i, Kaplan- Meier survival plot of C57B1 / 6 tumor-bearing mice injected with either wild type (Cas9) or Acss2-KO (sgAcss2) Brpkpl 10 cells or with wild type (Cas9) Brpkpl 10 cells that were treated daily from day 4 to day 31 (marked red triangles) with VY-3-135. P values are displayed on the graph and were generated using a log -rank (Mantel-Cox) test, n::= 8 mice per group, j, Representative western blot for ACSS2 expression in Brpkpl 10 wild type (Cas9 #1), sgAcss2, and sgAcss2 rev. k. Syngeneic allograft tumor growth of Brpkpl lO cells from panel j in C57B1 / 6 mice, n = 5 mice per group.

[0017] FIGS. 3A-I - T-cells and NK-cells are necessary' for suppression of Acss2-KO tumor growth a, Syngeneic allograft tumor growth of WT versus Acss2-KO Brpkpl lO tumors in C57B1 / 6 mice depleted of both CD41and CD8 ’ T-cells or treated with an isotype control IgG antibody. Values represent the mean tumor volume ± SEM. P values generated using Dunnetf s multiple comparisons test comparing all groups to Cas9 + IgG. n = 10 mice per group. b,c, Syngeneic allograft tumor growth of Brpkpl 10 WT or Acss2-KO cells grown in C57B1 / 6 mice individually depleted of ( D4 (b) or CD8" (c) T-cells or treated with an isotype control IgG antibody, n = 5 mice per group. Values represent the mean tumor volume ± SEM. P values generated using Dunnetf s multiple comparisons test comparing all groups to Cas9 + IgG. d. Syngeneic allograft tumor growth of A7C1 1 WT or Acss2-KO tumors in C57B1 / 6 mice depleted of both CD4+and CD8+T-cells or treated with an isotype control IgG antibody. Values represent the mean tumor volume ± SEM. P values generated using Dunnett’s multiple comparisons test comparing all groups to Cas9 + IgG. n:::5 io 10 mice per group, e. Syngeneic allograft tumor growth of Brpkpl 10 WT or Acs$2-KO tumors in C57BI / 6 mice depleted of NK- cells or treated with an isotype control IgG antibody. Values represent the mean tumor volume ± SEM. P values generated using Dunnetf s multiple comparisons test comparing all groups to Cas9 + IgG. n = 10 mice per group, f, final tumor weights from data in (e). Values represent the mean tumor volume ± SEM. P values generated using Dunnett’s multiple comparisons test comparing all groups to Cas9 + IgG. g, Schematic detailing tumor rechallenge experiments, hj. Syngeneic allograft tumor growth of WT Brpkpl 10 or T1 1 cells in C57B1 / 6 or BALB / c mice that had previously cleared Acss2-KO tumors or age-matched unchallenged mice, n = 5 mice per group. Values represent the mean tumor volume ± SEM. P values generated using two-sided Fisher’s exact test.

[0018] FIGS. 4A-D - Clearance of Acss2~KO tumors depends on the presence of functional T-cells, a. Representative flow plots of peripheral blood mononuclear cells collected from mice depleted of CD4 . CD8+, or CD4+and CD8+T-cells. b, Final tumor weights of WT or Acss2- KO BrpkpllO tumors grown in C57B1 / 6, C57B1 / 6 Rag2'A, or NSG mice. Values represent the mean tumor volume ± standard deviation (SD). P values generated using two-tailed Mann- Whitney U test, n = 4 to 12 mice per group. c,d, Representative flow plots of peripheral blood mononuclear cells collected from mice treated with IgG control (c) or an NK-cell depleting antibody against NK1.1 (d).

[0019] FIGS. 5A-F - ACSS2 is dispensable in T-cells for anti-tumor immune responses, a, Acss2_ / ’ mice possess a one base pair insertion into exon 3 at the indicated (red triangle) position causing a frameshift mutation, b. Representative sequences from WT and KO mice showing the adenine base pair insertion, c, ACSS2 protein expression in liver lysates from WT versus

[0020] Acss2-KO mice d, Immunohistochemistry staining for ACSS2 in WT and Acss2-KO mouse liver and colon e, sgAcss2 tumors are unable to grow in Acss2-KO mice, n = 8 mice per group.

[0021] £ Tumor growth studies in chimaera mice reconstituted with WT or Acss2-KO mouse bone marrow, n > 5 mice per group, g. Final tumor weights from the mice in panel f. P values displayed on the graph were generated using two-tailed Mann- Whitney U test. Values represent the mean tumor volume * SEM. n > 6 mice per group

[0022] FIGS. 6A-G - ACSS2 inhibitor treated tumors have gene signatures associated with increased immune surveillance and activation, a. Volcano plots showing differentially expressed genes (DEGs) in tumors from NSG or C57B1 / 6 mice treated with VY-3-135. NSG tumor-bearing mice were treated for 15 days. C57B1 / 6 mice were treated for 5 days b, Venn diagram illustrating the overlap of genes differentially regulated by VY-3-135 treatment in tumors grown in NSG versus C57B1 / 6 mice, c. Heat map of 64 genes in common from the data in panel a. d,e, GSEA of the top 20 most significantly activated functions in tumors after VY- 3-135 treatment in NSG and C57B1 / 6 mice. Heat mapping represents -log 10 FDR values. The number next to the bar displays the number of genes within that function that were altered. The x-axis shows the predicted Z score based on the gene expression differences, fig. IPA analysis of the top 20 significant regulators enriched by VY-3-135 treatment in NSG or C57B1 / 6 mice. Heat mapping on bar plots represents -logl 0 FDR values. The number next to the bar displays the number (N) of genes within that regulator or function gene set that were altered. The x-axis is the predicted Z score based on the gene expression differences

[0023] FIGS 7A-I - Inhibition of ACSS2 synergizes with chemotherapy in breast tumors, a, Seurat clustering of cell populations within BrpkpllO tumors highlighting the tumor ceil clusters 0 and 1. b, All upregulated genes in Brpkpl lO tumor cells treated with VY- 3-135 versus vehicle, c. Heatmapping of the relative gene expression of proliferation markers across ceil populations, d, Heat map illustrating top 10 gene markers for each tumor cell population from panel a. e, Average and percent expression of proliferative markers in cluster 0 versus cluster 1. f, GSEA of different functions between tumor cell cluster 0 and cluster 1. g-i, Syngeneic allograft tumor growth of BrpkpllO ceils in C57B1 / 6 mice treated with vehicle, palbociclib, doxorubicin, VY-3-135, paclitaxel, and combinations thereof starting at day 10 post injection. Values represent the mean tumor volume ± SEM. P values generated using 2- way ANOVA and Dunnett’s multiple comparisons test compared to vehicle.

[0024] FIGS. 8A-I - Single cell RNA sequencing identifies enhanced activation of tumorinfiltrating T-cells upon ACSS2 inhibition, a, Seurat clustering of cell populations within Brpkpl 10 tumors highlighting lymphocyte cluster 2. b,c, Upregulated genes in lymphocytes (b) and myeloid cells (c) from Brpkpl lO tumors treated with VY-3-135 for seven days, d, Seurat plot of cell populations following subclustering of lymphocytes from cluster 2. e. Heat map illustrating gene expression of the top five most highly expressed gene markers within each lymphocyte subcluster fi Volcano plot illustrating DEGs in VY-3-135 treated tumors of subcluster 0. g, IPA of the top 20 functions and regulators in subcluster 0 that were significantly activated by VY-3- 135 treatment, h. Volcano plot illustrating DEGs in subcluster 1 in VY -3- 135 treated tumors, i, GSEA of the top 20 functions and regulators in subcluster 1 that were significantly activated in ACSS2 inhibitor treated tumors. Heat mapping on bar plots represents -loglO FDR values. The number next to the bar displays the number (N) of genes within that regulator or function gene set that were altered. The x-axis is the predicted Z score based on the gene expression differences.

[0025] FIGS. 9A-G - Single cell RNA sequencing identifies enhanced activation of tumorinfiltrating myeloid cells after ACSS2 inhibition, a. Seurat plot of cell populations following subclustering of myeloid ceils from clusters 3 and 4 from Fig. 5a. b. Heat map illustrating gene expression of the top five most highly expressed gene markers within each subcluster c. Heatmapping of gene marker expression onto the myeloid cell subclusters, d, Volcano plot illustrating DEGs in VY-3- 135 treated tumors of subcluster 0. e, GSEA of the top 20 functions and regulators in subcluster 0 that were significantly activated in VY-3-135 treated tumors, f, Volcano plot illustrating DEGs in subcluster 1 in VY-3- 135 treated tumors, g, GSEA of the top 20 functions and regulators in subcluster 1 that were significantly- activated in VY -3-135 treated tumors. Heat mapping on bar plots represents -log 10 FDR values. The number next to the bar displays the number (N) of genes within that regulator or function gene set that were altered. The x-axis is the predicted Z score based on the gene expression differences

[0026] FIGS. 10A-C - Gene markers used to identify the cell types within the subclusters of T-cells and NK-cells. a,b. Heatmapping of the relative expression of canonical genes used to distinguish T- cells and NK-cells within all five lymphocyte subclusters from Fig. 5d. c. Predicted distribution of NK, NKT, and Treg cells within the subclusters.

[0027] FIGS. 11A-H - Phenotypic marker analysis of tumor-infiltrating leukocytes and splenocytes from VY-3-135 treated tumors, a, CCL5 protein expression in Brpkpl 10 tumors treated w ith VY-3-135. Bars represent the mean tumor volume ± S.D. P values generated using two-tailed Mann-Whitney U test, n = 5. b. Correlation of ACSS2 and CCL5 expression in TNBC patient survival Data obtained from METABRIC and TCGA databases. P values and hazard ratios (HR) are reported on the plots, c. Schematic of VY-3-135 treatment of tumorbearing mice for flow cytometry analysis, d-h. Flow cytometry analysis of phenotypic markers on tumor- infiltrating leukocytes and splenic leukocytes from Brpkpl 10 tumor-bearing mice treated with VY-3-135 for 7 days and 28 days, n = 3 to 5 mice per group. * = P < 0.05. ** = P <0.01. P values were generated using a t test. Values represent the mean fluorescence intensity' (MFI) ± Si -Al.

[0028] FIGS. 12A-L - CD8dT-cells find NK-cells readily oxidize acetate in a time and concentration dependent manner, a. Acetate uptake and release from Cas9 and sgAcss2 Brpkpl lO cells and Cas9 Brpkpl lO treated with VY-3-135. n = 3. P values generated using Dunnett’s multiple comparisons test comparing ail groups to Cas9 Values represent net acetate exchange ± SEM. b, Immunoblot for ACSS1 and ACSS2 expression in peripheral blood mononuclear cells (PBMC). Mouse li ve r lysates were used as a loading control for high ACSS2 low ACSS I expression, c. Diagram displaying ACSS1 -dependent13C2-acetate stable isotope tracing into citrate and expected isotoplogue labeling patterns, d-g. Stable isotope tracing of 0. 1 versus 0.5 mM '■’Cz-acetate into CD8+T-cells from wild type (VVT) versus Acss2-KO mice. Values represent the mass isotopologue distribution ± SEM forl3C2-acetate in metabolites at Ih and 2h after exposure. Where indicated, samples were treated with VY-3-135 (+Inh). h-k, Stable isotope tracing of 0.1 versus 0.5 mM13C?- acetate into NK-cells from C57B1 / 6 mice. Mass isotopologue distribution patterns ± SEM by3'Cc-acetate in metabolites at 3h after exposure.

[0029] FIGS. 13A-D - Lymphocytes express relatively high amounts of ACSS1 and oxidize acetate better than macrophages, a. Representative dot plots displaying enrichment for CD8+and CD41-T-cells from total splenocytes collected from mice. b. mRNA expression for macrophage markers of polarization from a representative ex vivo culture, c, ACSS1 mRNA expression in T-cells and macrophages. P values generated using Dunnell's multiple comparisons test compared CD8' T-cells, n.s. = not significant **** = P < 0.0001. d. Stable isotope tracing of 0.1 versus 0.5 mMl3C2-acetate into T-cells and macrophages for 1 h and LC-MS based analysis of citrate labeling. M+2, M+3, and M+4 plots show the percent labeling of citrate by ’ ‘Ci-acetate for each i sotopologue. P values generated using one-way ANOVA and Dunnett’s multiple comparisons test compared ( 1)8 T-cells + 0.5 mM! 3C'2-acetate. ** = P < 0.01. *** == P < 0.001 *** * == P < 0.0001.

[0030] FIGS. 14A-G - Acetate improves human CD8~ T-cell proliferation and functionality' when glucose is limited. a,b, CDS ' T-ceil proliferation in the presence of high (10 mM) and low (1 mM) glucose and high (0.5 mM) and low (0.1 mM) acetate c, Work flow for analysis of polyfunctionality in human CD8" T-cells, d. Expression of degranulation and activation markers on CD8 T-cells after stimulation in the presence of increasing concentrations of acetate and two different concentrations of glucose. Dotted line represents expression levels in standard culture medium with 10 mM glucose and 0 1 mM acetate. n = 2 in duplicate. Data are from a representative experiment. Values are plotted as percent of total CD8+T-cells ± S.D. e. Percent of CD8 T-cells displaying polyfunctionality after stimulation in different concentrations of acetate and glucose, f, Splenocyte cytotoxicity assay displaying the mean percent of Brpkpl lO cells remaining after 3 days of co-culiure with total splenocytes at the indicated ratios Black circles represent WT (Cas9) Brpkpl l O cells and red circles represent Acss2-KO (sgAcss2) Brpkpl lO. n = 8. P values generated using one-way ANOVA and Dunnett’s multiple comparisons test compared to 1: 10 splenocyte: Brpkpl lO ratio. *:::P < 0.05. ** = P < 0.01. g, Schematic cartoon illustrating how targeting acetate metabolism promotes tumor clearance. Blocking tumor acetate metabolism leads to enrichment of acetate in the tumor microenvironment T-cells and NK-cells then exploit acetate via ACSS1 as an alternative nutrient source to promote tumor clearance.

[0031] FIGS. 15A-E - Acetate increases expression of cytokines and activation markers in T- cells cultured in low glucose, a, ACSS1, ACSS2 and ACSS3 expression in lymphocytes and monocytes from one male and one female healthy human donor, b. ACS SI mRNA expression represented as normalized transcripts per kilobase million (nTPM) from a scRNAseq analysis of normal human breast tissue c, Expression of degranulation and activation markers in human CD A T-cells after stimulation in the presence of increasing concentrations of acetate and two different concentrations of glucose. Dotted line represents expression levels in standard culture medium, n:::2 in duplicate. Data are from a representative experiment. Values are plotted as percent of total CD8" T-cells ± S.D. d, Glucose and acetate consumption by human CD8hT- cells during polyfunctional assay. Data are expressed as change in concentration during overnight stimulation, n:::2.

[0032] FIG. 16 - Tumor volume after treatment with ACSS inhibitors, PD-l inhibitor, find a combination.

[0033] FIGS. 17A-C - SAR Development of ACSS2 Inhibitors FIG. 17A: The ACSS2 inhibitor VY-3-135 is a potent enzyme inhibitor with ADME drug-like properties shown. FIG. 17B: JS-134-S is an example of an inhibitor comprising a trifluoromethyl group. VY-4-289 is corresponding the racemic compound. FIG. 1 7C: JS-148 and JS- J 49 are additional illustrative inhibitor compounds, each of which have improved potency compared to JS-I34-S and VY-4- 289. All analogs in the senes bi-phenyl and trifluoromethyl series show good metabolic stability and oral bioavail ability.

[0034] FIG. 18 -Swiss Dock Binding Poses. VY-3-135, an illustrative ACSS2 inhibitor comprising a bi-phenyl group, preferentially docks in the Acetyl -AMP pocket (green), while VY -4-283, an illustrative ACSS2 inhibitor comprising a trifluoromethyl group, preferentially docks in the HS-CoA pocket (orange).

[0035] FIG, 19 - Plasma Clearance curves for Mouse Pharmacokinetic Study with VY-4-283. The plasma drug concentration was determined after a single injection at 0.25, 0.5, 1, 2, 4, and 6 hours by either IV (2 mg / kg), PO (30 mg / kg), or IP (10 mg / kg) in CD1 mice. The compound showed good exposures by all routes of administration, with oral bioavailability, %F ~ 95%.

[0036] FIGS. 20A & B - SAR for ACSS2 inhibition, Cell viability, and Induction of Apoptosis. FIG. 20A: ACSS2 Inhibition in the AMP-Glo assay, cell viability, and caspase activation was determined in triple negative breast cancer cell line, MDA-MB-468 and ER / PR- positive breast cancer cell line BTB474. FIG 20B: Dose response curves for MTB-8552, JS- 138, and JS-148.

[0037] FIG. 21 - Examples of dose response curves with and without JS148 suggesting possible synergy. Follow-up of compounds showing possible synergy will entail further crosstitration to the two compounds and statistical analysis of synergistic versus combination effects.

[0038] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0039] Provided herein are methods of use of compounds and compositions that may be used treat one or more conditions associated with cancers. In particular, described herein are small -molecule inhibitors that can be used to inhibit ACSSl -driven acetate metabolism. Acetate metabolism is an important metabolic pathway in many cancers and is controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer is well described, the consequences of blocking tumor acetate metabolism on the tumor microenvironment and anti-tumor immunity are unknown. The present disclosure demonstrates that blocking ACSS2 switches cancer cells from acetate consumers to producers of acetate. Tumorinfiltrating lymphocytes then use acetate as a fuel source due to expression of acetyl -Co A synthetase 1 (ACSSl). ACSSl-driven acetate metabolism metabolically bolsters lymphocyte effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or a small molecule inhibitor promotes an anti-tumor immune response and enhances the efficacy of chemotherapy in preclinical breast cancer models. Proposed is a paradigm for targeting acetate metabolism in cancer in which inhibition of ACSS2 dually acts to impair tumor cell metabolism and potentiate anti-tumor immunity. These and more details will be described below;

[0040] I. Acetyl-CoA Synthetase Inhibitors

[0041] The methods described herein may contemplate the use of one or more Acetyl-CoA synthetase inhibitors. There are three major types of Acetyl-CoA synthetase enzymes that are common in humans within the short chain family that convert short-chain fatty acids to short-chain acyl-CoA’s in mammalian cells. ACSS’s mainly differ in their subcellular localization and substrate selectivity. Acetyl-CoA synthetase 1 (ACSSl) and ACSS3 are found in the mitochondria (Fujino et al., 2001; Yoshimura et al., 2017). ACSSl were shown to have a high specific activity for both acetate and propionate, while ACSS3 greatly prefers propionate and has almost no activity towards acetate (Miller et al., 2021). ACSS2 is anucleo- cytosolic enzyme that strongly prefers acetate (Miller et al., 2021; Luong et al., 2000) Given that acetyl-CoA does not freely cross the mitochondrial membrane, ACSSl and ACSS2 control distinct subcellular pools of acetate-derived acetyl-CoA. Acetyl-CoA is a versatile metabolite that is oxidized in the tricarboxylic acid (TCA) cycle or used for fatty acid biosynthesis and protein (histone) acetylation (Schug el al., 2016). (ACSS) that convert short-chain fatty acids to short-chain acyl-CoA’s in mammalian cells. Therefore, the present methods may contemplate the use of a pan ACSS inhibitor. In other aspects, the methods may be an inhibitor that specifically inhibits one or more particular subtype of ACSS, such as ACSS2. In some aspects, the present methods may further comprise the use of an ACSS2 inhibitor.

[0042] The inhibition of ACSS2 may be performed by the use of a small molecule or through a biological molecule such as an antibody or inhibited through the use of inhibitor}' nucleotides. In particular, the present methods may include the use of small molecule inhibitors of ACSS2. Some non-limiting examples of ACSS2 inhibitors like MTB-9655 such as pyrazole inhibitors such as those described in WO 2020 / 230134, US 202.3 / 0084752, US 2019 / 0263758 and WO 2019 / 097515, imidazole-3 -oxide inhibitors such as those described in US 2023 / 0174607, quinoxaline based inhibitors such as those described in WO 2020 / 06903 and WO 2019 / 067528, benzimidazole based inhibitors such as those described in WO 2015 / 175845. urea substituted quinoline inhibitors such as those described in WO 2021 / 014415, and tetrazole inhibitors such as those described in WO 2021 / 185793. Additionally, the present methods may make use of ACSS2 inhibitors such as those described in WO 2020 / 252407. These ACSS2 inhibitors may have a formula such as the formula below: wherein :

[0043] Xi and X2 are each independently selected from N, S, or O;

[0044] X ' and XT are each independently selected from C orN;

[0045] Rs is amino, cyano, halo, hydroxy, nitro, alkyl(c<i2j, cycloalkyl(c-,i2), alkenyVc<i2), alkynyhczi?), aryl(c<j2), heteroajy1(c<i2>, heterocycloalkyl(c<i2), alkoxy (C<12), alkylamino(cci2), didkylamino(c<i2), acyloxy (c<i2), amido(c<i2), or a substituted version thereof: or -NR'R", -C(O)R', ~C(O)OR'. “C(O)NR'R'r, -S(O)sR'. wherein x is 0, 1. or 2; and R' and R" are each independently selected from alkyl^m, cycloalkyl(c<i2), alkenyl(c<i2), alkynyl<c<i2), aryLon?), or a substituted version thereof; n is 0, 1 , 2, or 3; Ri and Rf are each alkyl(c<i2>, cycloalkyl(c<t2), alkeiiyl(c<i2), alkynyl(c<i2), aryhc 12), heteroaryl(cu2). heterocy cloaiks d(c ■ 21, or a substituted version thereof;

[0046] R2 is ammo, hydroxy, alkoxy (cxi 2), substituted alkoxy ccn 2), alkylamino(c<i2), substituted alkyl amino^n), dialkylamino(c<i2), substituted dialkylarnino(c:;i2), acyloxy(c<i2), substituted acyloxy(C*;i2), amido(c<i2), or substituted amido(c<:2),

[0047] R3 is alkyl(c<i2) or substituted alkyl.yuz);

[0048] Yj is a covalent bond. O. C(O), C(O)NRa. or NR»; wherein Rais hydrogen. alkyl(c<6), or substituted alkykcxe,; and

[0049] Ra is alkvl(fx!2), cycloalkyl(c<i2), alkenyl(c<i 2), alkynyl(c^2), arykxi?), heteroaryl(c<i2), heterocycloalkyl(c<i2>, or a substituted version thereof; or -AA-R. / . wherein:

[0050] ¥2 is alkanediyl(c<i2). arenediyl(c<i2). heteroarenediyl<c<i2>, or heterocy cloalkanediyl(c<i 2), or a substituted version thereof; and

[0051] R4' is alkyl(Ci:i2), cycloalkyl(c<i2>, alkenyl(c<i2), alkynyl(c<i2>, aiyl(c<i2), heteroarvl(c<i2), heterocvcloalkyl(c<i2), or a substituted version thereof; ~C(O)R4", or -SCOJsR / ", wherein

[0052] R-i" and R'" are each independently amino, hydroxy, alky’l(c<i2> cycloalkyl(c<i2), aikenyl(c<i2), alkynyl<c<i2), aryl(c<i2). heteroaryl(c<i2), heterocy cloalkyl(c-:i2), alkoxy(c<i2), substituted alkoxytcxi 2), cycloalkoxy(c<i2), substituted cycloalkoxy (c<i2), alkylamino(c<i2>, substituted alkylamino(Cii2), dialky lamino<c:,i2), substituted dialkylamino(c<i2), cycloalkylammo(c<i2>, substituted cycloalkylamino(c<i2), dicycloalkylamino(c<i2), substituted dicycloalkylamino(c<i2), or a pharmaceutically acceptable salt thereof.

[0053] II. Compounds and Formulations Thereof

[0054] A. Compounds of the Present Disclosure

[0055] The compounds of the present disclosure include ACSS inhibitors like MTB-9655 such as pyrazole inhibitors such as those described in WO 2.020 / 230134, US 2023 / 0084752, US 2019 / 0263758 and WO 2019 / 097515. imidazole-3 -oxide inhibitors such as those described in US2023 / / 0174607, quinoxaline based inhibitors such as those described in WO 2020 / 06903 and WO 2019 / 067528, benzimidazole based inhibitors such as those described in WO 2015 / 175845, urea substituted quinoline inhibitors such as those described in WO 2021 / 014415, and tetrazole inhibitors such as those described in WO 2021 / 185793. Additionally, the present methods may make use of ACSS2 inhibitors such as those described in WO 2020 / 252407. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by a person skilled in the art Such principles and techniques are taught, for example, in Smith, March ’s Advanced Organic Chemistry: Reactions, Mechanisms, ana Structure, (2013), which is incorporated by reference herein. In addition, the synthetic methods may be further modified and optimized for preparative, pilot- or large-scale production, either batch or continuous, using the principles and techniques of process chemistry7as applied by a person skilled in the art Such principles and techniques are taught, for example, in Anderson, Practical Process Research & Development - A Guide for Organic Chemists (2012), which is incorporated by reference herein.

[0056] All the compounds of the present disclosure may in some embodiments be used for the prevention and treatment of one or more diseases or disorders discussed herein or otherwise. In some embodiments, one or more of the compounds characterized or exemplified herein as an intermediate, a metabolite, and / or prodrug, may nevertheless also be useful for the prevention and treatment of one or more diseases or disorders. .As such unless explicitly stated to the contrary, all the compounds of the present disclosure are deemed “active compounds” and “therapeutic compounds” that are contemplated for use as active pharmaceutical ingredients (.APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting the public health by assuring the safety, effectiveness, quality, and security of human and veterinary drugs, vaccines and other biological products, and medical devices.

[0057] In some embodiments, the compounds of the present disclosure have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, more metabolically stable than, more lipophilic than, more hydrophilic than, anchor have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than, and / or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.

[0058] Compounds of the present disclosure may contain one or more asymmetrically- substituted carbon or nitrogen atom and may be isolated in optically active or racemic form. Thus, all chiral, diastereo meric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present disclosure can have the S or the R configuration. In some embodiments, the present compounds may contain two or more atoms which have a defined stereochemical orientation.

[0059] Chemical formulas used to represent compounds of the present disclosur e will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups Regardless of which tautomer is depicted for a given compound, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.

[0060] In addition, atoms making up the compounds of the present disclosure are intended to include all isotopic forms of such atoms Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and w ithout limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include1 :C and14C.

[0061] In some embodiments, compounds of the present disclosure exist in salt or non-salt form. With regard to the salt form(s), in some embodiments the particular anion or cation forming a part of any salt form of a compound provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0062] B. Pharmaceutical Formulations and Routes of Administration

[0063] In another aspect, for administration to a patient in need of such treatment, pharmaceutical formulations (also referred to as a pharmaceutical preparations, pharmaceutical compositions, pharmaceutical products, medicinal products, medicines, medications, or medicaments) comprise a therapeutically effective amount of a compound disclosed herein formulated with one or more excipients and / or drug carriers appropriate to the indicated route of administration. In some embodiments, the compounds disclosed herein are formulated in a manner amenable for the treatment of human and / or veterinary patients. In some embodiments, formulation comprises admixing or combining one or more of the compounds disclosed herein with one or more of the following excipients: lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol. In some embodiments, e.g., for oral administration, the pharmaceutical formulation may be tableted or encapsulated. In some embodiments, the compounds may be dissolved or slurried in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and / or various buffers. In some embodiments, the pharmaceutical formulations may be subjected to pharmaceutical operations, such as sterilization, and / or may contain drug carriers and / or excipients such as preservatives, stabilizers, wetting agents, emulsifiers, encapsulating agents such as lipids, dendrimers, polymers, proteins such as albumin, nucleic acids, and buffers.

[0064] Pharmaceutical formulations may be administered by a variety of methods, e.g, orally or by injection (e.g. subcutaneous, intravenous, and intraperitoneal). Depending on the route of administration, the compounds disclosed herein may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. To administer the active compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. In some embodiments, the active compound may be administered to a patient in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water -in- oil-in-water CGF emulsions as well as conventional liposomes

[0065] ’The compounds disclosed herein may also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordmaiy conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0066] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal. and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or poly alcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0067] The compounds disclosed herein can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compounds and other ingredients may also be enclosed in a hard or soft-shell gelatin capsule, compressed into tablets, or incorporated directly into the patient’s diet. For oral therapeutic administration, the compounds disclosed herein may be incorporated with excipients and used in the form of mgestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the therapeutic compound in the compositions and preparations may, of course, be varied. The amount of the therapeutic compound in such pharmaceutical formulations is such that a suitable dosage will be obtained.

[0068] The therapeutic compound may also be administered topically to the skin, eye. ear, or mucosal membranes. Administration of the therapeutic compound topically may include formulations of the compounds as a topical solution, lotion, cream, ointment, gel, foam, transdermal patch, or tincture. When the therapeutic compound is formulated for topical administration, the compound may be combined with one or more agents that increase the permeability of the compound through the tissue to which it is administered. In other embodiments, it is contemplated that the topical administration is administered to the eye. Such administration may be applied to the surface of the cornea, conjunctiva, or sclera. Without wishing to be bound by any theory, it is believed that administration to the surface of the eye allows the therapeutic compound to reach the posterior portion of the eye. Ophthalmic topical administration can be formulated as a solution, suspension, ointment, gel, or emulsion. Finally, topical administration may also include administration to the mucosa membranes such as the inside of the mouth. Such administration can be directly to a particular location within the mucosal membrane such as a tooth, a sore, or an ulcer. Alternatively, if local delivery' to the lungs is desired the therapeutic compound may be administered by inhalation in a dry' -powder or aerosol formulation.

[0069] In some embodiments, it may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity' of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary' dosages for the patients to be treated; each unit containing a predetermined quantity' of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some embodiments, the specification for the dosage unit forms of the disclosure are dictated by and directly' dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a patient In some embodiments, active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition m a patient. For example, the efficacy of a compound can be evaluated in an animal model sy stem that may' be predictive of efficacy' in treating the disease in a human or another animal.

[0070] In some embodiments, the effective dose range for the therapeutic compound can be extrapolated from effective doses determined in animal studies for a variety’ of different animals. In some embodiments, the human equivalent dose (HED) in mg / kg can be calculated in accordance with the following formula (see, e.g., Reagan-Shaw el al.. FASEBJ, 22(3):659- 661, 2008, which is incorporated herein by reference):

[0071] HED (mg / kg) = Animal dosee (mg / kg) x (Animal Km / Human Kra)

[0072] If se of the Kmfactors in conversion results m HED values based on body surface area (BSA) rather than only on body mass. Kmvalues for humans and various animals are well known. For example, the Kr„ for an average 60 kg human (with a BSA of 1 6 m2) is 37, whereas a 20 kg child (BSA 0.8 m2) would have a Kmof 25. Kmfor some relevant animal models are also well known, including: mice Kmof 3 (given a weight of 0.02 kg and BSA of 0.007); hamster Km of 5 (given a weight of 0.08 kg and BSA of 0.02); rat Kmof 6 (given a weight of 0. 15 kg and BSA of 0.025) and monkey (given a weight of 3 kg and BSA of 0,24). Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are specific to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment and the potency, stability and toxicity of the particular therapeutic formulation.

[0073] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a patient mav be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. These factors may be determined by a skilled artisan The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual patient. The dosage may be adjusted by the individual physician in the event of any complication.

[0074] In some embodiments, the therapeutically effective amount typically will vary from about 0.001 mg / kg to about 1000 nig / kg, from about 0.01 mg / kg to about 750 mg / kg, from about 100 mg / kg to about 500 mg / kg, from about 1 mg / kg to about 250 mg / kg. from about 10 mg / kg to about 150 mg / kg in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above). Other suitable dose ranges include 1 nig to 10,000 mg per day, 100 mg to 10,000 mg per day, 500 mg to 10.000 mg per day, and 500 mg to 1,000 mg per day. In some embodiments, the amount is less than 10,000 rag per day with a range of 750 mg to 9,000 mg per day

[0075] In some embodiments, the amount of the active compound in the pharmaceutical formulation is from about 2 to about 75 weight percent. In some of these embodiments, the amount is from about 25 to about 60 weight percent.

[0076] Single or multiple doses of the agents are contemplated. Desired time intervals for delivery of multiple doses can be determined by one of ordinary skill in the art employing no more than routine experimentation. As an example, patients may be administered two doses daily at approximately 12-hour intervals In some embodiments, the agent is administered once a day. Hie agent(s) may be administered on a routine schedule. As used herein a routine schedule refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical, or which differ in length, as long as the schedule is predetermined For instance, the routine schedule may involve administration twice a day, every day, every' two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between. Alternatively, the predetermined routine schedule may' involve administration on a twice daily' basis for the first week, followed by a daily basis for several months, etc. In other embodiments, the disclosure provides that the agent(s) may be taken orally and that the timing of which is or is not dependent upon food intake. Thus, for example, the agent can be taken every' morning and / or every evening, regardless of when the patient has eaten or will eat.

[0077] II. Methods of Treatment and Combination Therapies

[0078] A. Methods of Treatment

[0079] In particular the compositions that may be used in treating a disease or disorder in a subject (e.g,. a human subject) are disclosed herein. The compositions described above are preferably administered to a mammal (e g. , rodent, human, non-human primates, canine, bovine, ovine, equine, feline, etc.) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., slowing, stopping, reducing or eliminating one or more symptoms or underlying causes of disease). Toxicity' and therapeutic efficacy of the compositions utilized in methods of the disclosure can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject’s size, body surface area, body weight, age, the particular composition to be administered, time and route of administration, general health, the clinical symptoms and other drugs being administered concurrently. In some embodiments, the amount of the compounds used is calculated io be from about 0.01 mg to about 10,000 nig / day. In some embodiments, the amount is from about 1 mg to about 1,000 mg / day. In some embodiments, the compounds may be administered for 1 day to 20 days. In further embodiments, it is contemplated that the compounds may be administered for 1 day, 2 days, 3 days, 4 days. 5 days, 6 days, 7 days. 8 days. 9 days. 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days. 16 days, 17 days, 18 days, 19 days, or 20 days, or any range derivable therein. In some embodiments, the compounds may be administered for between 3 and 5 days, inclusive. In some embodiments, the compounds may be administered once. It is also contemplated that in some embodiments, the compounds disclosed herein may be administered two or more times. In some embodiments, these dosings may be reduced or increased based upon the biological factors of a particular patient such as increased or decreased metabolic breakdown of the drug or decreased uptake by the digestive tract if administered orally. Additionally, the compounds may be more efficacious and thus a smaller dose is required to achieve a similar effect. Such a dose is typically administered once a day for a few weeks or until sufficient achieve clinical benefit.

[0080] The therapeutic methods of the disclosure (which include prophylactic treatment) in general include administration of a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like).

[0081] Combination Therapies

[0082] It is envisioned that the ACSS inhibitors described herein may be used in combination therapies with one or more cancer therapies or a compound which mitigates one or more of the side effects experienced by the patient. It is common m the field of cancer therapy to combine therapeutic modalities. The following is a general discussion of therapies that may be used in conjunction with the therapies of the present disclosure.

[0083] To treat cancers using the methods and compositions of the present disclosure, one would generally contact a tumor cell or subject with a compound and at least one other therapy. These therapies would be provided in a combined amount effective to achieve a reduction in one or more disease parameter. This process may involve contacting the cells / subjects with the both agents / lherapies at the same time, e.g., using a single composition or pharmacological formulation that includes both agents, or by contacting the cell / subject with two distinct compositions or formulations, at the same time, wherein one composition includes the compound and the other includes the other agent.

[0084] Alternatively, the ACSS inhibitors described herein may precede or follow the other treatment by intervals ranging from minutes to weeks. One would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapies would still be able to exert an advantageously combined effect on the cell / subject. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay time of only about 1-2 hours. In some situations, it may be desirable to extend the time period for treatment significantly; however, where several days (2, 3, 4, 5. 6 or 7) to several weeks (1, 2, 3. 4, 5. 6. 7 or 8) lapse between the respective administrations.

[0085] It also is conceivable that more than one administration of either the compound or the other therapy will be desired. Various combinations may be employed, where a compound of the present disclosure is “A,” and the other therapy is “B.” as exemplified below

[0086] A / B / A B / A / B B / B / A AA / B B / A / A A / B / B B / B / B / A B / B / A'B

[0087] A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A'B / A B / AA / B B / B / B / A

[0088] A / A / A'B B / A / AA A / B / AA AA / B / A A'B / B / B B, A / B / B B / B / AB

[0089] Other combinations are also contemplated. The following is a general discussion of cancer therapies that may be used combination with the compounds of the present disclosure.

[0090] 1. Chemotherapy

[0091] The term “'chemotherapy’' refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used io connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA. or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0092] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclospbosphamide; alkyl sulfonates such as busulfan. improsulfan and piposulfan: aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyl enimines and methylamelamines including altretamme, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and tnmethyiolomelamiiie; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryosiatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1 ); eleulherobin: pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide. estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carrnustine, chlorozotocin, fotemustine, lorausline, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g, calicheamicin. especially calicheamicin yl and calicheamicin ®1; dynemicin, including dynemicin A; uncialamycin and derivatives thereof; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin. azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzmophilin, chromomycinis, dactinomycin, daunorubicin. detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peploraycin, potfiromycin, puromycm. quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5 -FIT); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaplopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine. azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine. enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, niepitiostane, testolactone; anti- adrenals such as aminogluteihimide, mitotane, trilostane; folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside: aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate: defofamme; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid: gallium nitrate; hydroxyurea: lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol: nitraerine; pentostatin: phenamet; pirarubicin: losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxm; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridm A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C "); cyclophosphamide; thiotepa; taxoids. e.g., paclitaxel and docetaxel; chlorambucil; gemcitabine: 6-thioguanine: mercaptopurine; methotrexate: platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine, platinum; etoposide (VP-16); ifosfamide: mitoxantrone; vincristine; vinorelbine; novantrone, teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g.. CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; capeci tabine, cisplatin (CDDP), carboplatin, procarbazine, mechlorethaniine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, phcomycm. mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, famesy 1-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate and pharmaceutically acceptable salts, acids or derivatives of any of die above.

[0093] 2. Radiotherapy

[0094] Radiotherapy, also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly.

[0095] Radiation therapy used according to the present disclosure may include, but is not limited to, the use of y-rays, X-rays, and / or the directed delivety of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV- irradiation. It is most likely that all of these factors induce a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA. and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 12.9 to 51.6 raC / 'kg for prolonged periods of time (3 to 4 wk). to single doses of 0.516 to 1 .55 C / kg. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and ty pe of radiation emitted, and the uptake by the neoplastic cells.

[0096] Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins that are made by the body in response to the presence of antigens (substances recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large quantities of these antibodies can be made in the laboratory and attached to radioactive substances (a process known as radiolabeling). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. Tills approach can minimize the risk of radiation damage to healthy cells.

[0097] Conformal radiotherapy uses the same radiotherapy machine, a linear accelerator, as the normal radiotherapy treatment but metal blocks are placed in the path of the x-ray beam to alter its shape to match that of the cancer. This ensures that a higher radiation dose is given to the tumor. Healthy surrounding cells and nearby structures receive a lower dose of radiation, so the possibility' of side effects is reduced. A device called a multi -leaf collimator has been developed and may be used as an alternative to the metal blocks. The multi-leaf collimator consists of a number of metal sheets which are fixed to the linear accelerator. Each layer can be adjusted so that the radiotherapy beams can be shaped to the treatment area without the need for metal blocks. Precise positioning of the radiotherapy machine is very’ important for conformal radiotherapy- treatment and a special scanning machine may be used to check the position of internal organs at the beginning of each treatment.

[0098] High-resolution intensity modulated radiotherapy also uses a multi -leaf collimator. During this treatment the layers of the multi-leaf collimator are moved while the treatment is being given. This method is likely to achieve even more precise shaping of the treatment beams and allows the dose of radiotherapy to be constant over the whole treatment area.

[0099] Although research studies have shown that conformal radiotherapy and intensity modulated radiotherapy may reduce the side effects of radiotherapy treatment, it is possible that shaping the treatment area so precisely could stop microscopic cancer cells j ust outside the treatment area being destroyed. This means that the risk of the cancer coming back in the future may be higher with these specialized radiotherapy techniques.

[0100] Scientists also are looking for ways to increase the effectiveness of radiation therapy. Two types of investigational drugs are being studied for their effect on cells undergoing radiation. Radiosensitizers make the tumor cells more likely to be damaged, and radioprotectors protect normal tissues from the effects of radiation. Hy perthermia, the use of heat, is also being studied for its effectiveness in sensitizing tissue to radiation. 3. Immunotherapy

[0101] In the context of cancer treatment, immunotherapeutic agents, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin™) is such an example. The immune effector may be. for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.

[0102] In one aspect of immunotherapy, the tumor ceil must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present disclosure. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP. estrogen receptor, laminin receptor, erb B and pl 55. An alternative aspect of immunotherapy is to combine anti cancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL- 12, GM-CSF, y-IFN, chemokin es such as MIP-1, MCP-1 , IL-8 and growth factors such as FLT3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor has been shown to enhance anti-tumor effects (Ju et al.. 2000). Moreover, antibodies against any of these compounds may be used to target the anti-cancer agents discussed herein.

[0103] Examples of immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801.005 and 5.739,169: Hui and Hashimoto, 1998; Christodoulides, et al , 1998), cytokine therapy, e.g. , interferons a, p, and y; IL-1, GM-CSF and TNF (Bukowski, etal., 1998; Davidson, etal., 1998; Hellstrand, et al., 1998) gene therapy, e.g., TNF, IL-1, IL-2, p53 (Qin et al.. 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5.830,880 and 5,846,945) and monoclonal antibodies, e.g, anti-ganglioside GM2, anti-HER- anti-pl 85 (Pietras, et al., 1998; Hanibuchi, et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the gene silencing therapies described herein

[0104] In active immunotherapy, an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor ceil composition or ‘'vaccine’’ is administered, generally with a distinct bacterial adj uvant (Ravindranath and Morton, 1991 ; Morton, et al. , 1992; Mitchel] , et al. , ! 990; Mitchell, e AV . 1993).

[0105] In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro. activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg, el al., 1988; 1989).

[0106] I. Checkpoint Inhibitors

[0107] In some aspects, the present disclosure provides for using or administering one or more immune checkpoint inhibitors that can bind to and / or antagonize an immune checkpoint molecule, e.g., PD-1, PD-L1, CTLA-4, VISTA, PD-L2, IDO, ARG1, B7- H3, B7-H4, LAG3, 2B4, BTLA, TIM3, A2aR, and / or a KIR An agent that selectively binds to an immune checkpoint molecule without limitation can be, e.g., an antibody or an antigenbinding fragment thereof, a protein or peptide, a small molecule, or a nucleic acid. An immune checkpoint molecule that is a nucleic acid can be. e.g., an antisense molecule, asingle- or double-stranded DNA oligonucleotide, a single- or double-stranded RNA oligonucleotide, a peptide nucleic acid (PNA), a single- or double-stranded RNAi molecule, an shRNA, or an siRNA. A small molecule is an organic compound drug. .An agent that selectively binds to an immune checkpoint molecule can bind io nucleic acids or amino acids of the immune checkpoint molecule sequence. An agent that selectively binds an immune checkpoint molecule can bind to any region of the immune checkpoint molecule. a. Immune Checkpoints

[0108] Immune checkpoints refer to inhibitory pathways hardwired into the immune system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. Immune checkpoint molecules can be stimidatoiy or inhibitory to an immune checkpoint. The present disclosure and claims refer to inhibitory molecules of immune checkpoints as “immune checkpoint molecules”. Preliminary clinical findings with agents that block immune checkpoint molecules (e.g., PD1 or CTLA-4), suggest opportunities to enhance antitumor immunity with the potential to produce effective clinical responses. In some aspects, the present disclosure provides for combining immune checkpoint blockade using immune checkpoint inhibitor(s) with ACSS inhibitors to enhances treatment efficacy in a patient having a cancer. b. Immune Checkpoint inhibitors and Immune Checkpoint Blockade

[0109] An immune checkpoint inhibitor is a ty pe of drug that blocks the signaling of immune checkpoint molecule(s) made by some types of immune system cells, such as T cells and some cancer cells. Immune checkpoint inhibitors therefore can cause immune checkpoint blockade. Immune checkpoint molecules (e.g., PD1) help keep immune responses in check and can keep T cells from killing cancer cells. When these molecules are blocked, the “brakes” on the immune system are released (inhibition of the immune system is reduced or blocked) and T cells are able to kill cancer cells better. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4. In some embodiments, immune checkpoint molecules are proteins. In some embodiments, immune checkpoint molecules are nucleic acids that encode the proteins. In some embodiments, immune checkpoint inhibitors bind to and / or antagonize immune checkpoint molecules. In some embodiments, immune checkpoint inhibitors are used in combination with an .ACSS inhibitor to treat a subject having cancer

[0110] In some embodiments, a method for treating a disease or a subject having a disease comprises administering the ACSS inhibitors and administering an agent that binds to and / or antagonizes programmed death 1 (PD-l), programmed death ligand 1 (PD-L1), cytotoxic T- lymphocyte-associated Antigen 4 (CTLA-4), and / or V-domain Ig suppressor of T cell activation (VISTA)

[0111] An inhibitor or antagonist, as used herein, is a molecule that inhibits, reduces, or blocks activity of an immune checkpoint molecule to inhibit a suppressive effect that the immune checkpoint molecule has on the immune system. The inhibitor or antagonist can directly bind the immune checkpoint molecule, a molecule controlling the expression of the immune checkpoint molecule, or a ligand of the immune checkpoint molecule that mediates the activity of the immune checkpoint molecule. The inhibitor or antagonist may be an antibody (including a humanized antibody), a small molecule, a peptide, or a nucleic acid (e.g., an antisense molecule, or a single- or double-stranded RNAi molecule). Activity of the immune checkpoint molecule is referred to as its suppressive effect on an immune checkpoint. An immune checkpoint inhibitor can reduce or block the activity of an immune checkpoint molecule. c. Types of Immune Checkpoint Molecules and Antagonists

[0112] Programmed Death 1 (PD-1) In humans, programmed cell death protein 1 (PD-1 ) is encoded by the PDCD1 gene. PDCD1 has also been designated as CD279 (cluster of differentiation 279). This gene encodes a cell surface membrane protein of the immunoglobulin superfamily. PD-1 is a 288 ammo acid cell surface protein molecule. PD-1 is expressed on the stirface of activated T cells. B ceils, and macrophages. PD-1 is expressed in pro-B cells and is thought to play a role in their differentiation. See T. Shinohara et al.. Genomics 23 (3): 704-6 (1995). PD-1 is a member of the extended CD28CTLA-4 family ofT cell regulators. (Y. Ishida el al.. “EMBO J. 1 1 (11): 3887-95, (1992)). PD-1 may negatively regulate immune responses. PDi limits autoimmunity and the activity of T cells in peripheral tissues at the time of an inflammatory response to infection

[0113] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is upregulated on macrophages and dendritic cells (DC) in response to LPS and GM- CSF treatment, and on T cells and B cells upon TCR and B cell receptor signaling, whereas in resting mice, PD-L1 mRNA can be detected in the heart, lung, thymus, spleen, and kidney. PD- L1 is expressed on almost all murine tumor cell lines, including PA myeloma, PH815 mastocytoma, and B16 melanoma upon treatment with IFN-y. PD-L1 has been found to be highly expressed by several cancers and several PD-1 antagonists are being developed or are approved for treatment of cancer. PD-L2 expression is more restricted and is expressed mainly by DCs and a few tumor lines.

[0114] Programmed Death 1 (PD-1) antagonist. A PD-1 antagonist, as used herein is a molecule that binds to PD-1 protein or to a gene or nucleic acid encoding PD-1 protein and inhibits or prevents PD-1 activation. Without wishing to be bound by theory', it is believed that such molecules reduce or block the interaction of PD-1 with its ligand(s) PD-L1 and / or PD- L2. In some embodiments, a PD-1 antagonist may reduce PD-1 activity in a ceil or organism more when combined with an ACSS inhibitor, than when administered alone, as compared to a cell or organism that has not been exposed to the PD-1 antagonist.

[0115] PD-1 activity may be interfered with by antibodies that bind selectively to and block the activity' of PD-1. The activity of PD-1 can also be inhibited or blocked by molecules other than antibodies that bind PD-1. Such molecules can be small molecules or can be peptide mimetics of PD-L1 and PD-L2 that bind PD-1 but do not activate PD-1. Molecules that antagonize PD-1 activity-' include those described in U.S. Publications 20130280265. 20130237580, 20130230514, 20130109843. 20130108651, 20130017199, and 20120251537, 2011 / 0271358, EP 2170959B1 , the entire disclosures of which are incorporated herein by reference. See also M. A. Curran, et al., Proc. Nall. Acad. Sei. USA 107, 4275 (2010); S. L. Topalian. et al., New Engl. J. Med. 366. 2443 (2012); J. R. Brahmer, et al.. New Engl. J. Med. 366, 2455 (2012); and D. E Dolan et al . Cancer Control 21, 3 (2014), all incorporated by reference herein, in their entireties. Herein, exemplary PD-1 antagonists include: nivolumab, also known as BMS-936558, OPDIVO® (Bristol -Meyers Squibb, and also known as MDX- 1106 or ONO-4538), a fully human IgG4 monoclonal antibody against PD-1; pidilizumab, also known as CT-01 1 (CureTech), a humanized IgGl monoclonal antibody that binds PD-1; MK- 3475 (Merck, and also known as SCH 900475), an TgG4 antibody that binds PD-1 ; and pembrolizumab (Merck, also known as MK-3475, lambrolizumab, or KEYTRUDA®), a humanized lgG4-kappa monoclonal antibody that binds PD-1; MEDI-0680 (AstraZerieca / Medlmmune). a monoclonal antibody that binds PD-1 : and REGN2810 (Regeneron / Sanofi), a monoclonal antibody’ that binds PD-1. Another exemplary PD-1 antagonist is AMP -224 (Glaxo Smith Kline and Amplimmune), a recombinant fusion protein composed of the extracellular domain of the PD-1 ligand programmed cell death ligand 2 (PD- L2) and the Fc region of human IgGl, that binds to PD-I. Agents that interfere bind to the DNA or mRNA encoding PD-1 also can act as PD-1 inhibitors. Examples include a small inhibitory anti-PD-1 RNAi, an anti-PD-1 antisense RNA, or a dominant negative protein. PDL- 2 fusion protein AMP-224 (co-developed by Glaxo Smith Kline and Amplimmune) is believed to bind to and block PD-1. In some embodiments, anti-PD-1 antibodies may be used for treatment in combination with an ACSS inhibitor in further combination with additional immune checkpoint blockade, e.g., with anti-PD-L, anti-CTLA-4, and / or anti-VISTA treatment.

[0116] Programmed Death-Ligand 1 (PD-Ll). In humans, programmed death-ligand 1 (PD- L), also known as B7 homolog 1 (B7-H1) or cluster of differentiation 274 (CD274), is a 40 kDa type 1 transmembrane protein that is encoded by the CD274 gene. Foreign antigens normally induce an immune response triggering proliferation of antigen-specific T cells, such as antigen-specific CD8+ T cells. PD-Ll is an immune checkpoint inhibitor that may block or lower such an immune response PD-L1 may play a major role in suppressing the immune system during events such as pregnancy, tissue allografts, autoimmune disease, and other disease states, such as hepatitis and cancer. The PD-L1 ligand binds to its receptor. PD-1 , found on activated T cells, B cells, and myeloid cells, thereby modulating activation or inhibition In addition to PD-1, PD-L1 also has an affinity for the costimulatory molecule CD80 (B7-1). Upon IFN-T stimulation, PD-L1 is expressed on T cells, natural killer (NK) cells, macrophages, myeloid dendritic cells (DCs), B cells, epithelial cells, and vascular endothelial cells.

[0117] PD-L1 Antagonist A PD-L1 antagonist, as used herein, is a molecule that binds to PD- L1 protein or to a gene or nucleic acid encoding PD-L protein and inhibits or prevents PD-1 activation. Without wishing to be bound by theory, it is believed that such molecules reduce or block the interaction of PD-L 1 with PD-1. PD-L I activity may be blocked by molecules that selectively bind to and block the activity of PD-L1 In some embodiments, a PD-L1 antagonist may reduce PD-L1 activity in a cell or organism more when combined with an ACSS inhibitor treatment, than when administered alone, as compared to a cell or organism that has not been exposed to the PD-1 antagonist. Anti-PD-L antibodies block interactions between PD-L I and both PD-1 and B7-1 (also known as CD80). Block means inhibit or prevent the transmission of an inhibitory1signal mediated via such PD-L1 binding. PD-L1 antagonists include, for example: BMS-936559, also known as MDX-1105 (Bristol -Mey ers Squibb), a fully human, high affinity, immunoglobulin (Ig) G4 monoclonal antibody to PD-L; MPDL3280A, also known as RG7446 or atezolizumab (Genentech / Roche), an engineered human monoclonal antibody targeting PD-L1 ; MSB0010718C, also known as avelumab (Merck), a fully human IgGl monoclonal antibody that binds to PD-L1 ; and MEDI473 (AstraZeneca / Medlmmune), a human immunoglobulin (Ig) G1K monoclonal antibody that blocks PD-L1 binding to its receptors. Agents that bind to the DMA or mRNA encoding PD-L1 also can act as PD- L inhibitors, e g., small inhibitory-’ anti-PD-Ll RNAi, small inhibitory anti-PD-Ll RNA, anti- PD-L1 antisense RNA, or dominant negative PD-L1 protein. Antagonists of or agents that antagonize PD-L1, e.g., anti-PD-Ll antibodies and I’D-Ll antagonists, may include, but are not limited to those previously mentioned and any of those that are disclosed in Stewart ei al., 2015. 3(9): 1052-62; Herbst et aL, 2014. Nature Volume: 515:Pages: 563-567; Brahmer et al., N Engl J Med 2012: 366:2455-2465; U.S. Pat. No. 8,168,179; US20150320859; and / or US20130309250, all incorporated herein by reference. In clinical trials, treatment with anti- PD-Ll antibodies resulted in less adverse events than did treatment with anti-PD-1 antibodies. In some embodiments. anti-PD-Ll antibodies may be used for treatment in combination with an ACSS inhibitor in further combination with additional immune checkpoint blockade, e.g., with anti-PD-1 , anti-CTLA-4, and / or anti-VISTA treatment.

[0118] Cytotoxic T-Lymphocyte- Associated Antigen 4 (CTLA-4). CTLA-4 (also known as CTLA-4 or cluster of differentiation 152 (CD 152)), is a transmembrane glycoprotein that, in humans, is encoded by the CTLA-4 gene. CTLA-4 is a member of the immunoglobulin superfamily, which is expressed on the surface of helper T cells and is present in regulatory T cells, where it may be important for immune function. CTLA-4, like the homologous CD28, binds to B7 molecules, particularly CD80 / B7-1 and CD86 / B7-2 on antigen-presenting cells (APCs), thereby sending an inhibitory signal to T cells. CTLA-4 functions as an immune checkpoint that inhibits the immune system and is important for maintenance of immune tolerance

[0119] CTLA-4 antagonist. A CTLA-4 antagonist, as used herein, is a molecule that binds to CTLA-4 protein or to a gene or nucleic acid encoding CTLA-4 protein and inhibits or prevents CTLA-4 activation Without wishing to be bound by theory, it is believed that such molecules reduce or block the interaction of CTLA-4 with its ligands, e.g., B7 molecules CD80 / B7-I and CD86 / B7-2. CTLA-4 activity may be blocked by molecules that bind selectively to and block the activity of CTLA-4 or that bind selectively to its counter-receptors, e.g., CD80, CD86, etc. and block activity of CTLA-4. Blocking means inhibit or prevent the transmission of an inhibitory signal via CTLA-4. In some embodiments, anti-CTLA-4 antibodies may7be used for treatment in combination with an ACSS inhibitor in further combination with additional immune checkpoint blockade, e.g., with anti-PD-1, anti-PD-Ll, and / or anti-VISTA treatment.

[0120] CTLA-4 antagonists include, for example, inhibitory antibodies directed to CD80, CD86, and / or CTLA-4; small molecule inhibitors of CD80, CD86, and CTLA-4; antisense molecules directed against CD80, CD86, and / or CTLA-4; adnectins directed against CD80, CD86, and / or CTLA-4; and RNAi inhibitors (both single and double stranded) of CD80. CD86, and / or CTLA-4.

[0121] Suitable CTLA-4 antagonists and / or anti-CTLA-4 antibodies include humanized anti- CT'LA-4 antibodies. such as MDX-010 / ipilimumab (Bristol-Meyers Squibb), tremelimumab / CP-675,206 (Pfizer; AstraZeneca), and antibodies that are disclosed in PCT Publication No. WO 2001 / 014424, PCT Publication No. WO 2004 / 035607, U.S. Publication No. 2005 / 0201994, European Patent No. EP 1212422 Bl, U.S. Pat. Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 7,034,121, 8,475,790, U.S. Publication Nos. 2002 / 0039581 and / or 2002 / 086014, the entire disclosures of which are incorporated herein by reference. Other anti- CTLA-4 antibodies and / or CTLA-4 antagonists that can be used in a method of the present disclosure include, for example, those disclosed in Hurwitz et al.. Proc. Natl. Acad Sci. USA, 95(17): 10067-10071 (1998), Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr el al., Cancer Res., 58:5301-5304 (1998), and Lipson and Drake, Clin Cancer Res; 17(22) Nov. 15. 2011; U.S. Pat. No. 8,318,916; and / or EP12I2422B1, all of which are herein incorporated by reference, in their entireties.

[0122] V-domain Ig suppressor of T cell activation (VISTA). V-domain Ig suppressor of T cell activation (VISTA), (also known as PD-H1, PD-1 homolog, or Dies I), is a negative regulator of T cell function. VISTA is a 309 aa type 1 transmembrane protein that is composed of seven exons, it has one Ig-V like domain, and its sequence is similar to the Ig-V domains of members of CD28 and B7 families. VISTA is highly expressed in the tumor microenvironment (TME) and on hematopoietic cells. It is also expressed on macrophages, dendritic cells, neutrophils, natural killer cells, and naive and activated T cells. Its expression is highly regulated on myeloid antigen-presenting cells (APCs) and T cells, while lower levels are found on CD4+T cells, CD84T cells, and T®8ceils. VISTA shows some sequence homology to the PD-I ligand, PD-I..1. however the two immune checkpoint inhibitors are structurally different and have different signaling pathways. VISTA blockade has been shown to enhance antitumor immune responses in mice, while in humans, blockade of the related PD-1 pathway has shown great potential in clinical immunotherapy trials. VISTA is a negative checkpoint regulator that suppresses T-cell activation and its blockade may be an efficacious immunotherapeutic strategy for human cancer. (Wang et al.. 201 1. JEM. 208(3):577-92: Lines et al., 2014. Cancer Res. 74(7): 1924-32; Kondo et al. 2015. J. of Immuno. V194; WO2011120013; US20140105912; LIS20140220012; US20130177557, US20130177557, incorporated by reference herein, in their entireties).

[0123] VISTA Antagonist. A VISTA antagonist, as used herein, is a molecule that binds to VISTA protein or to a gene or nucleic acid encoding VISTA protein and inhibits or prevents VISTA activation. Without wishing to be bound by theory, it is believed that such molecules reduce or block the interaction of VISTA with its ligand(s). VISTA activity may be blocked by molecules that selectively bind to and block the activity of VISTA. Molecules or agents that are VIST A antagonists include peptides that bind VISTA, antisense molecules directed against VISTA, single- or double-stranded RN.Ai molecules targeted to degrade or inhibit VISTA, small molecule inhibitors of VISTA, anti-VISTA antibodies, inhibitory antibodies directed to VISTA, and humanized antibodies that selectively bind and inhibit VISTA. Antagonists of or agents that antagonize VISTA, e.g., anti-VISTA antibodies and VISTA antagonists, are not limited to, but may include any of those that are disclosed in Liu et al. 2015. PNAS. 112(21 );6682-6687; Wang et al., 2011. JEM. 208(3):577-92; Lines et al., 2014. Cancer Res. 74(7): 192.4-32: Kondo et al. 2015. J. of Immuno. V 194; WO2015097536, EP2552947, WO2011120013, US20I40056892, U S. Pat. No. 8,236,304, WO2.014039983, US20140105912, US20140220012, US20130177557, WO20I5I91881; US20140341920; CN105246507; and / or US20130I77557, all of which are incorporated by reference herein, in their entireties. In some embodiments, anti-VISTA antibodies may be used for treatment in combination with an ACSS inhibitor in further combination with additional immune checkpoint blockade, e.g., with anti-PD-1, anti-PD-LI , and / or anti-CTLA-4 treatment.

[0124] Antagonists of the inhibitory immune checkpoint molecules may also be used in combination with the ACSS inhibitors described herein. In some embodiments, an antagonist of any immune inhibitory molecule may be used in combination with the use of ACSS inhibitors for treatment of a subject having a disease, wherein the disease is a cancer. In some embodiments, an antagonist of PD-1, PD-L1, CTLA-4, VISTA, PD-L2. IDO. ARG1, B7-H3. B7-H4, LAG3, 2B4, BTLA, TIMS, A2aR, KIR. and / or STAT3 may be used for treatment in combination with ACSS inhibitors in further combination with additional immune checkpoint blockade, e.g., with anti-PD-1. anti-PD-LI, anti-CTLA-4. and / or anti-VISTA treatment.

[0125] 4. Surgery

[0126] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present disclosure, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0127] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery' includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs’ surgery). It is further contemplated that the present disclosure may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0128] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1 , 2, 3, 4, 5. 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or even' 1, 2. 3, 4, 5, 6. 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well

[0129] In some particular embodiments, after removal of the tumor, an adjuvant treatment with a compound of the present disclosure is believed to be particularly efficacious in reducing the recurrence of the tumor. Additionally, the compounds of the present disclosure cars also be used in a neoadjuvant setting.

[0130] 5, Other Agents

[0131] It is contemplated that other agents may be used with the present disclosure. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma. IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-ip, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand. DR4 or DR5 / TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing abilities of the present disclosure by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents may be used in combination with the present disclosure to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present disclosure. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present disclosure to improve the treatment efficacy. There have been many advances in the therapy of cancer following the introduction of cytotoxic chemotherapeutic drugs. However, one of the consequences of chemotherapy is the development / acqui sition of drug-resistant phenotypes and the development of multiple drug resistance. The development of drug resistance remains a major obstacle in the treatment of such tumors and therefore, there is an obvious need for alternative approaches such as gene therapy.

[0132] Another form of therapy for use in conjunction with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a procedure in which a patient’s tissue is exposed to high temperatures (up io 106°F). External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe, including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes.

[0133] A patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets. Alternatively, some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated. Whole-body heating may also be implemented in cases where cancer has spread throughout the body Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.

[0134] The skilled artisan is directed to "Remington's Pharmaceutical Sciences” 15th Edition, chapter 33. in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA’s Division of Biological Standards and Quality7Control of the Office of Compliance and Biologies Quality7.

[0135] It also should be pointed out that any7of the foregoing therapies may prove useful by themselves in treating cancer. III. Treatment of Cancers

[0136] In accordance with the present disclosure, provided are methods of treating cancers. The method may be an administration of a compound or composition described by the present disclosure performed multiple times (2, 3. 4, 5, 6. 7, 8, 9, 10 or more) to a subject as needed. The subject may be an infant, a pediatric patient, a juvenile, ayoung adult, and adult or a senior. The subject may be a human, male or female, or may be a non-human mammal.

[0137] In certain aspects the cancer tumor is a renal cell cancer, melanoma, prostate cancer, chronic lymphocytic leukemia, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestrne, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma: transitional cell carcinoma: papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary’ adenocarcinoma; chromophobe carcinoma: acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary’ and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma: skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma: papillary’ cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma: adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant, lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma: glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant. myxosarcoma, liposarcoma; leiomyosarcoma; rhabdomyosarcoma: embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant: mullerian mixed tumor; nephroblastoma: hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant, phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma: hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pineal oma, malignant; chordoma; glioma, malignant: ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary’ astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal: cerebellar sarcoma, ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory-’ neurogenic tumor, meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant: granular cell tumor, malignant: malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse, malignant lymphoma, follicular, mycosis fungoides; other specified non-Hodgkirfs lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma: immunoproliferative small intestinal disease: leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma ceil leukemia, myeloid leukemia; basophilic leukemia; eosinophilic leukemia.; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy7cell leukemia.

[0138] IV. Chemical Definitions

[0139] When used in the context of a chemical group: “hydrogen” means ~~H; “hydroxy” means -OH; “oxo” means =0; “carbonyl” means -C(::::0)-; “carboxy” means -C(:::O)OH (also written as -C00H or -COzH); “halo” means independently -F, -Cl, -Br or -I; “amino” means -NH2; “hydroxy amino” means -NHOH; “nitro" means -NO2; imino means =NH; “cyano” means -CN: “isocyanyi” means -N=C-O; “azido” means -Ny in a monovalent context “phosphate” means -0P(0)(0H)2 or a deprotonated form thereof; in a divalent context “phosphate’’ means -~OP(O)(OI 1)0 or a deprotonated form thereof; “mercapto” means SI I ; and “thio” means ~S; “thiocarbonyT’mens -C(:::S)-; “sulfonyl” means -~S(0)2~~; and “sulfinyl” means -S(0)-.

[0140] In the context of chemical formulas, the symbol means a single bond, ’ means a double bond, and means triple bond. The symbol represents an optional bond, which if present is either single or double. The symbol represents a single bond or a double bond. Thus, the formula covers, for example, . And it is understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that the covalent bond symbol when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Instead, it covers ail stereoisomers as well as mixtures thereof. The symbol when drawn perpendicularly for methyl) indicates a point of attachment of the group. It is noted that the point of attachment is typically only identified in this manner for larger groups in order to assist the reader in unambiguously identifying a point of attachment The symbol means a single bond where the group attached to the thick end of the wedge is “out of the page.” The symbol means a single bond where the group attached to the thick end of the wedge is “into the page”. The symbol means a single bond where the geometry' around a double bond (e.g., either A or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly' represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.

[0141] When a variable is depicted as a “floating group"' on a ring system, for example, the group “R” in the formula: then the variable may' replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed. When a variable is depicted as a “floating group” on a fused ring system, as for example the group “R” in the formula: then the variable may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hy drogen attached to group X, when X equals -CH---), so long as a stable structure is formed. In the example depicted, R may reside on either the 5-membered or the 6-membered ring of the fused ring system. In the formula above, the subscript letter “y” immediately following the R enclosed in parentheses, represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.

[0142] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: “Cn” or “C=n” defines the exact number (n) of carbon atoms in the group / class. “C<n” defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum number as small as possible for the group / class in question. For example, it is understood that the minimum number of carbon atoms in the groups “a1kyl(c<8)”, “cycloalkanediyl(c<8)”, “heteroaryl(c<8)”, and “acyl(c<s, / ’ is one, the minimum number of carbon atoms in the groups “alkenyfcss)”, “alkynyl(c<8)”, and “heterocycloalkyl(csg)’’ is two, the minimum number of carbon atoms in the group “cycloalkyl(c<8>” is three, and the minimum number of carbon atoms in the groups “aiykcssj” and “arenediyl<c<8)” is six. “Cn-ri” defines both the minimum (n) and maximum number (ri) of carbon atoms in the group. Thus, “alkyl(C2-io)” designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms “C5 olefin”, “C5-olefin”, “olefinccs ”, and “olefines” are all synonymous. Except as noted below, every carbon atom is counted to determine whether the group or compound falls with the specified number of carbon atoms. For example, the group dihexylamino is an example of a dialkylamino(c=i2> group: however, it is not an example of a dialkylamincvew.) group. Likewise, phenylethyl is an example of an aralkyl«>8) group. When any of the chemical groups or compound classes defined herein is modified by the term “substituted”, any carbon atom in the moiety replacing the hydrogen atom is not counted. Thus methoxyhexyl, which has a total of seven carbon atoms, is an example of a substituted alkyl(ci-o) Unless specified otherwise, any chemical group or compound class listed in a claim set without a carbon atom limit has a carbon atom limit of less than or equal to twelve.

[0143] The term “saturated” when used to modiiy a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of ketoenol tautomerism or imine / enamine tautomerism are not precluded. When the term “saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.

[0144] The term “aliphatic’" signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic compound or group. In aliphatic compounds / groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds / groups can be saturated, that is joined by single carboncarbon bonds (alkanes / alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes / alkenyl) or with one or more carbon-carbon triple bonds (alkynes / alkynyl).

[0145] The terra “aromatic” signifies that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4n +2 electrons in a fully conjugated cyclic 21 system. An aromatic compound or chemical group may be depicted as a single resonance structure; however, depiction of one resonance structure is taken to also refer to any other resonance structure. For example: Aromatic compounds may also be depicted using a circle to represent the delocalized nature of the electrons in the fully conjugated cyclic n system, two non-limiting examples of which are shown below:

[0146] The term "alkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of atachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen The groups -CH3 (Me), -CH2CH3 (Et), -CH2CH2CH3 (n-Pr or propyl), — CH(CH3)2(j-Pr, Tr or isopropyl), -CH2CH2CH2CH3 (n-Bu), -CH(CH3)CH2CH3 (sec-butyl), -CH2CH(CH3)2 (isobutyl), ~C(CH3)3(tert-butyl, t-butyl, z- Bu or Bu}. and -CH2C(CH3)3(neopentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of atachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH?- (methylene), -CH?CH?-, -CH2C(CZH3)2CH2-, and -CH2CH2CH?- are non-limiting examples of alkanediyl groups. The term “alkylidene” refers to the divalent group =CRR’ in which R and R’ are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include::::CH?, =CH(CH2CH3), and =C(CH3)2. An “alkane” refers to the class of compounds having the formula H-R. wherein R is alkyl as this term is defined above.

[0147] The term “cycloalkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH?)? (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permiting) attached to a carbon atom of the non- aromatic ring structure. The term “cycloalkanediyl” refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds. and no atoms other than carbon and hydrogen. The group is a non-limiting example of cycloalkanediyl group. A “cycloalkane” refers to the class of compounds having the formula H-R, wherein R is cycloalkyd as this term is defined above. lite term “alkenyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acy clic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CIT:CH2 (vinyl), ---CIT:::CHCH3, -CH=CHCH?CH3, ( H U ! H b (allyl), C! I -( H ( HCl ti. and -CH=CHCH=CH2. The term “alkenediyl” refers to a divalent unsaturated aliphatic group, with tw7o carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carboncarbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH~ -CH=C(CH3)CH2- ~CH=CHCH2~, and --CH2CH-CHCH2 - are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms “alkene” and “olefin” are synonymous and refer to the class of compounds having the formula H R. wherein R is alkenyl as this term is defined above. Similarly, the terms “terminal alkene” and “a-olefm” are synony mous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.

[0148] The term “alkynyl” refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carboncarbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromalic carbon-carbon double bonds. The groups “C^CH. -OCCH3. and --CH2CSCCH3 are non-limiting examples of alkynyl groups. The term “alkynediyl” refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon triple bond, no carbon-carbon double bonds, and no atoms other than carbon and hydrogen. The groups -C=C~, “-CsCCH2-, and -CH2C=CCH2“ are nonlimiting examples of alkynediyl groups. It is noted that while the alkynediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. An “alkyne” refers to the class of compounds having the formula H-R, wherein R is alkynyl.

[0149] The term “aryl” refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-]imiting examples of and groups include pheny] (Ph), methylphenyL (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e g, 4-phenylphenyl). The term “arenediyl” refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part, of one or more sixmembered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. Non-limiting examples of arenediyl groups include:

[0150] An “arene” refers to the class of compounds having the formula H-R. wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes.

[0151] The term “aralkyl” refers to the monovalent group --alkanediyl -aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.

[0152] The term “heteroaiyl” refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heteroaiyl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroaryl does not preclude the presence of one or more alky' 1 or ary l groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroaiyl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl. quinoxalinyl, triazinyi. tetrazolyl, thiazolyl, thienyl, and triazolyl. The term “A-heteroaryl” refers to a heteroaryl group with a nitrogen atom as the point of attachment. A “heteroarene” refers to the class of compounds having the formula H-R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.

[0153] The term “heterocydoalkyl” refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as ths point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heterocydoalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings are fused. As used herein, the term does not preclude the presence of one or more alky] groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocydoalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, tbiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl. tetrahydropyranyl. pyranyl. oxiranyl, and oxetanyl. The term ‘W-heterocycloalkyl’’ refers to a heterocydoalkyl group with a nitrogen atom as the point of attachment. A'-pyrrolidinyl is an example of such a group.

[0154] The term “acyl” refers to the group -~C(O)R, in which R is a hydrogen, alkyl, cycloalky], or aryl as those terms are defined above. The groups, -CHO, -C(O)CH3 (acetyl, Ac), C(O)CH2CH3, -C(O)CH(CH3)2, ('(O)CH(CI l.-i. . -C(O)C6HS, and -C(O)C6H4CH3 are nonlimiting examples of acyl groups. A “thioacyl” is defined in an analogous mariner, except that the oxygen atom of the group ~C(O)R has been replaced with a sulfur atom. -C(S)R. The term “aldehyde” corresponds to an alkyl group, as defined above, attached to a -CHO group.

[0155] The term “alkoxy” refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: OCI b (methoxy). -OCH2CH3 (ethoxy). — OCH2CH2CH3, — OCHfCHs)? (isopropoxy), or — OCfCHs);? (terf-butoxy) The terms “cycloalkoxy”, “alkenyloxy”, “alkynyloxy”, “aiyloxy”, “aralkoxy”, “heteroaiyloxy”, “heterocycloalkoxy”, and “acyloxy”, when used without the “substituted” modifier, refers to groups, defined as -OR. in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocydoalkyl, and acyl, respectively. The term “alkylthio” and “acylthio” refers to the group -SR. in which R is an alkyl and acyl, respectively. The term “’alcohol” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The term “ether” corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.

[0156] The term “alkylamino” refers to the group -NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: -NHCH3 and -NHCH2CH3. The term “dialky I amino” refers to the group -NRR'. in which R and R' can be the same or different alkyl groups Non-limiting examples of dialkylamino groups include: -N(CH3)2and -NfCHiXCHzCHa) The terms “cycloalkylamino”, “alkenylamino”, “alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylammo”, “heterocycloalkylamino”, and “alkoxyamino” when used without the “substituted” modifier, refers to groups, defined as -NHR. in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkoxy, respectively. A non-limiting example of an arylamino group is -NHCVH*. The terms “dicycloalkylamino”, “dialkenylamino”, “dialkynylamino”, “diarylamino”, “diaralkylamino”, “diheteroarylamino”, “diheterocycloalkylamino”, and “dialkoxyamino”, refers to groups, defined as -NRR'. in which R and R' are both cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkoxy, respectively. Similarly, the term alkyl (cycloalkyl)amino refers to a group defined as -NRR', in which R is alkyl and R' is cycloalkyl. The term “amido” (acylamino), when used without the “substituted” modifier, refers to the group -NHR, in which R is acyl, as that term is defined above A non-limiting example of an amido group is -NHC(O)CH3.

[0157] When a chemical group is used with the ‘"substituted” modifier, one or more hydrogen atom has been replaced, independently at each instance, by -OH, -F, -Cl, -Br, ‘I, -NHz, — NOz, -COzH, -CO2CH3, “CO2CH2CH3, -CN, -SH, -OCH3. -OCH2CH3, -C(O)CH3. -NHCH3. -NHCH2CH3. -N(CH3)2, -C(O)NH 2. -C(O)NHCH 3. ~CXO)N(CH3)2, ~OC(O )CH 3. “NHC(O)CH3, ~S(O)2OH, or -S(O)2NH2. For example, the following groups are non-limiting examples of substituted alkyl groups: -CH2OH. -CH2CI, -CF3, -CH2CN, -CHzC(O)OH, -CHZC(O)OCH3, -CH2C(O)NH2. -CH2C(O)CH3, -CH2OCH3. Cl 1 O(i ()i( H3. “CH2NH2, “CH2N(CH3)2, and -CH2CH2CI. The term “haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. --F, -Cl, -Br, or - I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, -CH2CI is a nonlimiting example of a haloalkyl. The term “fluoroalkyr is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups -CHzF, -CF3, and -CH2CF3 are non- limiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyI, and 2-chloro-2-phenyI-eth-l -yl. The groups, -CtOjCT-fcCF?,, --CO2H (carboxyl), -CO2CH3 (methyl carboxyl), -CO2CH2CH3, -C(0)NH2 (carbamoyl), and -CON(CHS)2, are non-limiting examples of substituted acyl groups. The groups -NHC(O)OCH3 and— NHCiOINHCHs are non-limiting examples of substituted amido groups.

[0158] As used herein, the term "functional group” refers to any chemical group or substituent covalently bound to a core structure Functional groups may include but are not limited to hydrogen, ail.} L alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, -OH, -F, -Cl, -Br, -I, -NH 2, -NO2, CO.-I L -CO2CH3, - CO2CH2CH3, -CN, ~SH, OCH ,. -OCH2CH3, -C(O)CH?, -NHCH3, -NFICH2CH3, XiCl k }.'. -C(O)NH2, -C(O)NHCH.3. — C(0)N(CH?)2, — OC(O)CH?, -NHC(O)CHS, -S(O)2OH, -S(0)?NH2 or a combination or substituted version of any of these groups.

[0159] The use of the word “a” or ‘"an,” when used in conjunction with the term “comprising” in the claims and / or the specification may mean ‘"one,” but it is also consistent with the meaning of ‘"one or more,” ‘'at least one,” and “one or more than one.”

[0160] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or patients.

[0161] An “active ingredient” (Al) or active pharmaceutical ingredient (API) (also referred to as an active compound, active substance, active agent, pharmaceutical agent, agent, biologically active molecule, or a therapeutic compound) is the ingredient in a pharmaceutical drug that is biologically active.

[0162] The terms ‘‘comprise,” “have” and '‘include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[0163] The term “effective,” as that term is used in the specification and / or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or '‘pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease.

[0164] An “excipient’’ is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug deli very system. Excipients may be used, for example, to stabilize the composition, to bulk up the composition (thus often referred to as “bulking agents,” “fillers,” or “diluents” when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharmaceutically acceptable versions of antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

[0165] As used herein, the term “IC50” refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.

[0166] An “isomer” of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs

[0167] As used herein, the term “patient” or “subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig. or transgenic species thereof. In certain embodiments, the patient or subject is a primate Non-limiting examples of human patients are adults, juveniles, infants and fetuses. As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are. within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity7, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0168] '‘Pharmaceutically acceptable salts’’ means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as L2-ethanedisulfonic acid, 2-hydroxyeihanesuJfonic acid, 2-naphthalenesulfonic acid. 3-phenylpropionic acid, 4,4’-methylenebis(3-hydroxy-2-ene- 1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenes ulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic, acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid. / Moluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butyl acetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, A’-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0169] A “pharmaceutically acceptable earner,” “drug carrier,” or simply ‘'carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and / or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and / or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery' to the specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-gly colic) acid), albumin microspheres, synthetic polymers, nanofibers, protem-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.

[0170] A “pharmaceutical drag” (also referred to as a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicine, medication, medicament, or simply a drug, agent, or preparation) is a composition used to diagnose, cure, treat, or prevent disease, which comprises an active pharmaceutical ingredient (API) (defined abov e) and optionally contains one or more inactive ingredients, which are also referred to as excipients (defined above).

[0171] “Prevention” or “preventing” includes: ( I ) inhibiting the onset of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display' any or all of the pathology or symptomatology' of the disease, and / or (2) slowing the onset of the pathology or symptomatology of a disease in a subj ect or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology' of the disease.

[0172] A “stereoisomer'’ or “optical isomer" is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. “Enantiomers” are stereoisomers of a given compound that are mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer. In organic compounds, the chiral center is Apically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g , tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed 2", where n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. It is contemplated that that for any stereocenter or axis of chirali ty for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and 5' forms, including racemic and non-racemic mixtures. As used herein, the phrase ‘'substantially free from other stereoisomers” means that the composition contains < 15%, more preferably < 10%, even more preferably < 5%, or most preferably < 1% of another stereoisomer(s).

[0173] ■‘Treatment” or ‘"treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and / or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g. , reversing the pathology and / or symptomatology), and / or (3) effecting any measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

[0174] The term "‘unit dose” refers to a formulation of the compound or composition such that the formulation is prepared in a manner sufficient to provide a single therapeutically effective dose of the active ingredient to a patient in a single administration. Such unit dose formulations that may be used include but are not limited to a single tablet, capsule, or other oral formulations, or a single vial with a syringeable liquid or other injectable formulations.

[0175] The above definitions supersede any conflicting definition in any reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the disclosure in terms such that one of ordinary skill can appreciate the scope and practice the present disclosure.

[0176] V. Examples

[0177] The following examples are included to demonstrate preferred embodiments of the disclosure. Il should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0178] Example 1 - Tumor growth suppression by ACSS2 inhibition enhanced in immune- competent mice versus immune-deficient mice.

[0179] To test the effectiveness of ACSS2 inhibition in immune-competent models, tumor growth was measured across four mouse breast cancer models (A7C11, Til, T12, Brpkpl 10) treated with VY-3-135 and injected into immune-competent mice versus immune- deficient mice (Fig. la). In all four models. VY-3-135 treatment caused stronger breast turner growth inhibition in immune-competent mice compared to immune- deficient mice (Fig. 1b- b). The presence of VY-3-135 in the tumors was confirmed by LC-MS based metabolomics (Fig. 2a). Likewise, the in vivo on-targel activity of VY-3-135 was confirmed by in vivo13C2- acetate stable isotope tracing (Fig. 2a). Importantly, there was no change in the proportion of citrate labeled by acetate indicating that VY-3-135 does not affect the activity of ACSS1 in the mitochondria (Fig. 2a). y4m'2-knockoiit C-lc5.«2-KO) mouse breast cancer cell hues were generated using a CRIPSR / Cas9 approach and tumor growth was followed in immune-deficient versus immune- competent mice to determine the effects of tumor-specific loss of ACSS2 (Fig. la). .4u«2-KO cell lines recapitulated the effects of VY-3-135, where depleting ACSS2 resulted in much stronger tumor growth inhibition in immune-competent mice (Fig. li-j and Fig. 2b- f). In many instances, knockout of Acss2 led to complete clearance of tumors in immune- competent mice leading to significant improvements in overall survival (Fig. Ij and Fig. 2b- k).

[0180] Tumor-bearing mice were also dosed for four weeks with VY-3-135 and then the dosing was stopped and the animals were monitored for an additional 120 days Significant improvement of survival was found in the inhibitor treated groups, with some VY-3-135 treated mice having undetectable tumors by the end of the study (Fig. 21). Importantly, reexpression of sgRNA-resistant mouse ACSS2 m Ac.vs'2-KO cells rescued tumor formation in immune-competent mice (Fig. 2g, j-k). Altogether, the VY-3-135 and rtc’.s'vz-KO tumor studies demonstrated that targeting tumor acetate metabolism is more efficacious when the tumor- bearing host is immune-competent. Example 2 - Depletion of T-celis or NK-cells restored growth of tumors that have loss oL4css2

[0181] The findings in immune-competent versus immune-deficient hosts led to the examination of whether host immunity may be controlling Acss2-KO tumor growth. WT versus A css2-K() tumors as syngenei c allografts were grown in immune-competent mice treated with immune cell-specific depletion antibodies Ac$s2-KO tumor growth significantly increased in mice depleted of CD4+, CD8+, or both CD4+and CD8!T-cells (Fig. 3a-d and Fig. 4a). The rescue ofAcss2-KO tumor growth was also seen in Rag2- KO mice, which lack mature T and B-cells (Fig. 4b). Depletion of natural killer (NK) cells was further found to allow Acss2-KO tumors to grow in otherwise immune-competent mice (Fig. 3e-f and Fig. 4c-d). Overall, loss of ACSS2 expression in mouse breast tumors promoted an anti-tumor immune response that involved T-cells and NK-celis.

[0182] Next, it was sought to determine if immune cell-mediated control of Acss2-KO tumors would lead to memory responses capable of preventing the growth of tumors with wild type ACSS2 expression (Fig. 3g). Acs.s'2-KO breast cancer cells were injected into immune- competent syngeneic mice and allowed to grow for 30 days, after which Acss2- KO tumors were no longer palpable. Acss2 WT breast cancer cells were then injected into the opposite side of the mouse and continued to follow tumor growth for 30 more days. All naive mice injected with Acss2 WT-cells effectively established tumors within weeks (Fig. 3h-i). However, all mice that had previously been exposed to Acss2-KO tumors completely prevented the growth of WT breast tumors (Fig. 3h-i). Together these data demonstrate that targeting ACSS2 within a tumor cell leads to enhanced clearance by host immunity with formation of memory' capable of controlling the growth of future insults by WT breast tumors

[0183] Example 2 - immune-competent Acss2'!mice are still capable of suppressing Aess2- KO tumor growth

[0184] A Acss2 whole body knockout (Ac.s-.y2''') mouse that harbors a single nucleotide insertion in exon 3 (Fig. 5a-d) was obtained. Tumor-bearing Acss 2" ' mice remained capable of clearing Acss2- KO tumors (Fig. 5e). This concept was also tested using bone marrow (BM) chimaeras, where WT or Acss 2"' donor BM was introduced into C57B1 / 6 mice conditioned with busulfan. Mice with donor BM fmmAcss2~'' mice controlled tumor growth as well as mice that had received BM from a wild type mouse (Fig. 5f-g). Overall, these studies highlight that systemic inhibition of ACSS2 in immune-competent mice by VY -3-

[0185] 135 or genetic knockout of Bcss2 does not impair the ability of mice to clear 4c5s2-K0 tumors.

[0186] Example 3 - ACSS2 inhibitor-treated tumors display immune cell activation gene expression signatures

[0187] QuantSeq 3' mRNA-sequencing was performed on bulk breast tumor tissue from vehicle versus VY-3-135 (ACSS2i, 15 days) treated NSG and C57B1 / 6 mice Surprisingly, there was upregulation of genes (163 genes; >1.3-fold change, FDR<5%) associated with antigen presentation {B2m, H2-K1) and immune cell migration, differentiation, and activation (CxcllO, Cxcl9. Ccl5) which likely reflects neutrophil and monocyte (i.e., innate immunity) infiltration into tumors in NSG mice. Indeed, the most highly enriched gene was Ly6c2 (+6.88), a marker for monocytes (Fig. 6a).

[0188] Gene expression analysis of breast tumors treated with VY-3-135 for five days in immune-competent mice found 64 genes in common between the models, including multiple MHC class I genes, Ccl5, CxcllO, and other genes associated with myeloid cell activation (430 genes: > 1.2-fold change, FDR<5%) (Fig. 6a-c). In contrast to tumors from immune- deficient mice, tumors from immune-competent mice additionally contained markers of T- cell activation (Prfl, Gzma, Gzmb, Cd2, Il2ra, Sell). Gene set enrichment analyses (GSEA) predicted significant increases (FDR<5%, Z score>2) in pathways associated with immune cell movement and infiltration (Fig. 6d-e). Interestingly, most pathways altered by VY-3- 135 treatment were associated with enhanced immune infiltration and activation and interferon signaling (Fig. 6d-e). Interferon-ip (IFNG) was predicted to be the most highly activated transcriptional regulator in the tumors following ACSS2 inhibition in immune- competent mice (Fig. 6f-g). ACSS2 inhibitors do not induce changes in gene transcription in tumor cells.

[0189] Single cell RNA sequencing (scRNAseq) was used to distinguish gene expression differences among cell types within the tumor. Seurat clustering identified five groups: two tumor ceil clusters (0 and 1) and three immune cell clusters (2, 3, and 4) (Fig. 7a). Surprisingly, very few genes in the tumor cells were differentially regulated after seven days of VY-3-135 treatment (11 up, 0 down, FO1.2, FDR<5%) (Fig. 7b). Cluster 1 had higher expression of proliferation markers (Fig. 7c-f). Ho wever, there was no difference in the ratio of proliferating versus non-proliferating tumor cells. VY-3-135 treatment was therefore tested to determine if it synergized with anti -proliferative chemotherapeutic agents. Combinatorial treatment of breast tumors with paibociclib, doxorubicin, or paclitaxel further enhanced tumor growth inhibition compared to monotherapy, suggesting ACSS2 inhibitors act as immunomodulators that enhance the effects of chemotherapies on breast tumor growth (Fig. 7g-i).

[0190] Example 4 - Single cell RNA sequencing of ACSS2 inhibitor treated tumors revealed activation of immune cells

[0191] Gene expression changes in tumor-infiltrating immune cells were much more extensive compared to tumor cells (Fig. 8a-c). Clusters 2. 3, and 4 showed significant upregulation of many chemokines and cytokines associated with immune cell recruitment and activation (Ccl4, Ccl5, Ccl6, Ccll2, Il lb, Ly6c2, CxcllO) as well as cytotoxic enzymes (Gzmb, Clq, Lyz2) (Fig. 8b-c). Further clustering identified subpopulations of tumor- associated monocytes and macrophages (TAMs; subcluster 0), dendritic cells (DCs: subcluster 1 ), granulocytes (subduster 2), mature DCs (subcluster 3), and mast-cells (subcluster 4) (Fig. 9a-c). Of these, TAMs and DCs exhibited the greatest number of differentially regulated genes upon VY-3-135 treatment. The TAMs from VY-3-135 treated tumors had higher expression of chemokines (Ccl4, Ccl7, and Cell 2) and many surface markers, receptors, and adhesion molecules (Ly6c2, Ly6a. Feris, Fcgrl, Cx3crl, Ccr5, Cd72) (Fig. 9d). GSEA predicted significant activation of functions and regulators associated with leukopoiesis and recruitment and chemotaxis of antigen- presenting cells (Fig. 9e). DCs in subcluster 1 displayed the highest number of upreguialed genes (1 199 genes, FC>1.2, FDR<5%) in VY-3-135 treated tumors DCs from VY-3-135 treated tumors had higher expression of Feerig, Illb, Cxcl2, Cxcll6, Irf7, Lyz2, and multiple complement proteins (Clq) (Fig. 9f). GSEA predicted activation of phagocytosis and cell engulfment in addition to cell movement and proliferation, which were likely driven by TNF and IFNip signaling in the tumor microenvironment (Fig. 9g).

[0192] Turnor-infiltraiing lymphocytes were further subclustered into six populations: CDS T-cells (cluster 0). CD4* T-cells (cluster I ), Tregs (cluster 2). NK-cells (cluster 3), proliferating T-cells (cluster 4). and NKT-cells (cluster 5) (Fig. 8d-e and Fig. lOa-c). CD8+and CD4!T-cells were the most functionally activated and possessed the largest number of differentially regulated genes in response to VY-3-135, while the NK-cell cluster had high expression of GzmB (Fig. lOa-c). CD8+T-cells showed strong suppression of Saib I, Lefl, Il7r. Tcf7. and Klf2, suggesting differentiation from a naive to active state (Fig. 8f). CxcllO (+3.6)8) and .Yc'Y (+3.16)) were the two most highly enriched genes in CD84T-cells from VY- 3-135 treated tumors (Fig. 8f). The upregulation of memory marker Ly6c2 (+2.40) on CD8+T-cells was also observed (Fig. 8f).

[0193] In the CD4 T-cells (subcluster 1) from VY-3-135 treated tumors, enrichment of multiple markers of activated T helper cells was seen, such as Ctla2a (+7.22), Bhlhe40 (+6.25), and Rora (+4.36) (Fig. 8h) (Tibbitt et al., 2019). CD4+T-cells also showed upregulation of Tnfrsf4 (+6.19), a receptor that promotes expression of apoptosis inhibitor BCL-xL (Bcl2ll, +2.1 1 ). thereby promoting cell survival (Fig. 8h) (Rogers et al.. 2001 ). GSEA predicted enhanced immune response, proliferation and viability driven by TNFa, IFNvp, and interleukin signaling (Fig. 8g, i). Thus, scRNA-seq analysts largely mimicked that of the bulk tumor RNA-seq where VY-3-135 promotes a local inflammatory response and activation of both innate and adaptive immune cells, but further highlighted antigen-presenting cells, T-cells, and NK-cells as the likely effectors of tumor control.

[0194] Example 5 - Immunophenotyping of splenic and tumor-infiltrating leukocytes indicates activation of lymphocytes and dendritic cells in response to ACSS2 inhibition

[0195] The most ubiquitously upregulated gene across all cell ty pes was CCL5, which was enriched in cancer cells (+1.97), lymphocytes (+2.20) and myeloid cells (+2.23) from VY-3- 135 treated tumors (Fig. 7b, Fig. 8b~e). This upregulation in VY-3- 135 treated Brpkpl 10 tumors was confirmed by ELISA (Fig. Ila). METABRIC (Curtis et al , 2012) and TCGA (Cancer Genome Atlas Research et al., 2013) breast cancer gene expression databases for correlation was also investigated between ACSS2 and CCL5 in TNBC patient survival. It was found that while low ACSS2 coupled with high CCL5, which would mimic VY-3-135 treated tumors, predicts better survival in TNBC patients (p=0.002; HR=1.86) from the METABRIC database, the same was not true for TCGA breast cancer patients (p::;:0.82; HR:=1.14) (Fig. 11b). The reason for this discrepancy is currently unclear and further work will be needed to clarify the relationship between ACSS2 and CCL5 in anti-tumor immunity.

[0196] Immunophenotyping of tumor-infiltrating and splenic leukocytes from mice treated with VY-3-135 for 7 days and 28 days (Fig. lie) were next performed. 7 days of VY-3-135 treatment showed activation of CD84T-cells (GranzB4) and DC (CD861' and CD 103;CD864) (Fig. lid). Analysis of spleens from seven-day treated tumor-bearing mice showed decreased expression of PDl in NK-cells, while macrophages had increased expression of CD86 (Fig. lie). The immunophenotypic changes found at 28 days further expanded the number of immune cell subsets affected by VY-3-135 treatment with activation of CD8 . CD4+, B-cells, and DCs in the tumor, but also a decrease in immunosuppressive Argl+TAMs (Fig. Ilf). Splenic CD4:T-cells, NK-cells, and B-cells from VY-3-135 treated mice had higher expression of CD44 (Fig. 11g). Higher percentages of TNFa and IFNtp double positive CD8+T-cells and CD4+T-cells in spleens were additionally observed from VY-3- 135 treated mice (Fig. lib). Interestingly, a marked decrease in the percent of myeloid derived suppressor cells (MDSC) in the spleen was noted (Fig. llh). In sum, the immunophenotyping and scRNAseq data strongly suggest that ACSS2 inhibitors potentiate a large-scale anti-tumor immune response with activation of numerous immune ceil subtypes. T-cells and NK-cells readily oxidize acetate Acss2-KO or VY-3-135 inhibition of ACSS2 switched breast cancer cells from being acetate consumers to acetate producers (Fig. 7a). A recent study also showed that knockdown of ACSS2 or an ACSS2 inhibitor switches kidney cancer cells from acetate consumers to acetate producers (Bacigalupa et al., 2022). Based on these findings, that inhibition of tumor acetate metabolism was predict that it might cause breast cancer cells to release acetate and that immune cells could then exploit it as a fuel source. Peripheral blood mononuclear cells (PBMC) have relatively higher expression of ACSS1 and lower expression of ACSS2 compared to hepatocytes (Fig. 12b). Furthermore, ACSS 1 mRNA expression in purified mouse splenic CD8+and CD4+T-cells was substantially higher than bone marrow derived macrophages (BMDM) as well as Ml and M2 macrophages (Fig. 13a-c).

[0197] To investigate the ability of T-cells to oxidize acetate, an ex vivo stable isotope tracer study was performed into mouse splenic CD8+T-ceils isolated from wild type and Aess2~'' mice that were exposed to physiologically relevant concentrations of 0.1 mM and 0.5 mM13C2~acetate (Fig. 12c). CDS T-cells readily oxidize acetate in a time and concentration dependent manner with 0.5 mMl3C2-acetate labelling -70% of the citrate pool within two hours (Fig. 12d). a -ketoglutarate, glutamate, and malate also showed substantialk'C2-acetate labelling (Fig. 12e-g). Neither knockout of Acs .'? 2 nor VY -3-135 affected citrate labeling by13Cz-acetate (Fig. 12d-g). These findings show that ACSS2 expression and activity do not influence ACSS1 activity in CD8’ T-cells and that VY-3- 135 does not inhibit ACSS1. Similari 3C2-acetate labelling patterns were produced in mouse splenic NK-cells (Fig. 12h- k). In addition, CD8’ T-cells oxidize acetate more efficiently than BMDM, Ml macrophages, or M2 macrophages, which paralleled their level of ACSSi expression (Fig. 13c-d). Altogether, the stable isotope tracer studies suggest that blocking tumor acetate metabolism with an ACSS2 inhibitor could potentially metabolically bolster T-cells by7freeing up acetate as an alternative nutrient source.

[0198] Example 6 - Acetate improves T-cefi proliferation and (poly)ftmctionalily

[0199] ACSSi, ACSS2, and ACSS3 protein expression in human CD8fT- ceils, CD4+T- cells, NK-celis, B-cells, and monocytes were compared. Lymphocytes in general had much higher expression of ACSSI compared to monocytes (Fig. 15a). ACSS2 expression was comparable across ceil types, while ACSS3 was not expressed at appreciable levels in any of the immune cells (Fig. 15a). A scRNAseq dataset was also examined that analyzed gene expression in normal human breast tissue (Bhat-Nakshatri el al. , 2022: Bhat-Nakshatri el al., 2021). Again. T-cells had the highest expression of ACSSI compared to all other identified cell types in normal human breast tissue (Fig. 15b).

[0200] It was then tested if acetate supplementation improves CDS T-cell proliferation and function. In standard culture medium containing 10 mM glucose, acetate did not affect the proliferation of human CDS T-cells (Fig. 14a). In contrast, increasing acetate from 0 1 to 0.5 mM improved proliferation of CD8^ T-cells in the presence of low glucose (1 mM), as shown by a decrease in the number of CD8+T-cells that never divided and increases in the number of T-cells that divided multiple times (Fig. 14b).

[0201] The ability7of acetate to improve T-cell function at different glucose concentrations (Fig. 8c) were next tested. Lack of glucose (0 mM) impaired expression of degranulation and cytotoxicity' markers in T-cells, suggesting that some level of glucose is necessary for T- cell activation in response to CD3 / CD28 stimulation (Fig. 14d-e and Fig. 12c). in contrast, acetate improved human CD8+T-cell function (Fig. 14d) and polyfunctionality (Fig. 8e) in a concentration dependent, manner in the presence of 1 mM glucose Importantly, neither glucose nor acetate was depleted from the medium in the overnight cultures (Fig. 15d). These results suggest that acetate can metabohcally bolster CD8+T-cell metabolism and, by extension, CD3 / CD28 stimulated degranulation and effector functions when glucose availability is limited. The ability of total mouse splenocytes to target and eliminate mouse breast cancer ceils in co-culture were tested. While splenocytes modestly decreased WT breast cancer cell survival, 4cs.y2-KO breast cancer cells were much more sensitive to co-culture with splenocytes with cytotoxic effects starting at a 2: 1 splenocyte to cancer cell ratio (Fig. 14f).

[0202] Since ACSS2 expression directly correlates with acetate consumption in cancer cells it is possible that targeting ACSS2 within a tumor leads to decreased acetate uptake by tumor cells, thereby increasing the local availability of acetate within the tumor microenvironment Likewise, blocking ACSS2 prevents cancer cells from recapturing acetate released by deacetylation reactions (Bulusu el al, 2017; Bacigalupa el al., 2022). .Acetate may then be more readily available and consumed by immune cells in the tumor microenvironment, such as T-cells and NK-cells, through ACSS1 that then helps fuel anti-tumor immune responses (Fig. 14g). It is likely that myeloid cells in the tumor microenvironment also contribute to the anti-tumor immune response, particularly DCs. The low expression of ACSS1 in monocytes and macrophages suggests innate immune cells might recognize and react to changes in acetate metabolism through mechanisms independent of ACSS 1.

[0203] Example 7 - ACSS Inhibitors Enhance the Effectiveness of Checkpoint Inhibitors

[0204] The ACSS inhibitor, VY-3-135, was combined with checkpoint inhibitors such as mPD- 1 (CD279) in the treatment of mice. Using BrpkpllO+GFP+Luciferase ceils to generate a tumor, the mice 'were treated with an IgG2a isotype control (InVivoMAb, rat), BE0146 mPD-1 (CD279). vehicle (PBS 70%. DMSO 10%. Solutol 20% with 0.5% Tween 20), or the ACSS2 inhibitor, VY-3-135 (100 mg / 'kg daily oral gavage, starting on day 12 continuing until day 40), or combinations thereof Die compounds were given to 32 mice with 7 getting the vehicle and IgG2a isotype, 8 with the anti-PD- J , 7 with the ACSS2 inhibitor, and 7 with the combination of the anti-PD-1 and the ACSS2 inhibitor. The size of the tumor was measured in the mice and the effects showing improved efficacy when combined with an ACSS2 inhibitor as shown in FIG. 16. While one tumor fully regressed with the anti-PD-1 treatment alone, two tumors were fully regressed when the ACSS2 inhibitor was administered. Similarly, the average tumor size was statistically lower than merely the anti-PD-1 treatment alone. Die statistical analysis is shown below' in Table 1 .

[0205] Table 1: Statistical Comparison of Different T reatment Formulations

[0206] Example 8 -ACSS2 Inhibitor Development and Evaluation of ACSS2 Inhibitor Binding

[0207] Described herein is the development an ACSS2 inhibitor series. Biochemical assays were also developed to evaluate ACSS2 enzyme inhibition. The biochemical ACSS2 enzyme reaction (Scheme 1) is a two-step mechanism that first catalyzes the formation of an activated acetyl -AMP ester while generating pyrophosphate (Scheme 1 A).

[0208] Scheme 1: Biochemical Mechanism of ACSS2. A) Acetate reacts with ATP to form the activated ester, Acetyl-AMP and releases inorganic phosphate, PPi. B) Nucleophilic attack by the sulfur of CoA on the activated carbonyl of Acetyl-AMP, via a tetrahedral intermediate, results m the formation of AcCOA and AMP. This is followed by the nucleophilic attack of the sulfur of the bound Coenzyme A to form a thioester bond between Coenzyme A and acetic acid, to form AcCoA. and release AMP (Scheme IB)

[0209] ’The ACSS2 enzyme activity will be evaluated using the TranScreener TRF AMP / GMP assay (Bellbrook labs). This assay uses an AMP-tracer and terbium conjugated AMP-antibody to generate the HTRF signal. Displacement of the AMP-tracer by the AMP released during the reaction disrupts FRET from the AMP-tracer-antibody-bound terbium chelate, forming a non- fluorescent AMP-antibody-bound terbium chelate, leading to a decrease in the HTRF signal. The far-red tracer used in this assay minimizes interference from fluorescent compounds and light scattering. Thus, small molecule inhibitors block the release of AMP and prevent the decrease in the fluorescent signal. 'This assay has limitations due to sensitivity and low signal to noise, therefore a more sensitive assay was evaluated using the Promega AMP -Gio assay format (Mon dal etaL, 2017). This is a highly sensitive enzyme-coupled bioluminescence assay platform that first removes residual ATP, then converts the AMP produced during the reaction back into ATP so it can be detected using the luciferase / luciferin reaction. The data from the two assay formats correlated well with less than a 2-3-fold difference between the assay s which is within normal biological variance The AMP-Glo assay was found to be much more robust and reliable. Thus, a robust enzyme inhibition assay was developed for use in a drug discovery program.

[0210] In the absence of a binding assay, the enzy me assay was used to develop a lead series, resulting in compound VY -3-135 (Miller et al., 2021). To expand the SAR for the VY -3-135 lead series, the three main regions of the molecule (FIG. 17) were modified by changing the right-hand side tail region, the left-hand side bi-phenyl head group, and the central benzimidazole core. A key objective was to modify the bi -phenyl head group (FIG. 17A; a) by removing one of the phenyl rings with an aim to reduce the lipophilicity, improve water solubility (FIG. 17B; a). Modification of the bi-phenyl head group shifted the SAR so smaller alkyl -alcohols on the tail were no longer potent enzyme inhibitors (FIG. 17A; a,b). Therefore, the tail region was modified through a series of analogs to identify the “meta-aryl-sulfone moiety'’ (FIG. 17B: b) which provided analogs which regained potency as ACSS2 enzyme inhibitors. Notably , this confirmed that the SAR for the trifluoromethyl series was distinct from the bi-phenyl series.

[0211] ’The trifluoromethyl series was represented by the single enantiomer, JS-134-S and racemic VY-4-289, (FIG. 17B) provided an orally bioavailable lead series with good drug-like properties. Interestingly, when the single enriched enantiomers i.e., JS-134-S (FIG. 17B) and JS-138-R were synthesized, they were observed to be equally potent and either single enantiomer inhibited the enzyme with an IC50 - 30 nM. The lack of enantioselectivity suggested, without being bound by theory, that there is significant room in this region w here the compounds must be binding to allow either enantiomer enough flexibility to negate any stereochemical preference. Therefore, the series was further modified to simplify the compounds by removing the chiral center to produce JS-148 and JS-149 (FIG. 17C). Notably, this modification resulted in a 5-10-fold improvement in enzyme inhibition potency The significant change to the head group region strongly suggests, without being bound by theory, that the head group, including the attachment of a suitable linker, can be modified to synthesize a binding probe or other bi -functional molecule. An example of a bi -functional molecrde that is formed according to the present methods is a a PROTA C (Chimomas el al.. 2023; Bekes et al, 2022). To develop an ACSS2 targeted PROTAC requires an ACSS2 ligand conjugated to an E3 recruiting ligand (Adams et al., 2023; De Dominici et al., 2020).

[0212] To understand how ACSS2 inhibitors may be binding to ACSS2, a docking experiment involving one inhibitor comprising a bi-phenyl group and one inhibitor comprising a trifluoromethyl group to the ACSS2 structure. The X-ray structure of acetyl-CoA synthetase from Salmonella enterica complexed with adenosine-5’-propylphosphate and CoA is known at 1 ,7 A resolution (Gulick et al. , 2003). This structure represents the enzyme conformation at the start of the second half-reaction, where the adenosine-5 ’-propylphosphate serves as a mimetic of the acetyl- AMP activated ester (Scheme I B) poised for nucleophilic attack by the sulfur of CoA bound in the active site to generate AcCoA. Swiss dock (Bitencourt-Ferreira & de Azevedo, 2019) was utilized to evaluate potential docking poses of the inhibitor comprising a biphenyl group (VY-3-135) and the inhibitor comprising a -CFs group (VY -4-283). The illustrative biphenyl VY-3-135 preferentially docks in the Acetyl-AMP pocket with the hydroxy pointing into the putative oxyanion hole (FIG. 18; square), suggesting, without being bound by theoiy, that this analog is mimicking the tetrahedral transition state conformation after attack by HSCoA on the acetate carbonyl. In contrast, VY -4-283, an illustrative trifluoromethyl inhibitor, without being bound by theoiy appears to prefer binding in the HSCoA binding pocket. These alternative binding poses may explain, without being bound by theoiy, why the SAR for the -CF3 series is distinct from the bi-phenyl series. For example, the “meta-aryl-sulfone moiety’’ right hand side tail of VY-4-283 seems preferred, and the series is less tolerant to other modifications, suggesting, without being bound by theory, that a hydrogen bond is important between a sulfone (-SO?-) oxygen with Arg584 normally involved in a flbond with the phosphate (-PO3-) group of CoA in the back end of the CoA pocket (FIG. 18; red circle). In addition, a Pi -Cation interaction between the benzimidazole scaffold of VY-4- 283 and Argl91, in the CoA pocket, can be observed suggesting, without being bound by theory, an electron rich scaffold may enhance this interaction. This docking model tracks with observed SAR and helps guide design of additional inhibitors, such as replacing the 4-fluoro phenyl group in VY-4-283 with a polar substituent to engage in a H-bond used by the CoA. The model seems to be, without being bound by theoiy, consistent with the modifications to the head group by removal of the chiral center to produce the more potent inhibitors, JS-148 and JS-149 (FIG. 17C). Thus, the docking suggests structure-based rational design approaches to improve these inhibitors. Importantly (FIG. 19), VY-4-283 is orally bioavailable and two illustrative -CF3 analogs VY-4-283 and VY-4-289 (racemic JS-134-S: Scheme IB) are 100- 200-fold selective for ACSS2 and inactive against ACSS1 or ACSS3. Thus, compounds comprising a -CF3 group are expected, without being bound by theory, to provide the immune enhancement due to ACSS2 inhibition in cancer cells. Notably, the exemplary inhibitors JS- 148 and JS149 are also inactive al ACSS1.

[0213] Over 50 compounds were synthesized to develop improved ACSS2 inhibitors and SAR for enzyme inhibition, effects on cell viability (FIG. 20), and induction of apoptosis as measured by caspase activation, were determined. The ACSS2 inhibitors disclosed herein were compared to two known ACSS2 inhibitors HY-148104 and MTB-8552. The enzyme inhibition potency for the compounds shown (FIG. 20) are similar, yet the effects on ceil viability range from low micromolar to >40 pM potency and JS-148 (CTG IC50 == 1.7 pM; CASP IC50 = 21.9 pM) was the only compound to induce apoptosis highlighting the importance of the cell-based SAR for the senes. VY-3-135. which inhibits cell viability with an IC50 - 8-9 pM (FIG. 20) was reported to inhibit, 3C-acetate incorporation most effectively in the low micromolar range (Miller el al.. 2021 ) suggesting, without being bound by theory, that effects on cell viability are due to inhibition of ACSS2. However, enzyme potency alone does not trend with the SAR for effects on cell viability7since MTB-8552 potently inhibited ACSS2 y7et poorly affected cell viability. Compare this to JS-148 which has the same enzyme inhibition potency, yet potently inhibited ceil viability and was the only compound to induce apoptosis in the MDA-MB-468 cells, albeit at 21 uM

[0214] Example 9 - Synthesis of an ACSS2 binding probe for use in a competitive displacement HTRF binding assay

[0215]

[0216] Scheme 2: Synthesis Scheme to make linker conjugates and Probes. Scheme 2A: This synthetic route was used to synthesize AP-10-101. The key step involved synthesis of the versatile benzimidazole intermediate, AP-10-093 which is further elaborated to produce the linker conjugate, AP-10-101, as shown. Scheme 2A: Proposed JS-148-FITC probe. Scheme 2A: Proposed JS-148-BODIPY 630 / 650 probe.

[0217] The synthesis used to make JS-148 has been modified to make the linker conjugated analog of JS-148, AP-10-101 (Scheme 2A). The modified analogs for ACSS2 enzyme inhibition were evaluated to see if they retained activity. Importantly, the methoxy analog, AP- 10-096, and the tri-PEG linker analog, AP-10-101 retain ACSS2 enzyme inhibition activity, albeit slightly weaker, where AP-10-101 inhibits ACSS2 with an IC50 ~ 200 nM. The modified inhibitor was linked to the FITC probe to form JS-148-FITC (Scheme 2B). A suitable probe is required to bind with a KD < 500 nM. Thus, the KD of JS-148-FITC to GST-tagged ACSS2 will be determined using HTRF format by incubating with a terbium conjugated anti-GST antibody (Degorce et al, 2009). Fluorescent resonance energy transfer occurs between the terbium donor and the fluorescein acceptor molecule when in close proximity to each other. A saturation binding experiment will be performed using various concentrations of protein and the probe to identify optimal conditions for the assay. The inventors have extensive experience in assay development (Adam et al., 2023; Vaziri-Gohar et al., 2022) and the HTRF assay format is used routinely. The linker trajectory may be modified in an iterative fashion by evaluating different linkage attachment points on the head group aromatic ring and to the N of the benzimidazole ring by replacing the ethyl alkyl chain. This will be readily achieved by replacing E1NH2 in intermediate, AP- 10-086 (Scheme 2A) with a suitable alkyl or PEG based linker. Thus, a linker conjugated ligand with suitable binding affinity for use as a competitive binding probe will be identified.

[0218] Example 10 - Generation of an ACSS2-nanoluc expressing cell line and an ACSS2 fluorescent binding probe for cellular target engagement assays

[0219] The ability to develop SAR for in-cell binding affinity for ACSS2 inhibitors is key to fully understanding their cell biology and pharmacology. Bioluminescence resonance energy transfer (BRET) is dependent upon energy transfer between a bioluminescent donor (luciferase oxidizing its substrate) and a fluorescent acceptor (an ACSS2 ligand tethered to a fluorescent dye; for example, JS-148-BODIPY 630 / 650 (Scheme 2C). BRET has become the proximity assay of choice for many researchers owing to its ease of use and capacity for real-time monitoring in live cells (Stoddart et al. , 2015). The assay requires ACSS2 tagged with NanoLuc luciferase on its extra-cellular N-terminus to be expressed in living cells (Hall et al., 2012). NanoLuc is a small luciferase subunit (19 kDa) from the deep-sea shrimp Oplophorus gracilirostris, that has been optimized for improved luminescence expression in cells when treated with its imidazopyrazinone substrate (furimazine) and produces glow-type luminescence (signal half-life >2 h) with a specific activity ~ 150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays (Hall et al. , 2012). The small size of the NanoLuc provides minimal perturbation or interference with the protein it is fused with.

[0220] A system will be developed to express N-terminally fused ACSS2-NanoLuc in a stable cell line using the molecular biology expertise in the Wistar Molecular Screening Core (Dr. Salvino Sci Director), OriGene Technologies, Inc (Rockville MD), and Epoch Life Sciences, Inc (Sugar Land, TX) for custom cloning and gene synthesis. The inventors have already developed several stable cell lines for PROTAC and degrader technologies. Thus, the constructs available from Origene Technologies will be used to develop a robust and reliable AAVS1 Safe Harbor Site streamlined plasmid system. The All-in-one Cas9 SmartNuclease™ and AAVS 1 gRNA Plasmid will be used to express both Cas9 and an AAVS 1 -targeting gRNA, followed by co-transfection with a homologous recombination (HR) Targeting Vector and selection for desired colonies. The AAVS 1 safe harbor site is a preferred target for gene knock- ins and provides consistent, robust transgene expression. Insertion at the AAVS1 site has been shown to be safe with no phenotypic effects reported, and the surrounding DNA appears to be kept in an open confirmation, enabling stable expression of a variety of transgenes. Alternatively, the inventors have experience using the FLP-IN system (Invitrogen) and will develop an N-terminally fused Protein-NanoLuc stably expressed in CHO cells. The FLP-IN system introduces a Flp Recombination Target (FRT) site into the genome of the mammalian cell line that allows integration and expression of the gene of interest in mammalian cells at the FRT site (O'Gorman et al., 1991). An expression vector containing the gene of interest will then be then integrated into the genome via Flp recombinase-mediated DNA recombination at the FRT site.

[0221] A challenging aspect of developing a nanoBRET assay is the identification of a suitable tracer which is designed based on an ACSS2 inhibitor. As noted above an HTFR binding assay will first be developed to confirm that a suitable ACSS2-fluorescent dye probe, such as an ACSS2-FITC probe, has been formed that can be readily displaced with unlabeled ACSS2 inhibitors. Then a BODIPY fluor (Pfister et al. , 2020), which is cell permeable, will be utilized for conjugation to the ACSS2 ligand. Thus, BODIPY 630 / 650 NHS ester will be conjugated to the PEG-amine linker to make, as an example, JS-148-BODIPY-630 / 650.

[0222] Alternatively, linker attachment to the N of the benzimidazole ring will be evaluated, and if required use alternative dyes to optimize the signal and provide long lasting fluorescence (Gratz et al., 2020; Keller et al., 2011). An interesting dye is the pyrylium dye, Py-1, (Keller et al. , 2011) which may offer some benefits such as reduced adsorption of the fluorescent probe to plastics.

[0223] Example 11 - Design of ACSS2 PROTACs

[0224] 4-hydroxytamoxifen (4-OHT) treatment, a selective estrogen receptor modulator, was shown to increase ACSS2 expression in estrogen receptor-a positive (ER+) and 4-OHT resistant breast cancer cells (Calhoun et al. , 2022). Long-term estrogen deprivation (LTED) was also shown to elevate levels of ACSS2 and increase sensitivity to ACSS2 inhibitors (Calhoun et al. , 2022). Additionally, siRNA depletion of ACSS2 had a pronounced effect on reduction of proliferation and viability in the LTED cells and tumors. These results suggest, without being bound by theory, that an ACSS2 PROTAC may mimic the ACSS2 siRNA result and provide a robust therapeutic response, perhaps better than an inhibitor. An ACSS2 PROTAC is a bifunctional molecule comprising an ACSS2 targeting ligand suitably linked to an E3 ligase recruiting ligand to recruit an E3 ligase complex such as cereblon (CRBN), Von Hippel-Lindau (VHL), Mouse double minute 2 homolog (MDM2), or Inhibitor of Apoptosis (cIAP) in close proximity to ACSS2 to ubiquitinate the protein and target it for degradation by the 26S proteasome (Chirnomas et al., 2023; Bekes et al., 2022; Burslem et al. , 2018). The inventors have experience in the design and synthesis of PROTACs (Adams et al., 2023; De Dominici et al. , 2020) for cancer treatment and have found these to have improved efficacy in vivo over the inhibitor alone. PROTACs also can be readily optimized to provide orally bioavailable molecules with in vivo absorption properties suitable for clinical advancement (Chirnomas et al. , 2023). It’s also interesting to note that in many cases after treatment with an inhibitor the cancer cells compensate by upregulating the protein, and ultimately this can become a resistance mechanism, such as in the case with the CDK4 / 6 inhibitor Palbociclib (Li et al., 2020). Also, inhibitor treatment leading to upregulation of the target protein, requiring longer and higher treatment concentrations may result in toxicity to normal cells, as seen in the clinic (Ray-Coquard et al., 2012), for example with MDM2 inhibitors, suggesting that degraders may also provide an improved therapeutic index. Since ACSS2 expression is increased after LTED (Calhoun et al., 2022) an approach to degrade ACSS2 may be superior compared treatment with an inhibitor compound alone.

[0225] The development of the probe as described in the section above will aid in the identification of a PROTAC. For a PROTAC with the linker attached off the head group aromatic ring, a series of possible PROTACs will be made using four complimentary E3 ligase recruiting ligands to recruit CRBN, VHL, MDM2, and cIAP (examples of PROTACs; Scheme 3).

[0226]

[0227] Scheme 3: Design of ACSS2 PROTACs showing representative examples. Scheme 3A: A CRBN recruiting ligand conjugated to an ACSS2 inhibitor. Scheme 3B: A VHL recruiting ligand conjugated to an ACSS2 inhibitor. Scheme 3C: An MDM2 recruiting ligand conjugated to an ACSS2 inhibitor. Scheme 3D:. A cIAP recruiting ligand conjugated to an ACSS2 inhibitor.

[0228] Importantly, the linker attachment and trajectory of the E3 ligase recruiting ligand can significantly influence the efficiency of a PROTAC for targeted degradation. To identify an PROTAC for therapeutic use, several analogs varying the linker and modifying its hydrophobicity and rigidity will be made. Binding to ACSS2 will be evaluated using the developed ACSS2 assay. The formation of a ternary complex will be evaluated using the Alpha Screen assay i.e., using differentially tagged ACSS2 and the CRBN or VHL complexes (Adams et al., 2023). Formation of a ternary complex is key to the mechanism of a PROTAC and optimizing this attribute is predictive of efficiency of degradation (Adams et al., 2023). To optimize for degradation, an In-Cell Western assay in an adherent cell line will be utilized (Lu & Salvino, 2023). ACSS2-HiBit assay will be developed to monitor ACSS2 degradation (Schwalm et al., 2023). This assay uses a cell which expresses an ACSS2 fusion protein with a small nanoluciferase fragment which has high affinity to the complementary large bit nanoluciferase fragment so when the cell is lysed, and the large bit is added to the lysis buffer along with luciferase substrate there is bioluminescence, ACSS2 degradation by a PROTAC results in a decreased bioluminescence signal. The fusion protein will be generated in a similar manner as the ACSS2-nanoluc for the BRET assay as described above.

[0229] Example 12 - Combination studies to identify potential synergistic drug combinations using an unbiased approach

[0230] ACSS2 inhibitors will be evaluated in the MDA-MB-468 breast cancer cell line. Its ICso value will be calculated in a Cell Titer Gio cell viability assay and the -ICso will be used in combination with 4 concentrations of -3000 known cancer drugs with known pathways and mechanisms of action. This assay has been optimized by the inventors in other contexts and will be efficiently run in 1536 well format. Analysis of the results of this assay will allow the calculation of an estimated IC50 value and comparison of the ratio of the IC50 for DMSO treated to ACSS2 treated samples. Combinations which have a ratio of greater than 2 will then be cherry picked using the ECHO 650 acoustic liquid handler in an automated fashion to generate a cherry-picked set of compounds for follow-up. A screen using the IC30 of the ACSS2 inhibitor with 8 concentrations of the cancer drug identified from screening will be done, starting at concentrations based on their IC50 values in the MDA-MB-468 cells. As an example, after the first round of combination screening with JS-148, 47 compounds were identified that had improved potency compared to JS-148 alone. The 47 compounds were selected from the library and a further screening at 8 concentrations was carried out, which confirmed that 10 compounds appear to improve potency of JS- 148 in the Cell Titer Gio cell viability assay. The ten compounds will be ordered as fresh powder samples and additional cross-titrations will be carried out to vary the concentrations of both compounds to collect enough data to analyze this using the BLISS Index score (Demidenko & Miller, 2019; Liu et al., 2018). Examples of dose response curves show a leftward shift in the dose response curve in combination (FIG. 5). Thus, potential synergistic combinations useful for evaluation in in vivo efficacy studies to enhance the potency of ACSS2 inhibitor treatment will be identified. This information will complement work involving immunotherapies, immunogenic therapies, and radiation therapy.

[0231] Example 13 - Determine the ability of ACSS2 inhibitors to decrease tumor hypoxia and increase immune cell infiltration and functionality

[0232] To test the ability of ACSS2 inhibitors to target and eliminate tumor hypoxic regions from forming, Brpkpl lO and A7C11 tumor-bearing mice will be treated with an ACSS2 inhibitor (ACSS2i) (50 mg / kg; daily PO) for up to 4 weeks. Five mice per week per group from vehicle and ACSS2i-treated groups will be euthanized. Prior to euthanasia mice will be injected with pimonidazole (Li et al., 2022). Pimonidazole is a compound that binds to thiol-containing proteins specifically in hypoxic cells in tumors in vivo. The protein adducts can then be stained with antibodies by immunohistochemistry. Three serial sections (150 mm apart) from each tumor will be co-stained with anti-pimonidazole antibodies (Hypoxyprobe) (Li et al. , 2018) and by H&E. Digital images of the slides will be acquired and a mask will be generated for regions positive for pimonidazole. Images will be split into individual channels and an autothreshold determined by ImageJ software will be applied as well as smoothed edges and filled holes to reduce noise. The mean grey value per pixel will be measured to determine scores and ratios between hypoxic versus normoxic regions in vehicle versus ACSS2i-treated tumors. Additional sections will be cut to evaluate changes in immune cell infiltration through multiplex staining for markers of CD4+T-cells, CD8+T-cells, Tregs, NK-cells, macrophages, dendritic cells, and myeloid-derived suppressor cells. Immune cell types that are altered by ACSS2 inhibition will be further assessed for functionality by staining for markers of activation and / or differentiation. In this way, the formation of tumor hypoxia over time in tumors will be defined, along with its impact on immune cell infiltration, and its sensitivity to ACSS2 inhibitors.

[0233] Example 14 - Metabolomics as a pharmacodynamic readout of on-target activity of ACSS2 inhibitors in tumors An in vivo13C-acetate tracer approach will be used to determine the on-target activity of ACSS2 inhibitors (ACSS2i) in tumors (A7C11 and Brpkpl lO; n = 3 per treatment group), as previously described (Miller et al., 2021). In brief, mice will be provided drinking water supplemented with 2%13C-acetate. Tumor tissue will be flash frozen and then pulverized to powder at liquid N2 temperatures using a CryoMill (Retsch) before fatty acid extraction (i.e., Folch extraction followed by saponification). Fatty acids will be separated by Cl 8 chromatography and analyzed on a Q Exactive HF-X Orbitrap high resolution mass spectrometer (ThermoScientific). Decreased13C-labeling of fatty acids (i.e., palmitate, stearate, oleate) will be used as a readout of on-target inhibition of ACSS2 in tumors.

[0234] Example 15 - Chemotherapy-induced immunogenic responses will enhance the antitumor immune effects caused by ACSS2 inhibitors to create long-lasting immunity

[0235] To determine the ability of low-dose cyclophosphamide (CTX) and doxorubicin (DOX) to create immunogenic responses that synergize with ACSS2 inhibitors in tumors, T11 and T12 TNBC mouse models will be used. T11 and T12 tumor-bearing mice (BALB / c) will be treated with vehicle, CTX (100 mg / kg; once weekly IP injection), DOX (5 mg / kg; once weekly IP injection), or ACSS2 inhibitor (50 mg / kg; daily PO). The individual combination of CTX and DOX with ACSS2 inhibitors will also be tested, as well as “triple combination” of CTX+DOX+ACSS2 inhibitors. The treatment effect of ACSS2 inhibitors will be determined with or without combination of the second therapy (cyclophosphamide or doxorubicin). A 2x2 factorial design will be applied to the 4 groups (Vehicle, ACSS2 inhibitor, the 2nd therapy, and ACSS2 inhibitor + 2nd therapy) to determine the treatment effect of ACSS2 inhibitor, the 2nd therapy, and their combined effect. The combination effect of inhibiting tumor growth will be tested using two-way ANOVA with interaction. A significant interaction effect will indicate that the combination effect could be synergistic if it produces a level of tumor inhibition greater than the sum of what each single drug could achieve independently. Eight animals will be used per group. When an F test is used to test two factors interaction, such sample size will have 80% power at a two-sided type I error rate of 0.05 to detect an effect size of > 0.52 in a study outcome. Here effect size will refer to the ratio of standard deviation of the group means over the subject-to-subject standard deviation. It will also have 80% power at a two-sided type I error rate of 0.008 (Bonferroni’s adjustment for 6 comparisons in a 2x2 factorial experiment) when comparing any of the two treatment groups to detect a difference of > 2.0 times pooled standard deviation in a study outcome. Similar analysis will be performed for tumor weight. The effect of a triple combination (ACSS2 inhibitor + cyclophosphamide + doxorubicin) treatment in comparison to any combination of two drugs will be examined. One-way ANOVA will be applied. 8 animals per group will have >80% power at a two-sided type I error rate of 0.017 (Bonferroni’s adjustment for 3 group comparison) to detect an effect size of 1.8 in a study outcome.

[0236] Breast tumor material will then be split for flow cytometry -based immunophenotyping (i.e., markers of differentiation, activation, exhaustion) and digital spatial profiling by transcriptomics (GeoMx system from NanoString; Wistar Institute). For immunophenotyping, tumor tissue will be dissociated into single cell suspensions and flow cytometry-based analysis of tumor infiltrating leukocytes will be performed as previously described (Miller et al. , 2023). Conjugated-antibodies for use in these experiments will include (Zombie Aqua Viability Dye, TruStain FcX PLUS (anti-mouse CD16 / 32), CD3, CD19, CD45, MHC-I, CD206, F4 / 80, CDl lb, GR1, MHC-II, CD40, PDL1, CD86 , Ly6C, Ly6G, CDl lc, CD103, TNFa, IL-6, Argl, IL-12p40, IL-10, CD8a, CD4, CD44, PD1, CD62L, GranzB, TNFa, Ki67, CD44, IFNg, FoxP3, and CD25). The set will be expanded to include CXCL10, XCL1, Ly6A, CCL5, and Ly6C2 based on strong upregulation of these targets in scRNAseq dataset from vehicle versus ACSS2 inhibitor treated tumors (Miller et al., 2023). Marker expression will be compared across groups using t tests IP < 0.05). The impact of ACSS2 inhibitors on tumor-infiltrating CD8+ T- cells and NK-cells effector function will be evaluated. As an unbiased approach and to account for effects of CTX and DOX on tumor-infiltrating immune cells, digital spatial profiling (DSP), including whole transcriptomics, will be performed using the GeoMx system (NanoString) at the Wistar Institute. Multiple regions of interest (ROIs) will be selected across tumor sections representing immune cell-enriched (i.e., TILs) as well as hypoxic (i.e., pimonidazole positive) and normoxic tumor areas. DSP barcodes will be released by UV and then collected and sequenced through standard NGS procedures or counted using the nCounter® Analysis System. In collaboration with the Bioinformatics shared resource at the Wistar Institute, gene expression changes (FDR < 5%) will be evaluated and gene set enrichment analyses (Ingenuity; Qiagen) will be used to identify pathways for further intervention and targeting in combination with ACSS2 inhibitors.

[0237] Example 16 - Radiation therapy and ACSS2 inhibitors will induce potent immunogenic responses that promote anti-tumor immunity and tumor clearance

[0238] To test the ability of radiation therapy to create immunogenic responses that synergize with ACSS2 inhibition in tumors, Brpkpl 10 and A7C11 mouse breast cancer models will be used. For radiation therapy studies, the optimal dose and fraction of radiation as previously reported will be used (Dewan et al., 2009; Twyman-Saint Victor et al., 2015; Lee et al., 2009; Benci et al., 2019; Benci et al., 2016; Weichselbaum et al., 2008). All irradiation will be performed using the Small Animal Radiation Research Platform (S ARRP) housed in the Wistar Institute Animal Facility and overseen by the Imaging Facility Shared Resource at Wistar. Tumor-bearing mice (C57B1 / 6) will be treated with an ACSS2 inhibitor (50 mg / kg; daily PO from Day 1) and radiation therapy (8 Gy on three consecutive days starting on day 10 post tumor implantation). The combination of radiation therapy and ACSS2 inhibitor where radiation is first given on day 10 and then daily ACSS2 inhibitor treatment is initiated thereafter will be tested. Tumor growth will be monitored and analyzed as described above. In situations where complete tumor regression is observed, treatment will cease, and mice will continue to be monitored for up to 3 additional months for recurrence. All mice will be injected with an in vivo hypoxia probe called pimonidazole. Brpkpl lO model does form lung metastases (Rutkowski et al., 2014) and therefore lungs will be collected and blindly scored by a board- certified pathologist for tumor burden. Breast tumor material will be split for flow cytometrybased immunophenotyping (i.e., markers of differentiation, activation, exhaustion, etc) and digital spatial profiling (DSP) as described above.

[0239] Example 17 - Combination of ACSS2 inhibitors with potential synergistic drug combinations

[0240] In addition to the immunogenic therapies testing described in Examples 15 and 16, the combination of ACSS2 inhibitors with potential up to two synergistic therapies that will be identified through the unbiased screen described in Example 12 will be tested. For instance, Plinabulin, is an immunomodulating microtubule binding agent (Natoli et al., 2021) that is contemplated for use as an immunogenic chemotherapy in combination with ACSS2 inhibitors. Likewise, two different glycogen synthase kinase 3 (GSK3) inhibitors (LY2090314 and BIO) as potentially synergistic and are contemplated for use with ACSS2 inhibitors. Similar statistical considerations will be done as described in Example 15.

[0241] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. REFERENCES

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Claims

WHAT IS CLAIMED:

1. A method of modulating the immune response to a cancer cell in a patient comprising administering to the patient a therapeutically effective amount of an inhibitor of acetyl- CoA synthetase short-chain (ACSS).

2. The method of claim 1, wherein the ACSS is ACSS2.

3. The method of either claim 1 or claim 2, wherein the immune response is the increased production of one or more immune cells.

4. The method of claim 3, wherein the immune cell is a T cell.

5. The method of claim 4, wherein the T cell is CD4+T cell.

6. The method of claim 4, wherein the T cell is CD8+T cell.

7. The method of claim 3, wherein the immune cell is a natural killer cell.

8. The method according to any one of claims 1-7, wherein the immune response is a change in the amount of an immune cell of at least 10% over the amount before administration of the ACSS inhibitor.

9. The method of claim 8, wherein the change is at least 25%.

10. The method of either claim 8 or claim 9, wherein the change is at least 50%.

11. The method according to any one of claims 1-10, wherein the patient exhibits a normal immune response.

12. The method according to any one of claims 1-11, wherein the cancer cell is a part of a solid tumor.

13. The method according to any one of claims 1-12, wherein the cancer cell is a cancer cell that shows increased expression of one or more ACSS enzymes.

14. The method according to any one of claims 1-13, wherein the cancer cell is a breast cancer cell, an ovarian cancer cell, a brain cancer cell, a prostate cancer cell, a pancreatic cancer cell, a sarcoma cancer cell, a lung cancer cell, a colorectal cancer cell, a skin cancer cell, a bone or connective tissue cancer cell, a blood cancer cell, or a liver cancer cell.The method according to any one of claims 1-14, wherein the cancer cell comprises one or more genetic markers that impair the cancer cell’s effectiveness to one or more treatments. The method according to any one of claims 1-15, wherein the cancer cell is a breast cancer cell, a glioblastoma cell, an ovarian cancer cell, a prostate cancer cell, a melanoma cancer cell, a fibrosarcoma cancer cell, a multiple myeloma cancer cell, or a hepatocellular carcinoma cancer cell. The method according to any one of claims 1-16, wherein the patient is a mammal. The method of claim 17, wherein the mammal is a human. The method according to any one of claims 1-18, wherein the ACSS inhibitor is further defined as:wherein:R1and R1' are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Xi is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(O), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c≤6), or substituted alkyl(c≤6);Y3 is N, O, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4'", wherein 4'" and R4'" are each independently amino, hydroxy, alkyl(c≤12), substituted alkyl(c≤12), cycloalkyl(c≤12), substituted cycloalkyl(c≤12), alkenyl(c≤12), substituted alkenyl(c≤12), alkynyl(c≤12), substituted alkynyl(c≤12), aryl(c≤12), substituted aryl(c≤12), heteroaryl(c≤12), substituted heteroaryl(c≤12), heterocycloalkyl<c≤12), substituted heterocycloalkyl(c≤12), alkoxytc≤12), substituted alkoxy(c≤12), cycloalkoxy<c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof.The method according to any one of claims 1-19, wherein the ACSS inhibitor is further defined as:wherein:R1and R1' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R2 is amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12)!Y1 is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c≤6), or substituted alkyl(c≤6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4"', whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c≤12), or a pharmaceutically acceptable salt thereof.The method of either claim 19 or claim 20, wherein the compound is further defined as:wherein:R1and R1' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R3 is alkyl(c≤12) or substituted alkyl(c≤12);Y1 is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c≤6), or substituted alkyl(c≤6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4"', whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy<c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c≤12), or a pharmaceutically acceptable salt thereof.The compound according to any one of claims 19-21 further defined as:wherein:R1and R1' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R3 is alkyl(c≤12) or substituted alkyl(c≤12); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4"', whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c≤12), or a pharmaceutically acceptable salt thereof.The method according to any one of claims 19-22 further defined as:wherein:R1and R1' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4"', whereinR4'1and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c≤12), or a pharmaceutically acceptable salt thereof. The method according to any one of claims 19-23, wherein Ri is aryl(c≤12) or substituted aryl(c≤12). The method of claim 24, wherein Ri is aryl(c≤12). The method of claim 25, wherein Ri is phenyl.The method according to any one of claims 19-26, wherein Rf is aryl(c≤12) or substituted aryl(c≤12). The method of claim 27, wherein Ri ' is aryl(c≤12). The method of claim 28, wherein Ri ' is phenyl. The method according to any one of claims 19-29, wherein R2 is hydroxy. The method according to any one of claims 19-30, wherein R3 is alkyl(c≤12) or substituted alkyl(c≤12)- The method of claim 31, wherein R3 is alkyl(c≤12). The method of claim 32, wherein R3 is ethyl. The method according to any one of claims 19-30, wherein Y 1 is -C(O)NRa-, whereinRais hydrogen. The method according to any one of claims 19-30, wherein R4 is alkyl(c≤12) or substituted alkyl(c≤12). The method of claim 35, wherein R4 is substituted alkyl(c≤12). The method of claim 36, wherein R4 is 2-hydroxypropyl. The method according to any one of claims 19-37, wherein the ACSS inhibitor is further defined as:or a pharmaceutically acceptable salt thereof. A method of treating cancer comprising administering to the patient in need thereof a therapeutically effective amount of an ACSS inhibitor and a therapeutically effective amount of a therapeutic modality, wherein the therapeutic modality is a treatment for the cancer.The method of claim 39, wherein the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The method of claim 39, wherein the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid. The method according to any one of claims 39-41, wherein the therapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. The method of claim 42, wherein the therapeutic modality is a chemotherapeutic agent. The method of claim 42, wherein the therapeutic modality is an immunotherapy. The method according to any one of claims 39-44, wherein the patient is a mammal. The method of claim 45, wherein the mammal is a human. The method according to any one of claims 39-46, wherein the ACSS inhibitor is administered no more than 12 hours before the therapeutic modality. The method of claim 47, wherein the ACSS inhibitor is administered no more than 24 hours before the therapeutic modality. The method of claim 48, wherein the ACSS inhibitor is administered no more than 3 days before the therapeutic modality. The method according to any one of claims 39-49, wherein the patient is immunocompromised. The method according to any one of claims 39-49, wherein the patient has a healthy immune system. The method according to any one of claims 39-51 , wherein the ACSS inhibitor is further defined as:wherein:R1and R1' are each independently absent, halo; oralkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);X1 is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Y1 is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c≤6), or substituted alkyl(c≤6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4'", whereinR4''" and R4'" are each independently amino, hydroxy, alkyl(c≤12), substituted alkyl(c≤12), cycloalkyl(c≤12), substituted cycloalkyl(c≤12), alkenyl(c≤12), substituted alkenyl(c≤12), alkynyl(c≤12), substituted alkynyl(c≤12), aryl(c≤12), substituted aryl(c≤12), heteroaryl(c≤12), substituted heteroaryl(c≤12), heterocycloalkyl<c≤12), substituted heterocycloalkyl(c≤12), alkoxytc≤12), substituted alkoxy(c≤12), cycloalkoxy<c≤12), substitutedcycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof.The method according to any one of claims 39-52, wherein the ACSS inhibitor is further defined as:wherein:R1and R1' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R2 is amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Y1 is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c≤6), or substituted alkyl(c≤6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(O)R4", or -S(O)2R4'', whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c≤12), or a pharmaceutically acceptable salt thereof. The method according to any one of claims 39-53 , wherein the ACSS inhibitor is further defined as:or a pharmaceutically acceptable salt thereof. A method of treating cancer in a patient who has been administered an ACSS inhibitor comprising administering to the patient a therapeutically effective amount of a therapeutic modality that is a treatment for the cancer. The method of claim 55, wherein the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The method of claim 55 , wherein the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid.The method according to any one of claims 55-57, wherein the therapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. The method of claim 58, wherein the therapeutic modality is a chemotherapeutic agent. The method of claim 58, wherein the therapeutic modality is an immunotherapy. The method according to any one of claims 55-60, wherein the patient is a mammal. The method of claim 61, wherein the mammal is a human. The method according to any one of claims 55-62, wherein the ACSS inhibitor has been administered no more than 12 hours before the therapeutic modality. The method of claim 63, wherein the ACSS inhibitor has been administered no more than 24 hours before the therapeutic modality. The method of claim 64, wherein the ACSS inhibitor has been administered no more than 3 days before the therapeutic modality. The method according to any one of claims 55-65, wherein the patient is immunocompromised. The method according to any one of claims 55-65, wherein the patient has a healthy immune system. The method according to any one of claims 55-67, wherein the ACSS inhibitor is further defined as:wherein:R1and R1' are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy<c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy<c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);X1is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c≤6), or substituted alkyl(c≤6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Yi is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4'", whereinR4" and R4'" are each independently amino, hydroxy, alkyl(c≤12), substituted alkyl(c≤12), cycloalkyl(c≤12), substituted cycloalkyl(c≤12), alkenyl(c≤12), substituted alkenyl(c≤12), alkynyl(c≤12), substituted alkynyl(c≤12), aryl(c≤12), substituted aryl(c≤12), heteroaryl(c≤12), substituted heteroaryl(c≤12), heterocycloalkyl(c≤12), substituted heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof.The method according to any one of claims 55-68, wherein the ACSS inhibitor is further defined as:wherein:R1and R1' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R2 is amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c≤6), or substituted alkyl(c≤6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4"', whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substitutedcycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c<i 2), or a pharmaceutically acceptable salt thereof. The method according to any one of claims 55-69, wherein the ACSS inhibitor is further defined as:or a pharmaceutically acceptable salt thereof. A method of modulating the effectiveness of a chemotherapeutic modality comprising administering to a patient an ACSS inhibitor and the chemotherapeutic modality. The method of claim 71, wherein the method results in a 10% increase in the effectiveness of the chemotherapeutic modality as measured in tumor size. The method of claim 72, wherein the effectiveness is a 25% increase. The method according to any one of claims 71-73, wherein the ACSS inhibitor is administered before the chemotherapeutic modality. The method according to any one of claims 71-73, wherein the ACSS inhibitor is administered concurrently with the chemotherapeutic modality. The method according to any one of claims 71-73, wherein the ACSS inhibitor is administered within 3 hours after with the chemotherapeutic modality. The method according to any one of claims 71-76, wherein the chemotherapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. The method of claim 77, wherein the chemotherapeutic modality is a chemotherapeutic agent.The method of claim 77, wherein the chemotherapeutic modality is an immunotherapy. The method according to any one of claims 71-79, wherein the patient is a mammal. The method of claim 80, wherein the mammal is a human. The method according to any one of claims 71-81, wherein the ACSS inhibitor has been administered no more than 12 hours before the chemotherapeutic modality. The method of claim 82, wherein the ACSS inhibitor has been administered no more than 24 hours before the chemotherapeutic modality. The method of claim 83, wherein the ACSS inhibitor has been administered no more than 3 days before the chemotherapeutic modality. The method according to any one of claims 71-84, wherein the patient is immunocompromised. The method according to any one of claims 71-84, wherein the patient has a healthy immune system. The method according to any one of claims 71-86, wherein the ACSS inhibitor is further defined as:wherein:R1and R1' are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);X1 is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Y1 is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl <c<6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4'", whereinR4" and R4'" are each independently amino, hydroxy, alkyl(c≤12), substituted alkyl(c≤12), cycloalkyl(c≤12), substituted cycloalkyl(c≤12), alkenyl(c≤12), substituted alkenyl(c≤12), alkynyl(c≤12), substituted alkynyl(c≤12), aryl(c≤12), substituted aryl(c≤12), heteroaryl(c≤12), substituted heteroaryl(c≤12), heterocycloalkyl(c≤12), substituted heterocycloalkyl(c≤12), alkoxy(C<i2), substituted alkoxy(c≤12), cycloalkoxy<c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c<i 2) , substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof.The method according to any one of claims 71-87, wherein the ACSS inhibitor is further defined as:wherein:R1 and R1' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R2 is amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Y1 is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl(c<6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4"', whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substitutedcycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c<i 2), or a pharmaceutically acceptable salt thereof. The method according to any one of claims 71-88, wherein the ACSS inhibitor is further defined as:or a pharmaceutically acceptable salt thereof. The method according to any one of claims 71-89, wherein the patient is a mammal. The method of claim 90, wherein the mammal is a human. A method of modulating the effectiveness of a chemotherapeutic modality comprising administering to a patient who has been administered an ACSS inhibitor the chemotherapeutic modality. The method of claim 92, wherein the method results in a 10% increase in the effectiveness of the chemotherapeutic modality as measured in tumor size. The method of claim 93, wherein the effectiveness is a 25% increase. The method according to any one of claims 92-94, wherein the ACSS inhibitor is administered before the chemotherapeutic modality. The method according to any one of claims 92-94, wherein the ACSS inhibitor is administered concurrently with the chemotherapeutic modality. The method according to any one of claims 92-94, wherein the ACSS inhibitor is administered within 3 hours after with the chemotherapeutic modality.The method according to any one of claims 92-97, wherein the chemotherapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. The method of claim 98, wherein the chemotherapeutic modality is a chemotherapeutic agent. The method of claim 98, wherein the chemotherapeutic modality is an immunotherapy. The method according to any one of claims 92-100, wherein the patient is a mammal. The method of claim 101 , wherein the mammal is a human. The method according to any one of claims 92-102, wherein the ACSS inhibitor has been administered no more than 12 hours before the chemotherapeutic modality. The method of claim 103, wherein the ACSS inhibitor has been administered no more than 24 hours before the chemotherapeutic modality. The method of claim 104, wherein the ACSS inhibitor has been administered no more than 3 days before the chemotherapeutic modality. The method according to any one of claims 92-105, wherein the patient is immunocompromised. The method according to any one of claims 92-105, wherein the patient has a healthy immune system. The method according to any one of claims 92-107, wherein the ACSS inhibitor is further defined as:wherein:Ri and Rf are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12),substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R.3 is alkyl(c≤12) or substituted alkyl(c≤12);Xi is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl;c<6), or substituted alkyl(c<6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4'", whereinR4'1and R4'" are each independently amino, hydroxy, alkyl(c≤12), substituted alkyl(c≤12), cycloalkyl(c≤12), substituted cycloalkyl(c≤12), alkenyl(c≤12), substituted alkenyl(c≤12), alkynyl(c≤12), substituted alkynyl(c≤12), aryl(c≤12), substituted aryl(c≤12), heteroaryl(c≤12), substituted heteroaryl(c≤12), heterocycloalkyl(c≤12), substituted heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy<c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12);or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof.The method according to any one of claims 92-108, wherein the ACSS inhibitor is further defined as:wherein:Ri and Ri' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R2 is amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Yi is a covalent bond, 0, C(O), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl<c<6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(O)R4", or -S / CkhRY'', whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substitutedalkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c<i 2), or a pharmaceutically acceptable salt thereof. The method according to any one of claims 92-109, wherein the ACSS inhibitor is further defined as:or a pharmaceutically acceptable salt thereof. The method according to any one of claims 92-110, wherein the patient is a mammal. The method of claim 111, wherein the mammal is a human. A pharmaceutical composition comprising:(A) an ACSS inhibitor; and(B) a chemotherapeutic agent or an immunotherapy. The pharmaceutical composition of claim 113, wherein the ACSS inhibitor and the chemotherapeutic agent or the immunotherapy are formulated into a single unit dose. The pharmaceutical composition of either claim 113 or claim 114, wherein the pharmaceutical composition comprises a chemotherapeutic agent. The pharmaceutical composition of either claim 113 or claim 114, wherein the pharmaceutical composition comprises an immunotherapy. The pharmaceutical composition according to any one of claims 113-116, wherein the ACSS inhibitor is further defined as:wherein:Ri and Rf are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c<t2);Xi is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl(c<6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4'", whereinR4'1and R4'" are each independently amino, hydroxy, alkyl(c≤12), substituted alkyl(c≤12), cycloalkyl(c≤12), substitutedcycloalkyl(c≤12), alkenyl<c≤12), substituted alkenyl(c≤12), alkynyl(c≤12), substituted alkynyl(c≤12), aryl(c≤12), substituted aryl(c≤12), heteroaryl(c≤12), substituted heteroaryl(c≤12), heterocycloalkyl(c≤12), substituted heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to any one of claims 113-117, wherein theACSS inhibitor is further defined as:wherein:Ri and Ri' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R2 is amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl <c<6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4"', whereinR4'1and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c<i 2), or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to any one of claims 113-118, wherein the ACSS inhibitor is further defined as:or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to any one of claims 113-119, wherein the immunotherapy is a checkpoint inhibitor. The pharmaceutical composition of claim 120, wherein the checkpoint inhibitor is an anti-PDl checkpoint inhibitor.The pharmaceutical composition of claim 121, wherein the anti-PDl checkpoint inhibitor in an anti-PDl antibody. A method of treating cancer in a patient who has been treated with a checkpoint inhibitor with a therapeutically effective amount of an ACSS inhibitor of the formula:wherein:Ri and Rf are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c<i2), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Xi is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c<i2), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl(c<6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c<i2), alkynyl(c<i2), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c<i2), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S / CThR / '', whereinR-4" and R4'" are each independently amino, hydroxy, alkyl(c≤12), substituted alkyl(c≤12), cycloalkyl(c≤12), substituted cycloalkyl(c≤12), alkenyl(c≤12), substituted alkenyl(c≤12), alkynyl(c≤12), substituted alkynyl(c≤12), aryl(c≤12), substituted aryl(c≤12), heteroaryl(c≤12), substituted heteroaryl(c≤12), heterocycloalkyl(c≤12), substituted heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. The method of claim 123, wherein the ACSS inhibitor is further defined as:wherein:R1 and R1' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R2 is amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl(c<6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenybc n., alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4"', whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy<c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c<i 2), or a pharmaceutically acceptable salt thereof.A method of treating cancer in a patient in need thereof with a therapeutically effective amount of a checkpoint inhibitor and a therapeutically effective amount of an ACSS inhibitor of the formula:wherein:Ri and Ri' are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Xi is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl(c<6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Yi is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4”, or -S(O)2R4'”, whereinR4" and R4'" are each independently amino, hydroxy, alkyl(c≤12), substituted alkyl(c≤12), cycloalkyl(c≤12), substituted cycloalkyl(c≤12), alkenyl(c≤12), substituted alkenyl(c≤12), alkynyl(c≤12), substituted alkynyl(c≤12), aryl(c≤12), substituted aryl(c≤12), heteroaryl(c≤12), substituted heteroaryl(c≤12), heterocycloalkyl<c≤12), substituted heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substitutedcycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or a pharmaceutically acceptable salt thereof. The method of claim 125, wherein the ACSS inhibitor is further defined as:wherein:Ri and Ri' are each alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof;R2 is amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy<c≤12), substituted acyloxy(c≤12), amido<c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl(c<6); andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenybc n., alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version thereof; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; -C(0)R4", or -S(O)2R4'", whereinR4" and R'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy<c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substitutedcycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c<i 2), or a pharmaceutically acceptable salt thereof. The method according to any one of claims 123-126, wherein the ACSS inhibitor is further defined as:or a pharmaceutically acceptable salt thereof. The method according to any one of claims 123-127, wherein the patient is a mammal. The method of claim 128, wherein the mammal is a human. The method according to any one of claims 123-129, wherein the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The method according to any one of claims 123-129, wherein the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid. The method according to any one of claims 123-131, wherein the ACSS inhibitor is administered no more than 12 hours before the checkpoint inhibitor. The method of claim 132, wherein the ACSS inhibitor is administered no more than 24 hours before the checkpoint inhibitor.The method of claim 133, wherein the ACSS inhibitor is administered no more than 3 days before the checkpoint inhibitor. A compound of Formula (I):wherein:Ri and Ri' are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2is hydrogen, halo, amino, hydroxy, alkoxy(c<i2), substituted alkoxy(c<i2), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Xi is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alky 1 (c<6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version thereof; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(O)R4", or -S / CfhR / '', whereinR4" and R4'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or salt thereof.

136. The compound of claim 135, wherein Y3 is achiral.

137. The compound of claim 135, wherein Y 1 is C(O)NRa.

138. The compound of claim 135, wherein Rais hydrogen.

139. The compound according to any one of claims 135-138, wherein R4 is -Y2-R4'; whereinY2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4"', whereinR4" and R4'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicy cloalky lamino(c< 12) .

140. The compound according to any one of claims 135-139, wherein Y2 is arenedi yfc i 2) or substituted arenediyl(c≤12).

141. The compound according to any one of claims 135-140, wherein Y2 is arenediyl(c≤12).

142. The compound according to any one of claims 135-141, wherein Y is benzenediyl.

143. The compound according to any one of claims 135-142, wherein R4' is -SCO^RT", whereinR4'" is amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy(c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), or substituted dicy cloalky lamino(c< 12) .

144. The compound according to any one of claims 135-143, wherein R4"' is alkyl(c≤12)-145. The compound according to any one of claims 135-144, wherein R4'" is methyl.

146. The compound according to any one of claims 135-145, wherein Y3 is C or N.

147. The compound according to any one of claims 135-146, wherein Y3 is C.

148. The compound according to any one of claims 135-146, wherein Y3 is N.

149. The compound according to any one of claims 135-148, wherein Ri or Ri' is aryl(c≤12) or substituted aryl(c≤12).

150. The compound according to any one of claims 135-149, wherein Ri and Rf are both aryl(c≤12) or substituted aryl(c≤12).

151. The compound according to any one of claims 135-150, wherein Ri and Rf are both aryl(c≤12).

152. The compound according to any one of claims 135-151, wherein Ri and Rf are both phenyl.

153. The compound according to any one of claims 135-149, wherein Ri and Rf are not both aryl(c≤12) or substituted aryl(c≤12).

154. The compound according to any one of claims 135-149 and 153, wherein Ri and Rf are not both aryl(c≤12).

155. The compound according to any one of claims 135-149, 153, and 154, wherein Ri and Ri ' are not both phenyl.The compound according to any one of claims 135-155, wherein Ri is aryl(c≤12) or substituted aryl(c≤12). The compound according to any one of claims 135-149, wherein Ri is aryl(c≤12). The compound according to any one of claims 135-151, wherein Ri is phenyl. The compound according to any one of claims 135-158, wherein Rf is aryl(c≤12) or substituted aryl(c≤12). The compound according to any one of claims 135-159, wherein Rf is aryl(c≤12)- The compound according to any one of claims 135-160, wherein Rf is phenyl. The compound according to any one of claims 135-149, wherein Ri or Ri' is substituted aryl(c≤12). The compound according to any one of claims 135-149, wherein Ri or Ri' is 4- fluorophenyl. The compound according to any one of claims 135-163, wherein Ri is aryl(c≤12) and R4 is 3-methylsulfonylphenyl. The compound according to any one of claims 135-164, wherein the compound is further defined as:or a tautomer, solvate, and / or salt of any of these formulae. A pharmaceutical composition comprising:(a) a compound according to any one of claims 135-165; and(b) an excipient. A method of treating a disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of a compound according to any one of claims 135-165 or a pharmaceutical composition according to any one of claims 166. The method of claim 167, wherein the disease or disorder is cancer. The method of claim 168, wherein the cancer is a breast cancer, an ovarian cancer, a brain cancer, a prostate cancer, a pancreatic cancer, a sarcoma cancer, a lung cancer, a colorectal cancer, a skin cancer, a bone or connective tissue cancer, a blood cancer, or a liver cancer. A method of modulating the immune response to a cancer cell in a patient comprising administering to the patient a therapeutically effective amount of a compoundaccording to any one of claims 135-165 or a pharmaceutical composition according to any one of claims 166. The method according to claim 170, wherein the cancer cell is a part of a solid tumor. The method according to any one of claims 170-171, wherein the cancer cell is a cancer cell that shows increased expression of one or more ACSS enzymes. The method according to any one of claims 135-172, wherein the cancer cell is a breast cancer cell, an ovarian cancer cell, a brain cancer cell, a prostate cancer cell, a pancreatic cancer cell, a sarcoma cancer cell, a lung cancer cell, a colorectal cancer cell, a skin cancer cell, a bone or connective tissue cancer cell, a blood cancer cell, or a liver cancer cell. The method according to any one of claims 135-173, wherein the cancer cell comprises one or more genetic markers that impair the cancer cell’s effectiveness to one or more treatments. The method according to any one of claims 135-174, wherein the cancer cell is a breast cancer cell, a glioblastoma cell, an ovarian cancer cell, a prostate cancer cell, a melanoma cancer cell, a fibrosarcoma cancer cell, a multiple myeloma cancer cell, or a hepatocellular carcinoma cancer cell. The method according to any one of claims 135-175, wherein the patient is a mammal. The method of claim 176, wherein the mammal is a human. A method of treating cancer comprising administering to the patient in need thereof a therapeutically effective amount of a compound according to any one of claims 135- 165 or a pharmaceutical composition according to any one of claims 166 and a therapeutically effective amount of a therapeutic modality, wherein the therapeutic modality is a treatment for the cancer. The method of claim 178, wherein the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The method of claim 178, wherein the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid.The method according to any one of claims 178-180, wherein the therapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. The method of claim 181, wherein the therapeutic modality is a chemotherapeutic agent. The method of claim 181, wherein the therapeutic modality is an immunotherapy. The method according to any one of claims 178-183, wherein the patient is a mammal. The method of claim 184, wherein the mammal is a human. The method according to any one of claims 178-185, wherein the compound or composition is administered no more than 3 days before the therapeutic modality. The method of claim 186, wherein the compound or composition is administered no more than 24 hours before the therapeutic modality. The method of claim 187, wherein the compound or composition is administered no more than 12 hours before the therapeutic modality. A method of treating cancer in a patient who has been administered a compound according to any one of claims 135-165 or a pharmaceutical composition according to any one of claims 166 comprising administering to the patient a therapeutically effective amount of a therapeutic modality that is a treatment for the cancer. The method of claim 189, wherein the cancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The method of claim 189, wherein the cancer is of the bladder, blood, bone, brain, breast, central nervous system, cervix, colon, endometrium, esophagus, gall bladder, genitalia, genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, testicle, or thyroid. The method according to any one of claims 189-191, wherein the therapeutic modality is a chemotherapeutic agent, an immunotherapy, a radiation therapy, or surgery. The method of claim 192, wherein the therapeutic modality is a chemotherapeutic agent. The method of claim 192, wherein the therapeutic modality is an immunotherapy. The method according to any one of claims 189-194, wherein the patient is a mammal.

96. The method of claim 195, wherein the mammal is a human.

97. The method according to any one of claims 189-196, wherein the compound or composition is administered no more than 3 days before the therapeutic modality.

98. The method of claim 197, wherein the compound or composition is administered no more than 24 hours before the therapeutic modality.

99. The method of claim 198, wherein the compound or composition is administered no more than 12 hours before the therapeutic modality.

00. An ACSS-PROTAC conjugate comprising an acetyl-CoA synthetase short chain (ACSS) targeting ligand covalently linked to an E3 ligase recruiting ligand.

01. The conjugate of claim 200, wherein the ACSS targeting ligand is of formula (I):wherein:Ri and Rf are each independently absent, halo; or alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;R2 is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy<c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c<t2);Xi is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl(c<6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4'", whereinR4'1and R4'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy<c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substituted cycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or salt thereof.

202. The conjugate according to either claim 200 or claim 201 , wherein the ACSS targeting ligand is further defined as:or a tautomer, solvate, and / or salt of any of these formulae.The conjugate according to any one of claims 200-202, wherein the E3 ligase recruiting ligand is selected from among a CRBN recruiting ligand, a VHL recruiting ligand, an MDM2 recruiting ligand, or a cIAP recruiting ligand. The conjugate according to any one of claims 200-203, wherein the E3 ligase recruiting ligand is selected from amongThe conjugate according to any one of claims 200-203, wherein the linker is a polymer comprising 2 to 20 repeating units. The conjugate of claim 204, wherein the linker is a polyethylene glycol linker. An ACSS binding probe comprising an acetyl-CoA synthetase short chain (ACSS) targeting ligand covalently linked to a fluorescent moiety. The probe of claim 206, wherein the ACSS targeting ligand is of formula (I):wherein:Ri and Rf are each independently absent, halo; oralkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups;Rz is hydrogen, halo, amino, hydroxy, alkoxy(c≤12), substituted alkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), acyloxy(c≤12), substituted acyloxy(c≤12), amido(c≤12), or substituted amido(c≤12);R3 is alkyl(c≤12) or substituted alkyl(c≤12);Xi is N or CRb, wherein Rb is selected from among hydrogen, hydroxy, halo, cyano, alkyl(c≤12), cycloalkyl(c≤12), alkoxy(c≤12), alkylamino(c≤12), dialkylamino(c≤12), or a substituted version of any of these groups;Yi is a covalent bond, 0, C(0), C(0)NRa, or NRa; wherein Rais hydrogen, alkyl(c<6), or substituted alkyl(c<6);Y3 is N, 0, or C; andR4 is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; or -Y2-R4', wherein:Y2 is alkanediyl(c≤12), arenediyl(c≤12), heteroarenediyl(c≤12), or heterocycloalkanediyl(c≤12), or a substituted version of any of these groups; andR4' is alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), or a substituted version of any of these groups; -C(0)R4", or -S(O)2R4'", whereinR4'1and R4'" are each independently amino, hydroxy, alkyl(c≤12), cycloalkyl(c≤12), alkenyl(c≤12), alkynyl(c≤12), aryl(c≤12), heteroaryl(c≤12), heterocycloalkyl(c≤12), alkoxy(c≤12), substituted alkoxy<c≤12), cycloalkoxy(c≤12), substituted cycloalkoxy(c≤12), alkylamino(c≤12), substituted alkylamino(c≤12), dialkylamino(c≤12), substituted dialkylamino(c≤12), cycloalkylamino(c≤12), substitutedcycloalkylamino(c≤12), dicycloalkylamino(c≤12), substituted dicycloalkylamino(c≤12); or a tautomer, solvate, and / or salt thereof. . The conjugate according to either claim 207 or claim 208, wherein the ACSS targeting ligand is further defined as:or a tautomer, solvate, and / or salt of any of these formulae. . The probe according to any one of claims 206-209, wherein the fluorescent moiety is FITC. . The probe according to any one of claims 206-209, wherein the fluorescent moiety is a BODIPY dye. . The probe according to claim 211, wherein the fluorescent moiety is BODIPY / 650. . The probe according to any one of claims 206-209, wherein the fluorescent moiety is a pyrylium dye. . The probe according to claim 213, wherein the fluorescent moiety is Py-1. . The probe according to any one of claims 206-214, wherein the probe binds to ACSS with a KD of about 500 nM. . The probe according to any one of claims 206-214, wherein the probe binds to ACSS with a KD less than 500 nM. . The probe according to any one of claims 206-214, wherein the probe binds to ACSS2 with a KD of about 500 nM. . The probe according to any one of claims 206-214, wherein the probe binds to ACSS2 with a KD less than 500 nM. . A method for determining the binding affinity of a compound to an ACSS enzyme comprising:(A) contacting an ACSS enzyme with a probe according to any one of claims 206- 214; and(B) determining the amount of luminescence generated from the probe. The method of claim 219, wherein the binding affinity is determined in vivo. The method of either claim 219 or claim 220, wherein the ACSS enzyme is ACSS2. The method according to any one of claims 219-221, wherein the ACSS enzyme is a modified ACSS enzyme. The method of claim 222, wherein the modified ACSS enzyme is modified with luciferase subunit. The method of either claim 222 or claim 223, wherein the modified ACSS enzyme is an ACSS enzyme with a 19 kDA luciferase subunit. The method according to any one of claims 222-224, wherein the modified ACSS enzyme is an ACSS enzyme tagged with a 19 kDa subunit derived from Oplophorus gracilirostris.

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