How to Use STING Agonists to Treat Cancer

Abstract

The invention relates, in part, to methods for polarizing tumor-promoting macrophages into anti-tumor macrophages in a subject with cancer using STING agonists, e.g., to improve the efficacy of PARP inhibition.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 148,426, filed February 11, 2021, which is incorporated by reference herein in its entirety.

[0002] STATEMENT OF GOVERNMENT RIGHTS This invention was made with Government support under Grant Nos. P50CA168504, P50CA165962, and R35CA210057 awarded by the National Institutes of Health, and Grant No. W81XWH-20-1-0118 awarded by the Department of Defense. The Government has certain rights in this invention. [Background technology]

[0003] Homologous recombination (HR) deficiency confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), which are utilized therapeutically in both ovarian and breast tumors harboring loss-of-function mutations in HR pathway genes, most commonly BRCA1 and BRCA2 (BRCA1 / 2). Based on substantial progression-free survival (PFS) benefits, three PARPi have received FDA approval for BRCA-mutated ovarian cancer in both the adjuvant and metastatic settings. Recently, maintenance treatment with olaparib was shown to confer unprecedented overall survival benefit to patients with BRCA-mutated recurrent ovarian cancer. However, compared to ovarian cancer, PARPi therapy appears to be less effective in BRCA-mutated breast cancer. Nevertheless, the FDA has approved two PARPi (olaparib and talazoparib) as monotherapy for patients with germline BRCA1 / 2 mutations and HER2-negative advanced breast cancer.

[0004] Although these PARPis significantly improved PFS, recent results from the OlympiAD and EMBRACA clinical trials suggest a lack of overall survival benefit with both olaparib and talazoparib in patients with advanced breast cancer harboring germline BRCA1 / 2 mutations, highlighting the need to understand why BRCA-mutated breast cancer is more refractory to PARPi in order to develop strategies to improve response to PARPi.

[0005] Osimertinib (AZD9291) is a third-generation EGFR tyrosine kinase inhibitor (TKI) for patients with non-small cell lung cancer (NSCLC) harboring EGFR activating mutations or acquired T790M mutations resistant to previous generation EGFR-TKIs. Despite its remarkable efficacy, the emergence of resistance to osimertinib is inevitable, and overcoming such resistance remains a significant challenge in clinical practice, and therefore novel therapies to overcome such resistance are needed. Summary of the Invention [Means for solving the problem]

[0006] The present invention is based at least in part on the discovery that STING agonists reprogram M2-like pro-tumor macrophages to M1-like anti-tumor macrophages in a macrophage STING-dependent manner. This discovery can be utilized in a variety of ways, for example, to treat certain cancers that are rich in M2-like macrophages, or to improve the efficacy of PARP inhibition, TK inhibitors, and / or inhibitors of DNA synthesis in some cancers. This discovery can be used to overcome or prevent drug resistance in any cancer where the resistance is characterized by an increase in the level or amount of M2-like pro-tumor macrophages in the tumor or tumor microenvironment. In some embodiments, drug resistance can be characterized by the recruitment of M2-like pro-tumor macrophages to the cancer or tumor. This discovery can also be used to overcome drug resistance characterized by activation of STAT3 signaling as a result of drug administration. Finally, this discovery can also be used to overcome drug resistance characterized by secretion of hepatocyte growth factor (HGF) in a feed-forward manner in cancer. Further details regarding HGF and its role in drug resistance can be found in Dong N, et al. M2 macrophages mediate sorafenib resistance by secreting HGF in a feed-forward manner in hepatocellular carcinoma. Br J Cancer. 2019 Jul;121(1):22-33, which is incorporated by reference in its entirety.

[0007] In some embodiments, a method of improving the efficacy of PARP inhibition in a subject having cancer comprises administering to the subject an effective amount of a STING agonist in combination with an effective amount of a PARP inhibitor.

[0008] Syngeneic genetically engineered mouse (GEM) models of lung cancer driven by mutant EGFR show that EGFR mutant tumors are highly sensitive to osimertinib in the early stages of tumor growth in a T cell-dependent manner, but become resistant as they progress. Thus, the present invention is also based in part on the determination that the presence of immunosuppressive tumor-associated macrophages (TAMs) renders tumors resistant to osimertinib. Depletion of TAMs in these tumors rescues the efficacy of osimertinib. Reprogramming of TAMs with the newly developed STING agonist MSA-2 reactivates antitumor immunity and, when combined with osimertinib, leads to sustained regression of resistant tumors in mice. The results presented herein show that an inhibitory tumor immune microenvironment can drive resistance to osimertinib in EGFR mutant tumors. Thus, a new strategy is provided herein to overcome resistance and improve treatment outcomes.

[0009] Also provided herein is a method of improving the efficacy of tyrosine kinase inhibitor (TKI) inhibition in a subject having cancer by administering to the subject an effective amount of a STING agonist in combination with an effective amount of a tyrosine kinase inhibitor (TKI).

[0010] In certain embodiments, the method of polarizing tumor-promoting macrophages into anti-tumor macrophages in a subject with cancer comprises administering to the subject an effective amount of a STING agonist. Also provided herein is a method of preventing or reversing drug resistance in a subject with cancer, the drug resistance being the result of polarizing anti-tumor macrophages into tumor-promoting macrophages, comprising administering to the subject an effective amount of a STING agonist. The drug resistance can be resistance to PARP inhibition. The drug resistance can be resistance to TK inhibition. The drug resistance can be resistance to DNA synthesis inhibitors.

[0011] Also provided herein is a method of improving the efficacy of a DNA synthesis inhibitor in a subject having cancer by administering to the subject an effective amount of a STING agonist in combination with an effective amount of a DNA synthesis inhibitor. Exemplary DNA synthesis inhibitors include, but are not limited to, gemcitabine, sapacitabine, cytidine analogs, cytarabine, tezacitabine, troxacitabine, DMDC, CNDAC, ECyD, clofarabine, or nucleoside analogs such as decitabine. Additional information regarding resistance in gemcitabine can be found in Bulle A, et al. Gemcitabine Recruits M2-Type Tumor-Associated Macrophages into the Stroma of Pancreatic Cancer. Transl Oncol. 2020 Mar;13(3):100743, which is incorporated herein by reference in its entirety.

[0012] Numerous embodiments are further provided that may be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in some embodiments, the STING agonist activates STING signaling in macrophages. In some embodiments, the STING agonist (e.g., tumor cell cytoplasmic dsDNAs / cGAMP or intratumorally delivered STING agonist) does not activate STING signaling in intratumoral dendritic cells. In some embodiments, the subject has a defect in the STING signaling pathway in tumor cells. In some embodiments, the tumor-promoting macrophages are M2-like. In some embodiments, the anti-tumor macrophages are M1-like.

[0013] Similarly, in some embodiments, administration includes systemic delivery of the STING agonist. It is known that early generation STING agonists are not suitable for systemic delivery. In some embodiments, administration is oral, intravenous, or intraperitoneal administration. In some embodiments, the STING agonist is a modified nucleotide STING agonist. In some embodiments, the STING agonist is selected from DMXAA, MSA-2, SR-717, FAA, CMA, α-mangostin, BNBC, DSDP, diABZI, bicyclic benzamides, and benzothiophenes. In some embodiments, the PARP inhibitor is selected from olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, CEP9722, E7016, AG014699, MK4827, BMN-673, iniparib, and 3-aminobenzamide. In some embodiments, the STING agonist and the PARP inhibitor are administered together. In some embodiments, the co-administering comprises administering a STING agonist before a PARP inhibitor. In some embodiments, the co-administering comprises administering a STING agonist simultaneously with a PARP inhibitor. In some embodiments, a STING agonist and a TK inhibitor are co-administered. In some embodiments, the co-administering comprises administering a STING agonist before a TK inhibitor (e.g., an EGFR-TK inhibitor, any other TK inhibitor disclosed herein). In some embodiments, the co-administering comprises administering a STING agonist simultaneously with a TK inhibitor (e.g., an EGFR-TK inhibitor). The TK inhibitor (e.g., an EGFR-TK inhibitor) may be selected from afatinib, dacomitinib, osimertinib (AZD9291), rociletinib (CO-1686), olmutinib (HM61713), nazartinib (EGF816), nacotinib (ASP8273), mavereltinib (PF-0647775), almonertinib, TY-9591, gefitinib, erlotinib, and AC0010.

[0014] The TK inhibitors disclosed herein can be vascular endothelial growth factor receptor (VEGF) TK inhibitors, epidermal growth factor (EGF) receptor TK inhibitors, platelet-derived endothelial growth factor receptor (PDGF) TK inhibitors, or the TK inhibitors can be fibroblast growth factor (FGF) receptor TK inhibitors. Examples of TK inhibitors include axitinib, dasatinib, erlotinib, imatinib, nilotinib, pazopanib, sorafenib, bosutinib, avapritinib, capmatinib, pemigatinib, ripretinib, selpercatinib, selumetinib, tucatinib, entrectinib, erdafitinib, fedratinib, pexidartinib, tenosynovium, upadacitinib, zanubrutinib, baricitinib, binimetinib, dacomitinib, fostamatinib, gilteritinib, and lapatinib. It may be rotrectinib, lorlatinib, acalabrutinib, brigutinib, midostaurin, neratinib, alectinib, cobimetinib, lenvatinib, osimertinib, ceritinib, nintedanib, afatinib, ibrutinib, trametinib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, tofacitinib, crizotinib, ruxolitinib, vandetanib, pazopanib, lapatinib, gefitinib, or sunitinib.

[0015] In some embodiments, the STING agonist and the DNA synthesis inhibitor are co-administered. In some embodiments, co-administering comprises administering the STING agonist prior to the DNA synthesis inhibitor. In some embodiments, co-administering comprises administering the STING agonist simultaneously with the DNA synthesis inhibitor.

[0016] Similarly, in some embodiments, the cancer is characterized by an M2 enrichment score greater than 0.27 (e.g., 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, , 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80 or higher, or any range therebetween (including boundaries, e.g., 0.27 to 0.60). In some embodiments, the cancer has an M2 enrichment score greater than 0.15 (e.g., 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97 0.6, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or higher).In some embodiments, the cancer comprises a tumor having an M2 enrichment score of 0.15-0.25, 0.20-0.30, 0.25-0.3, 0.3-0.40, 0.35-0.45, 0.40-0.50, 0.45-0.55, 0.50-0.60, 0.55-0.65, 0.60-0.70, 0.65-0.75, 0.70-0.80, 0.75-0.85, 0.80-0.90, or any range therebetween (including boundaries, e.g., 0.15-0.90, 0.30-0.85, etc.).

[0017] In some embodiments, the cancer comprises a tumor with an M2 / M1 ratio greater than 1.0 (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 1.95, or more (including boundaries), e.g., 1.1-1.3, 1-1.2, 1.2-1.4, 1.3-1.5, 1.4-1.6, 1.5-1.7, 1.6-1.8, 1.7-1.9, 1.8-2.0, 1.0-1.5, 1.5-1.0, 1.2-1.7, 1.4-1.9, 1.5-2.0, or any range therebetween (including boundaries), e.g., 1.1-2.0, 1.3-1.8, etc.). In some embodiments, M1 cells are characterized as CD45+CD11b+F4 / 80+MHCIIhighCD206low, or CD45+CD11b+F4 / 80+MHCIIhighCD163low, and / or M2 cells are characterized as CD45+CD11b+F4 / 80+MHCIIlowCD206high, or CD45+CD11b+F4 / 80+MHCIIlowCD163high. In some embodiments, the cancer comprises head and neck squamous cell carcinoma (HNSC), lung cancer such as non-small cell lung cancer (NSCLC), liver cancer such as lung squamous cell carcinoma (LUSC), hepatocellular carcinoma (HCC), colon cancer, prostate cancer, pancreatic cancer, cutaneous melanoma (SKCM), glioblastoma multiforme (GBM), breast invasive carcinoma (BRCA), lung adenocarcinoma (LUAD), renal clear cell carcinoma (KIRC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), diffuse large B-cell lymphoma (DLBC), gastric adenocarcinoma (STAD), ovarian cancer such as high-grade serous ovarian cancer (HGSOC) or homologous recombination (HRP) ovarian cancer, or any homologous recombination (HRP) ovarian cancer. The cancer can be any homologous recombination deficient (HRD) cancer (e.g., HRD ovarian cancer). The cancer may be an HRD cancer or tumor comprising a mutation in the RAD51, PALB2, ATM, ATR, CHEK2, or FANC gene. The cancer may be an HRD cancer or tumor associated with aberrant expression of a protein encoded by ... genetic mutation that upregulates STAT3 signaling and / or converts tumor-associated macrophages into M2-like macrophages (e.g., KRAS G12DThe cancer may be any cancer that contains a genetic mutation that polarizes it to a cancer with a mutation in the KRAS gene, such as a KRAS mutation. G12D Further details regarding STAT3 signaling and its association with STAT3 signaling can be found in Dai E et al. Autophagy-dependent ferroptosis drives tumor-associated macrophage polarization via release and uptake of oncogenic KRAS protein. Autophagy. 2020;16(11):2069-2083, which is incorporated by reference in its entirety. In some embodiments, the cancer comprises breast cancer carrying a BRCA mutation. In some embodiments, the cancer comprises advanced breast cancer carrying a germline BRCA1 / 2 mutation.

[0018] STING agonists can also enhance the therapeutic efficacy of PARPi in naive BRCA1 tumors with a ratio of M2 to M1 less than 1.0. In certain embodiments, the subject has BRCA naive breast cancer or ovarian cancer. In some embodiments, the cancer comprises breast cancer that does not carry a BRCA mutation. In some embodiments, the cancer does not comprise advanced breast cancer that carries a germline BRCA1 / 2 mutation. In some embodiments, the cancer comprises a tumor with an M2 / M1 ratio of less than 1.0 (e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1, or more (including boundaries), e.g., 0.1-0.3, 0.2-0.4, 0.3-0.5, 0.4-0.6, 0.5-0.7, 0.6-0.8, or 0.7-0.9, or any range therebetween (including 0.1-0.9, 0.3-0.8, etc.).

[0019] In some embodiments, the cancer comprises a lung cancer harboring an EGFR mutation, such as an EGFR activating mutation or a T790M mutation. In some embodiments, the cancer comprises a lung cancer harboring an exon 19 deletion mutation. In some embodiments, the cancer comprises a lung cancer harboring a single point substitution mutation L858R in exon 21. In some embodiments, the cancer comprises a non-small cell lung cancer harboring an EGFR mutation, such as an EGFR activating mutation or a T790M mutation.

[0020] Similarly, in some embodiments, the cancer is characterized by an M2 enrichment score greater than 0.27 (e.g., 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 0.10, 0.104, 0.105, 0.106, 0.107, 0.108, 0 5, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, or more, or any range therebetween (including boundaries, e.g., 0.27 to 0.60). In some embodiments, the cancer has an M2 enrichment score greater than 0.15 (e.g., 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97 0.6, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or higher).In some embodiments, the cancer comprises a tumor having an M2 enrichment score of 0.15-0.25, 0.20-0.30, 0.25-0.3, 0.3-0.40, 0.35-0.45, 0.40-0.5, 0.45-0.55, 0.50-0.60, 0.55-0.65, 0.60-0.70, 0.65-0.75, 0.70-0.80, 0.75-0.85, 0.80-0.90, or any range therebetween (including boundaries, e.g., 0.15-0.90, 0.30-0.85).

[0021] In some embodiments, the cancer comprises a tumor that has achieved an M2 enrichment score of greater than 0.27. In some embodiments, the cancer comprises a tumor that has achieved an M2 enrichment score of greater than 0.15 (e.g., 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.6 3, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0 In some embodiments, the subject is a rodent, primate, human, or animal model of cancer, and optionally, the subject is a human. In some embodiments, the subject has a defect in activation of STING signaling in intratumoral dendritic cells.

[0022] Similarly, in some embodiments, the M2 enrichment score is determined by GSEA analysis of RNA sequencing data of tumors and tumor microenvironment (e.g., RNA sequencing analysis of bulk tumor, pleural effusion, or ascites; single cell RNA sequencing analysis of tumor-infiltrating immune cells or immune cells in any type of effusion in cancer patients). In some embodiments, the ratio of M2 / M1 is detected by cyclic immunofluorescence (CyCIF) or conventional immunohistochemistry analysis of any type of tumor tissue (e.g., paraffin-embedded tumor tissue, frozen tumor tissue). In some embodiments, the ratio of M2 / M1 is detected by flow cytometry analysis or time-of-flight cytometry (CyTOF) analysis of tumor-infiltrating immune cells or immune cells in any type of effusion in cancer patients. In some embodiments, M1 and M2 macrophages are determined using any type of phenotypic marker listed in the literature (e.g., macrophage markers: CD11b, F4 / 80, and CD68; M1 markers: CD80, CD86, MHC-II, TLR2, TLR4, iNOS, SOCS3, IFN-β, TNF-α, CCL2, CCL3, CCL4, CCL5, CCL8, CCL9, CCL10, CCL11; M2 markers: CD163, CD206, CD200R, ARG-1, Ym1 / 2, Fizz1, IL-6, IL-10, TGF-β, VEGF, CCL17, CCL22, CCL24, CCR2).

[0023] Similarly, in some embodiments, the PARP inhibitor is administered at a dosage of at least 10 mg / kg body weight, at least 15 mg / kg body weight, at least 20 mg / kg body weight, at least 25 mg / kg body weight, at least 30 mg / kg body weight, at least 35 mg / kg body weight, at least 40 mg / kg body weight, at least 45 mg / kg body weight, 50 mg / kg body weight, at least 55 mg / kg body weight, at least 60 mg / kg body weight, at least 65 mg / kg body weight, at least 70 mg / kg body weight, at least 75 mg / kg body weight, at least 80 mg / kg body weight, at least 85 mg / kg body weight, at least 90 mg / kg body weight, at least 95 mg / kg body weight, or at least 100 mg / kg body weight per dose. In some embodiments, the EGFR-TK inhibitor is administered at a dosage of at least 1 mg / kg body weight, at least 2 mg / kg body weight, at least 3 mg / kg body weight, at least 4 mg / kg body weight, at least 5 mg / kg body weight, at least 6 mg / kg body weight, at least 7 mg / kg body weight, at least 8 mg / kg body weight, at least 9 mg / kg body weight, at least 10 mg / kg body weight, at least 15 mg / kg body weight, at least 20 mg / kg body weight, at least 25 mg / kg body weight, at least 30 mg / kg body weight, at least 35 mg / kg body weight, at least 40 mg / kg body weight, at least 45 mg / kg body weight, 50 mg / kg body weight, at least 55 mg / kg body weight, at least 60 mg / kg body weight, at least 65 mg / kg body weight, at least 70 mg / kg body weight, at least 75 mg / kg body weight, at least 80 mg / kg body weight, at least 85 mg / kg body weight, at least 90 mg / kg body weight, at least 95 mg / kg body weight, or at least 100 mg / kg body weight per dose. Dosages may be administered twice daily, once daily, twice daily, twice weekly, once weekly, three times monthly, twice monthly, or monthly.In some embodiments, the STING agonist is administered at a dosage of at least 1 mg / kg body weight, at least 2 mg / kg body weight, at least 3 mg / kg body weight, at least 4 mg / kg body weight, at least 5 mg / kg body weight, at least 6 mg / kg body weight, at least 7 mg / kg body weight, at least 8 mg / kg body weight, at least 9 mg / kg body weight, at least 10 mg / kg body weight, at least 15 mg / kg body weight, at least 20 mg / kg body weight, at least 25 mg / kg body weight, at least 30 mg / kg body weight, at least 35 mg / kg body weight, at least 40 mg / kg body weight, at least 45 mg / kg body weight, 50 mg / kg body weight, at least 55 mg / kg body weight, at least 60 mg / kg body weight, at least 65 mg / kg body weight, at least 70 mg / kg body weight, at least 75 mg / kg body weight, at least 80 mg / kg body weight, at least 85 mg / kg body weight, at least 90 mg / kg body weight, at least 95 mg / kg body weight, or at least 100 mg / kg body weight per dose. Dosages may be administered twice daily, once daily, twice daily, twice weekly, once weekly, three times monthly, twice monthly, or monthly. In some embodiments, the STING agonist is administered two to three times. In some embodiments, the method includes an additional therapy. In some embodiments, the additional therapy includes radiation therapy. In some embodiments, the additional therapy includes chemotherapy (including, for example, paclitaxel, platinum-based drugs (e.g., cisplatin, oxaliplatin), inhibitors of topoisomerase activity such as topoisomerase II (e.g., etoposide), DNA intercalators (e.g., doxorubicin), and / or DNA alkylating agents (e.g., temozolomide)). In some embodiments, the additional therapy includes a DNA damage response (DDR) targeting agent (including, for example, ATMi, ATRi, CHK1 / 2i, or Wee1i).

[0024] In some embodiments, a method of selecting a subject having cancer for treatment with a STING agonist comprises detecting an M2 enrichment score for a tumor from the subject and selecting the subject if the score is greater than 0.27. In some embodiments, the cancer has an M2 enrichment score greater than 0.15 (e.g., 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97 0.6, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or higher). In some embodiments, a method of selecting a subject having cancer for treatment with a STING agonist comprises detecting the M2 / M1 ratio in a tumor from the subject and selecting the subject if the score is greater than 1.0 (i.e., greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 1.95).

[0025] As described above, a number of embodiments are further provided that can be applied to any aspect of the present invention and / or can be combined with any other embodiment described herein.For example, in some embodiments, cancer comprises head and neck squamous cell carcinoma (HNSC), lung cancer such as lung squamous cell carcinoma (LUSC) or non-small cell lung cancer (NSCLC), liver cancer such as hepatocellular carcinoma (HCC), colon cancer, prostate cancer, pancreatic cancer, cutaneous melanoma (SKCM), glioblastoma multiforme (GBM), breast invasive carcinoma (BRCA), lung adenocarcinoma (LUAD), renal clear cell carcinoma (KIRC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), diffuse large B-cell lymphoma (DLBC), gastric adenocarcinoma (STAD), ovarian cancer such as high-grade serous ovarian cancer (HGSOC) or homologous recombination (HRP) ovarian cancer, or any homologous recombination (HRP) cancer. The cancer may be any homologous recombination deficient (HRD) cancer (e.g., HRD ovarian cancer). The cancer may be an HRD cancer or tumor comprising a mutation in the RAD51, PALB2, ATM, ATR, CHEK2, RAD51, or FANC gene. The cancer may be an HRD cancer or tumor associated with aberrant expression of a protein encoded by the RAD51, PALB2, ATM, ATR, CHEK2 ... genetic mutation that upregulates STAT3 signaling and / or converts tumor-associated macrophages into M2-like macrophages (e.g., KRAS G12DThe cancer may be any cancer that contains a genetic mutation that polarizes the cancer to a BRCA mutation (e.g., a cancer with a mutation in the KRAS gene, such as a mutation). In some embodiments, the cancer includes breast cancer carrying a BRCA mutation (e.g., advanced breast cancer carrying a germline BRCA1 / 2 mutation). In certain embodiments, the subject has BRCA-naive breast cancer or ovarian cancer. In some embodiments, the subject is a rodent, primate, human, or animal model of cancer, and optionally, the subject is a human. In some embodiments, the subject has a defect in activating STING signaling in tumor cells. In some embodiments, the subject has a defect in the STING signaling pathway in tumor cells, and thus, intratumoral STING agonists (e.g., dsDNAs / cGAMP released from tumor cells or intratumoral delivered STING agonists) cannot activate intratumoral dendritic cells and macrophages.

[0026] In one aspect, a method of treating a subject with advanced breast cancer harboring a germline BRCA1 / 2 mutation, wherein the cancer comprises a tumor with an M2 enrichment score greater than 0.27 or a ratio of M2 / M1 greater than 1.0, comprises systemically administering to the subject about 10 mg / kg body weight of a STING agonist in combination with about 50 mg / kg body weight of a PARP inhibitor.

[0027] Also provided herein is a method of treating a subject having non-small cell lung cancer harboring a germline EGFR mutation, wherein the cancer comprises a tumor having an M2 enrichment score greater than 0.27, comprising administering to the subject a STING agonist in combination with an EGFR-TK inhibitor.

[0028] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0029] [Figure 1A]We show that Brca1-deficient mammary tumors with immunosuppressive TAMs have a moderate response to olaparib in vivo. We show generation of syngeneic GEMMs of Brca1-deficient mammary tumors by intraductal injection of adenovirus expressing Cre recombinase (Ad-Cre) directly into the lumen of the mammary gland. Data are presented as mean ± SEM (Fig. 1C and Fig. 1G), mean ± SEM (Fig. 1F), or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Fig. 1C). Two-tailed unpaired t-test (Fig. 1D-G). ns: not significant. *P<0.05, **P<0.005, ****P<0.0001. [Figure 1B] Figure 1 shows that Brca1-deficient mammary tumors with immunosuppressive TAMs have a moderate response to olaparib in vivo. Tumor-free survival of Brca1L / L Trp53L / L mice with or without intraductal injection of Ad-Cre (n=6). Data are presented as mean ± SEM (Figure 1C and Figure 1G), mean ± SEM (Figure 1F), or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Figure 1C). Two-tailed unpaired t-test (Figure 1D-Figure 1G). ns: not significant. *P<0.05, **P<0.005, ****P<0.0001. [Figure 1C] Figure 1 shows that Brca1-deficient mammary tumors with immunosuppressive TAMs have a moderate response to olaparib in vivo. Figure 1 shows tumor growth of Brca1- / - Trp53- / - (BP) allografts in FVB mice treated with olaparib or anti-PD-1 as monotherapy or in combination. Control: n=8, anti-PD-1: n=8, olaparib: n=6, olaparib + anti-PD-1: n=10. Data are presented as mean ± SEM (Figure 1C and Figure 1G), mean ± SEM (Figure 1F), or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Figure 1C). Two-tailed unpaired t-test (Figure 1D-Figure 1G). ns: not significant. *P<0.05, **P<0.005, ****P<0.0001. [Figure 1D]Figure 1 shows that Brca1-deficient mammary tumors with immunosuppressive TAMs have a moderate response to olaparib in vivo. Analysis of BP tumors from FVB mice treated with or without olaparib for 21 days is shown. Tumor-associated macrophages (TAMs, 7AAD-CD45+CD11b+F4 / 80+) were plotted as CD206 versus MHC-II to identify M1-like (MHC-II high CD206-) and M2-like (MHC-II low CD206+) polarized phenotypes. Each dot represents a result from a single tumor. Data are presented as mean ± standard error (Figure 1C and Figure 1G), mean ± standard deviation (Figure 1F), or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Figure 1C). Two-tailed unpaired t-test (Figure 1D-Figure 1G). ns: not significant. *P<0.05, **P<0.005, ****P<0.0001. [Figure 1E] Figure 1 shows that Brca1-deficient mammary tumors with immunosuppressive TAMs have a moderate response to olaparib in vivo. Analysis of tumors from FVB mice bearing mammary tumors or ovarian tumors of Brca1-deficient mice 28 days after tumor cell implantation. Each dot represents the result from a single tumor. Data are presented as mean ± standard error (Figure 1C and Figure 1G), mean ± standard deviation (Figure 1F), or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Figure 1C). Two-tailed unpaired t-test (Figure 1D-Figure 1G). ns: not significant. *P<0.05, **P<0.005, ****P<0.0001. [Figure 1F]Figure 1 shows that Brca1-deficient breast tumors with immunosuppressive TAMs have a moderate response to olaparib in vivo. Analysis of the TCGA cohort for the M2 immunosuppressive gene signature in patients with BRCA1 mutated (mut) ovarian cancer (n=29) and patients with BRCA1 mutated breast cancer (n=30). Data are presented as mean ± SEM (Figure 1C and Figure 1G), mean ± SEM (Figure 1F), or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Figure 1C). Two-tailed unpaired t-test (Figure 1D-Figure 1G). ns: not significant. *P<0.05, **P<0.005, ****P<0.0001. [Figure 1G] Figure 1 shows that Brca1-deficient mammary tumors with immunosuppressive TAMs have a moderate response to olaparib in vivo. Analysis of cytokine production of CD8+ T cells co-cultured for 2 days with TAMs sorted from BP tumors (n=4-8). Data are presented as mean ± SEM (Figure 1C and Figure 1G), mean ± SEM (Figure 1F), or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Figure 1C). Two-tailed unpaired t-test (Figure 1D-Figure 1G). ns: not significant. *P<0.05, **P<0.005, ****P<0.0001. [Figure 2A] Brca1L / LTrp53L / L mouse model of breast tumors induced by intraductal injection of Ade-Cre. Left panel shows RT-qPCR analysis of Brca1 in mouse mammary epithelial cells (MMEC) and breast tumor cells of Brca1- / - Trp53- / - (BP) mice. Right panel shows RNA-seq analysis of Trp53 in BP and MMEC (n=3-5). Data are presented as mean ± SEM or median with quartiles (violin plot). Two-tailed unpaired t-test. ns: not significant. *P<0.05, ***P<0.0005, ****P<0.0001. [Figure 2B]Brca1L / LTrp53L / L mouse model of breast tumors induced by intraductal injection of Ade-Cre. H&E staining of breast tumors from Brca1L / LTrp53L / L mice is shown. Data are presented as mean ± SEM or median with quartiles (violin plot). Two-tailed unpaired t-test. ns: not significant. *P<0.05, ***P<0.0005, ****P<0.0001. [Figure 2C] Figure 1 shows a Brca1L / LTrp53L / L mouse model of breast tumors induced by intraductal injection of Ade-Cre. Analysis of BP tumors from FVB mice with or without olaparib treatment is shown, with assessment of intratumoral CD8+ T cells after 7 days of treatment as well as tumor-associated macrophages (TAM) after 21 days of treatment. Each dot represents the result from a single tumor. Data are presented as mean ± SEM or median with quartiles (violin plot). Two-tailed unpaired t-test. ns: not significant. *P<0.05, ***P<0.0005, ****P<0.0001. [Figure 2D] Brca1L / LTrp53L / L mouse model of breast tumors induced by intraductal injection of Ade-Cre. Analysis of effector cell percentage (CD44highCD62Llow) and surface expression of CD25 of CD8+ T cells co-cultured for 2 days with TAMs (7AAD-CD45+CD11b+F4 / 80+) sorted from BP tumors (n=4-8). Data are presented as mean±sem or median with quartiles (violin plot). Two-tailed unpaired t-test. ns: not significant. *P<0.05, ***P<0.0005, ****P<0.0001. [Figure 2E] Brca1L / LTrp53L / L mouse model of breast tumors induced by intraductal injection of Ade-Cre. Gating strategy for flow cytometry analysis described in Fig. 1D, Fig. 1E, and Fig. 2C. Data are presented as mean ± SEM or median with quartiles (violin plot). Two-tailed unpaired t-test. ns: not significant. *P<0.05, ***P<0.0005, ****P<0.0001. [Figure 3A] Figure 3 shows that BRCA1-deficient breast tumor cells induce M2-like macrophage polarization in vitro. Diagram of workflow. Top: Mouse bone marrow-derived macrophages (BMDMs) co-cultured with BP tumor cells with or without Olaparib treatment. Bottom: BMDMs were incubated with 50% conditioned medium (CM) from Olaparib-treated or DMSO-treated BP tumor cells. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figure 3B, Figure 3D, and Figure 3E). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 3B] Figure 3 shows that BRCA1-deficient breast tumor cells induce M2-like macrophage polarization in vitro. Flow cytometry analysis of BMDMs co-cultured with BP tumor 1 cells. BMDMs (CD11b+) were plotted as CD206 versus MHC-II to identify M1-like (CD206-MHC-II high) and M2-like (CD206+MHC-II low) polarized phenotypes (n=3-6). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 3B, 3D, and 3E). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 3C] Figure 3 shows that BRCA1-deficient breast tumor cells induce M2-like macrophage polarization in vitro. Heatmap of gene expression for anti-tumor and tumor-promoting genes in BMDMs incubated with DMSO vehicle control, olaparib (OL, 5 μM), 50% BP-CM, or 50% OL-treated BP (BP / OL)-CM for 24 hours. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 3B, 3D, and 3E). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 3D]Figure 3 shows that BRCA1-deficient mammary tumor cells induce M2-like macrophage polarization in vitro. RT-qPCR analysis of mouse BMDMs incubated with control medium, 50% BP-CM, or 50% BP / OL-CM for 24 h (n = 3–5). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 3B, 3D, and 3E). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 3E] Figure 3 shows that BRCA1-deficient breast tumor cells induce M2-like macrophage polarization in vitro. RT-qPCR analysis of THP-1 human macrophages incubated with control medium, 50% tumor cell CM, or 50% CM of olaparib-treated tumor cells for 24 h (n = 3–6). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 3B, 3D, and 3E). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 4A] Figure 1 shows that BRCA1-deficient mammary tumor cells induce tumor-promoting macrophage polarization in vitro. Analysis of mouse BMDMs incubated for 2 days with control medium or 50% conditioned medium (CM) from BP cells or olaparib-treated BP cells (BP / OL). BMDMs (CD11b+) were plotted as CD206 versus MHCII to identify M1-like (CD206-MHCIIhigh) and M2-like (CD206+MHCIIlow) polarized phenotypes (n=4-7). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005. [Figure 4B]Figure 1 shows that BRCA1-deficient mammary tumor cells induce tumor-promoting macrophage polarization in vitro. Analysis of mouse BMDMs incubated for 2 days with control medium or 50% conditioned medium (CM) from BP cells or olaparib-treated BP cells (BP / OL). BMDMs (CD11b+) were plotted as CD206 versus MHCII to identify M1-like (CD206-MHCIIhigh) and M2-like (CD206+MHCIIlow) polarized phenotypes (n=4-7). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005. [Figure 5A] Figure 5 shows that TEM suppresses Olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs. Diagram of workflow for Figure 5B-F. Conditioned medium from control BMDMs or tumor-educated macrophages (TEMs) was added to tumor cell cultures followed by treatment with Olaparib. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5B] TEM shows that olaparib-induced DNA damage in BRCA1-deficient breast tumor cells is suppressed and STING activation in DCs is abolished. Two days after olaparib treatment, BP tumor cells were stained using DAPI and anti-dsDNA antibody. The intensity of cytoplasmic dsDNA was quantified. Scale bar: 50 μm (n=10-26 fields from two independent experiments). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5C]TEM shows that olaparib-induced DNA damage in BRCA1-deficient breast tumor cells is suppressed and STING activation in DCs is abolished. BP cells were stained with anti-H2AX phospho (Ser139) antibody and analyzed by flow cytometry. MFI: median fluorescence intensity (n=3). Data are presented as mean ± standard error. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5D] TEM shows that olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs. MDA-MB-436 cells were stained with anti-H2AX phospho (Ser139) antibody and analyzed by flow cytometry. MFI: median fluorescence intensity (n=3). Data are presented as mean ± standard error. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5E] Figure 2 shows that TEM suppresses olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs. BP cells were analyzed for apoptosis (Annexin V+7-AAD-) (n=3-6). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5F] Figure 2 shows that TEM suppresses olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs. MDA-MB-436 cells were analyzed for apoptosis (Annexin V + 7-AAD-) (n = 3-6). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5G]Figure 2 shows that TEM suppresses olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs. Apoptosis analysis of BP cells co-cultured with or without tumor-associated macrophages (TAMs) sorted from BP tumors and subsequently treated with olaparib for 3 days (n=3). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5H] Figure 5 shows that TEM suppresses Olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs. Diagram of workflow in Figure 5I and Figure 5J. Tumor cells were incubated with control medium or conditioned medium from BMDMs or TEMs, followed by treatment with Olaparib. Two days after treatment, Olaparib was washed out and mouse bone marrow-derived dendritic cells (DCs) were added to BP cells and co-cultured for 24 hours. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5I] TEM suppresses olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs. Flow cytometry analysis of STING pathway activation (p-TBK1+1p-IRF3+) in DCs co-cultured with olaparib-treated or DMSO-treated BP cells (n=4). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 5J]Figure 2 shows that TEM suppresses olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs. RT-qPCR analysis of Ifnb, Ccl5, and Cxcl10 expression in DCs isolated from co-cultures (n=4-9). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 6A] Tumor cell-educated macrophages (TEMs) suppress the synthetic lethal response of BRCA1-deficient breast tumor cells to Olaparib. Analysis of MDA-MD-436 cells co-cultured with THP-1 human macrophages with or without 16-hour treatment with Olaparib. MDA-MD-436 tumor cells (CD45-) in co-culture were analyzed by flow cytometry for phosphorylation of histone H2AX at Ser139 (n=6). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 6B] Tumor cell-educated macrophages (TEMs) suppress the synthetic lethal response of BRCA1-deficient mammary tumor cells to olaparib. Analysis of BP cells co-cultured with mouse BMDMs with or without olaparib treatment for 3 days. BP tumor cells (CD11b-) were analyzed for apoptosis (Annexin V+7-AAD-) by flow cytometry (n=5-6). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 6C]Tumor cell-educated macrophages (TEMs) suppress the synthetic lethal response of BRCA1-deficient breast tumor cells to Olaparib. Analysis of MDA-MB-436 cells co-cultured with THP-1 human macrophages with or without Olaparib treatment for 3 days. MDA-MB-436 tumor cells (CD45-) were analyzed for apoptosis by flow cytometry (n=3). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 6D] Tumor cell-educated macrophages (TEMs) suppress the synthetic lethal response of BRCA1-deficient mammary tumor cells to olaparib. Gating strategy for flow cytometry analysis of STING pathway activation (p-TBK1+p-IRF3+) in DCs co-cultured with BP cells as shown in Figure 3I. Data are presented as mean ± standard error. One-way analysis of variance (ANOVA). *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 6E] Tumor cell-educated macrophages (TEMs) suppress the synthetic lethal response of BRCA1-deficient breast tumor cells to olaparib. RT-qPCR analysis of BP tumor cells in control medium, 50% naive BMDM-conditioned medium (CM), or 50% TEM-CM with or without olaparib treatment for 2 days (n=5-6). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7A]Figure 1 shows that STING agonists reprogram BMDMs to an M1-like state and promote the activation of DCs by tumor cells in the presence of TEMs and olaparib. Mouse BMDMs were treated with DMSO vehicle control, olaparib (OL, 5 μM), DMXAA (0.05 mg / mL), 50% BP-CM with or without DMXAA, or 50% BP / OL-CM with or without DMXAA for 24 hours and then subjected to transcriptome analysis. Heatmap of antitumor and tumor-promoting gene expression in BMDMs. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7B] Figure 1 shows that STING agonists reprogram TEMs to an M1-like state and promote activation of DCs by tumor cells in the presence of TEMs upon olaparib. Mouse BMDMs were treated for 24 hours with DMSO vehicle control, olaparib (OL, 5 μM), DMXAA (0.05 mg / mL), 50% BP-CM with or without DMXAA, or 50% BP / OL-CM with or without DMXAA, and then subjected to transcriptome analysis. Left: Volcano plot showing the significance and magnitude of changes in gene expression in BMDMs treated with BP-CM / DMXAA compared to BP-CM / DMSO. Right: Top ranked Gene Ontology (GO) terms that were up- and downregulated in BMDMs treated with BP-CM / DMXAA. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: no significant difference. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7C]Figure 1 shows that STING agonists reprogram BMDMs to an M1-like state and promote the activation of DCs by tumor cells in the presence of TEMs when o-a-rapid. Analysis of control naive BMDMs and BP TEMs with or without DMXAA (0.05 mg / mL) treatment for 2 days. M1-like (CD11b+CD206-MHC-II high) vs. M2-like (CD11b+CD206+MHC-II low) ratios were analyzed by flow cytometry (n=4). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7D] We show that STING agonists reprogram TEMs to an M1-like state and promote the activation of DCs by tumor cells in the presence of TEMs when OAPR. Analysis of STING pathway activation (p-TBK1+p-IRF3+) in control naive BMDMs and BP TEMs with or without DMXAA treatment for 24 hours (n=3-4). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7E] We show that STING agonists reprogram TEMs to an M1-like state and promote the activation of DCs by tumor cells in the presence of TEMs when o-rapidly administered. BMDMs isolated from wild-type (WT) or STING knockout (STING- / -) C57 / BL6J mice were incubated with control medium or 50% BP-CM to generate control naïve BMDMs and TEMs, respectively. Cells were then treated with or without DMXAA for 2 days. The M1 to M2 ratio of the costimulatory molecule CD86 was analyzed by flow cytometry (n=4). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7F]We show that STING agonists reprogram TEMs to an M1-like state and promote the activation of DCs by tumor cells in the presence of TEMs when o-a-rapid. BMDMs isolated from wild-type (WT) or STING knockout (STING- / -) C57 / BL6J mice were incubated with control medium or 50% BP-CM to generate control naïve BMDMs and TEMs, respectively. Cells were then treated with or without DMXAA for 2 days. Surface levels of the costimulatory molecule CD86 were analyzed by flow cytometry (n=4). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7G] We show that STING agonists reprogram TEMs to an M1-like state and promote the activation of DCs by tumor cells in the presence of TEMs when o-a-rapid. Expression of M2 (CD163) and M1 (CD86) markers in control THP-1 macrophages or MDA-MB-436 tumor cell-educated THP-1 macrophages (TEM-436) with or without ADU-S100 (10 μM) treatment for 2 days (n=3). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7H] Figure 7I shows that STING agonists reprogram BMDMs to an M1-like state and promote the activation of DCs by tumor cells in the presence of TEMs and olaparib. Diagram of workflow in (Figure 7I). Tumor cells were incubated with control medium or conditioned medium from naive BMDMs, TEMs, or DMXAA-treated TEMs and subsequently treated with olaparib. After 2 days of treatment, the drug was removed and tumor cells 1 were co-cultured with DCs for 24 hours. Co-cultured cells were harvested for flow cytometry. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 7I]We show that STING agonists reprogram TEMs to an M1-like state and promote tumor cell-induced DC activation in the presence of TEMs and olaparib. Top: DNA damage response of BP tumor cells to olaparib was analyzed by staining for Ser139 phosphorylated H2AX (n=3-6). Bottom: Flow cytometry analysis of STING pathway activation (p-TBK1+p-IRF3+) in DCs co-cultured with BP tumor cells (n=4). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA). ns: not significant. **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 8A] We show that STING agonists reprogram tumor cell-educated macrophages (TEMs) to an M1-like state in vitro. Mouse BMDMs were exposed to 50% conditioned medium (CM) from olaparib-treated BP tumor cells (BP / OL) with or without DMXAA (0.05 mg / mL) for 24 hours and then subjected to transcriptome analysis. Left: Volcano plot showing the significance and magnitude of gene expression changes comparing BP / OL-CM / DMXAA-treated macrophages with BP / OL-CM / DMSO-treated macrophages. Right: Top ranked Gene Ontology (GO) terms that were up- and downregulated in BP / OL-CM / DMXAA-treated macrophages. [Figure 8B] Figure 1 shows that STING agonists reprogram tumor cell-educated macrophages (TEMs) to an M1-like state in vitro.Representative images of mouse BMDMs after 24 h incubation with media containing IL4 (20 ng / mL), LPS (100 ng / mL), IFNg (20 ng / mL), olaparib (5 μM), DMXAA (0.05 mg / mL), 50% BP-CM with or without DMXAA, or 50% BP / OL-CM with or without DMXAA. [Figure 9A]9A-9C show that STING agonists improve the therapeutic response of orthotopic BP tumors to olaparib in syngeneic immunocompetent mice in vivo. Growth of BP tumors in FVB mice treated with olaparib (50 mg / kg, intraperitoneally, daily) or intratumoral injection of DMXAA (10 mg / kg, once weekly for 3 weeks [total of 3 doses]) as monotherapy or combination therapy. Control: n=8, olaparib: n=9, DMXAA: n=6, olaparib+DMXAA: n=7. Each dot represents the result from a single tumor. Data are presented as mean ± standard error (tumor volume) or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (FIG. 9A). One-way analysis of variance (FIG. 9B and FIG. 9C). ns: no significant difference. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 9B] Figure 9 shows that STING agonists improve the therapeutic response of orthotopic BP tumors to olaparib in syngeneic immunocompetent mice in vivo. Analysis of BP tumors after 21 days of treatment for effector cytokine production by intratumoral CD8+ and CD4+ T cells. Each dot represents the result from a single tumor. Data are presented as mean ± SEM (tumor volume) or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Figure 9A). One-way analysis of variance (Figure 9B and Figure 9C). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 9C]Figure 9 shows that STING agonists improve the therapeutic response of orthotopic BP tumors to olaparib in syngeneic immunocompetent mice in vivo. Analysis of BP tumors after 21 days of treatment for effector cytokine production by intratumoral CD8+ and CD4+ T cells. Each dot represents the result from a single tumor. Data are presented as mean ± SEM (tumor volume) or median with quartiles (violin plot). Two-way analysis of variance (ANOVA) (Figure 9A). One-way analysis of variance (Figure 9B and Figure 9C). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 10A] Figure 10 shows that TAM depletion improves the therapeutic response of orthotopic BP tumors to olaparib in syngeneic immunocompetent mice in vivo. Tumor growth of Brca1- / - Trp53- / - (BP) allografts in FVB mice treated with olaparib or anti-CSF1R as monotherapy or in combination. Control: n=10, olaparib: n=10, anti-CSF1R: n=10, olaparib + anti-CSF1R: n=10. Each dot represents the result from a single tumor. Data are presented as mean ± standard error (Figure 10A) or median with quartiles (violin plots, Figures 10B-D). Two-way analysis of variance (ANOVA) (Figure 10A). One-way analysis of variance (Figures 10B-D). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 10B] Figure 10 shows that TAM depletion improves the therapeutic response of orthotopic BP tumors to olaparib in syngeneic immune competent mice in vivo. Flow cytometry analysis of intratumoral immune cells of BP tumors after 21 days of treatment. Each dot represents the result from a single tumor. Data are presented as mean ± SEM (Figure 10A) or median with quartiles (violin plots, Figures 10B-D). Two-way analysis of variance (ANOVA) (Figure 10A). One-way analysis of variance (Figures 10B-D). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 10C] Figure 10 shows that TAM depletion improves the therapeutic response of orthotopic BP tumors to olaparib in syngeneic immune competent mice in vivo. Flow cytometry analysis of intratumoral immune cells of BP tumors after 21 days of treatment. Each dot represents the result from a single tumor. Data are presented as mean ± SEM (Figure 10A) or median with quartiles (violin plots, Figures 10B-D). Two-way analysis of variance (ANOVA) (Figure 10A). One-way analysis of variance (Figures 10B-D). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 10D] Figure 10 shows that TAM depletion improves the therapeutic response of orthotopic BP tumors to olaparib in syngeneic immune competent mice in vivo. Flow cytometry analysis of intratumoral immune cells of BP tumors after 21 days of treatment. Each dot represents the result from a single tumor. Data are presented as mean ± SEM (Figure 10A) or median with quartiles (violin plots, Figures 10B-D). Two-way analysis of variance (ANOVA) (Figure 10A). One-way analysis of variance (Figures 10B-D). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11A] Figure 11 shows that systemic delivery of STING agonist sensitizes STING-null BP tumors to olaparib in vivo. Western blots of STING and vinculin in CRISPR / Cas9 control and STING knockout BP tumor cells (BP-sg control and BP-sgSTING). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 11C-11F). Two-way ANOVA for tumor growth (Figures 11G, 11H, 11I, and 11J). Log-rank Mantel-Cox test for survival (Figures 11G and 11H). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11B]Figure 11 shows that systemic delivery of STING agonist sensitizes STING-null BP tumors to Olaparib in vivo. CellTiter-Glo® analysis showing cell viability of BP-sg control and BP-sgSTING cells after 3 days of treatment with serial dilutions of Olaparib (n=3). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 11C-11F). Two-way ANOVA for tumor growth (Figures 11G, 11H, 11I, and 11J). Log-rank Mantel-Cox test for survival (Figures 11G and 11H). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11C] Figure 11 shows that systemic delivery of STING agonists sensitizes STING-null BP tumors to olaparib in vivo. Flow cytometry analysis of mouse BMDMs treated with DMSO vehicle control, olaparib (OL, 5 μM), 50% BP-sgSTING-CM, or 50% BP-sgSTING / OL-CM for 2 days (n=3). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 11C-F). Two-way ANOVA for tumor growth (Figures 11G, 11H, 11I, and 11J). Log-rank Mantel-Cox test for survival (Figures 11G and 11H). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11D]Figure 11 shows that systemic delivery of STING agonist sensitizes STING-null BP tumors to Olaparib in vivo. ELISA analysis of IFNβ in media from BP-sg control or BP-sgSTING cells with or without olaparib treatment for 2 days (n=7). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 11C-F). Two-way ANOVA for tumor growth (Figures 11G, 11H, 11I, and 11J). Log-rank Mantel-Cox test for survival (Figures 11G and 11H). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11E] Figure 11 shows that systemic delivery of STING agonist sensitizes STING-null BP tumors to Olaparib in vivo. RT-qPCR analysis of Ccl5 in BP-1sg control and BP-sgSTING cells with or without olaparib treatment for 2 days (n=3-4). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figure 11C-F). Two-way ANOVA for tumor growth (Figure 11G, H, I, and J). Log-rank Mantel-Cox test for survival (Figure 11G and H). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11F] Figure 11 shows that systemic delivery of STING agonist sensitizes STING-null BP tumors to Olaparib in vivo. RT-qPCR analysis of Cxcl10 in BP-1sg control and BP-sgSTING cells with or without olaparib treatment for 2 days (n=3-4). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figure 11C-F). Two-way ANOVA for tumor growth (Figure 11G, H, I, and J). Log-rank Mantel-Cox test for survival (Figure 11G and H). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11G]Figure 11 shows that systemic delivery of STING agonists sensitizes STING-null BP tumors to olaparib in vivo. Tumor growth (left) and survival (right) of BP-sg control tumor-bearing FVB mice treated with olaparib (50 mg / kg, i.p., daily), DMXAA (10 mg / kg, i.p.), or olaparib+DMXAA. Median survival times are shown in brackets. Left - control: n=13, olaparib: n=7, DMXAA: n=9; olaparib+DMXAA: n=14. Right - control: n=8, olaparib: n=5, DMXAA: n=6, olaparib+DMXAA: n=9. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 11C-11F). Two-way ANOVA for tumor growth (Figures 11G, 11H, 11I, and 11J). Log-rank Mantel-Cox test for survival (Figures 11G and 11H). ns: no significant difference. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11H] Figure 11 shows that systemic delivery of STING agonists sensitizes STING-null BP tumors to olaparib in vivo. Tumor growth (left) and survival (right) of BP-sgSTING tumor-bearing FVB mice treated with olaparib (50 mg / kg, i.p., daily), DMXAA (10 mg / kg, i.p.), or olaparib + DMXAA. Median survival times are shown in brackets. Left - control: n=24, olaparib: n=11, DMXAA: n=9, olaparib + DMXAA: n=19. Right - control, n=13: olaparib, n=7, DMXAA: n=6, olaparib + DMXAA: n=11. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figure 11C-F). Two-way ANOVA for tumor growth (Figures 11G, 11H, 11I, and 11J). Log-rank Mantel-Cox test for survival (Figures 11G and 11H). ns: no significant difference. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11I]11C-11F). Two-way ANOVA for tumor growth (FIGS. 11G, 11H, 11I, and 11J). Log-rank Mantel-Cox test for survival (FIGS. 11G and 11H). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 11J] Figure 11 shows that systemic delivery of STING agonist sensitizes STING-null BP tumors to olaparib in vivo. BP-sgSTING tumor growth in FVB mice treated with olaparib + DMXAA with or without anti-CD8 or anti-IFNAR1 neutralizing antibodies (n=6 per condition). Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) (Figures 11C-11F). Two-way ANOVA for tumor growth (Figures 11G, 11H, 11I, and 11J). Log-rank Mantel-Cox test for survival (Figures 11G and 11H). ns: not significant. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. [Figure 12A] Figure 12 shows that systemic delivery of STING agonist sensitizes one STING-null BP tumor to olaparib in vivo. Analysis of bone marrow-derived dendritic cells (DCs) co-cultured with DMSO vehicle control-treated or olaparib-treated BP-sg control or BP-sgSTING tumor cells for 24 hours (n=3). Data are presented as mean±SEM or median with quartiles (violin plot). One-way analysis of variance (ANOVA) (Figure 12A). Two-way analysis of variance (Figure 12B). ns: not significant. *P<0.05, **P<0.005. [Figure 12B]Figure 12 shows that systemic delivery of STING agonists sensitizes one STING-null BP tumor to olaparib in vivo. Tumor growth of BP-sgSTING allografts in FVB mice treated with intratumoral injection (IT) of DMXAA (10 mg / kg, given once weekly) either as single agent treatment or in combination with olaparib (50 mg / kg, intraperitoneally, daily). Control: n=4 tumors, DMXAA: n=4 tumors, DMXAA+olaparib: n=4 tumors. Data are presented as mean ± SEM or median with quartiles (violin plot). One-way analysis of variance (ANOVA) (Figure 12A). Two-way analysis of variance (Figure 12B). ns: not significant. *P<0.05, **P<0.005. [Figure 13] We show that harnessing antitumor immunity with STING agonists overcomes immune suppression and resistance to PARP inhibition in BRCA1-deficient breast cancer. BRCA1-deficient mammary tumors induce the polarization of tumor-promoting macrophages via paracrine activation of macrophage M2-like phenotype. In turn, these tumor-educated macrophages not only exhibit suppressive activity against T cells but also attenuate PARPi-mediated synthetic lethality and production of cytoplasmic double-stranded DNA (dsDNA), thus abolishing the activity of the DNA-sensing adaptor STING, rendering BRCA1-deficient mammary tumors resistant to PARPi therapy. Exogenous agonists of the STING pathway reprogram macrophages, triggering activation of innate immunity in both macrophages and DCs, and inducing adaptive immune responses that synergize with PARPi therapy to induce DNA damage in tumor cells and resensitize tumors to PARPi therapy. [Figure 14A] We show that systemic delivery of STING agonist MSA-2 sensitizes PARPi-resistant tumors to olaparib in vivo. Generation of PARPi-resistant ovarian cancer tumors. PBM is the control group, and PBM-R is the recurrent tumor (olaparib-resistant group) upon long-term olaparib treatment. [Figure 14B]Shows that systemic delivery of the STING agonist MSA-2 sensitizes PARPi-resistant tumors to olaparib in vivo. M2-like TAMs are enriched in PBM-R tumors. Flow cytometry analysis of intratumoral and ascites M2-like TAMs in PBM and PBM-R tumors. Two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. [Figure 14C] We show that systemic delivery of the STING agonist MSA-2 sensitizes PARPi-resistant tumors to olaparib in vivo. STING agonists reprogram PBM-R medium educated macrophages (TEMs) to an M1-like state in vitro. Analysis of control naive BMDMs and PBM-R TEMs with or without treatment with ADU-S100 (10uM) or MSA-2 (5ug / ml) for 2 days. M1-like (CD11b+CD206-MHC-II high) vs. M2-like (CD11b+CD206+MHC-II low) ratios were analyzed by flow cytometry (n=3). Two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. [Figure 14D] Figure 1 shows that systemic delivery of the STING agonist MSA-2 sensitizes PARPi-resistant tumors to olaparib in vivo. Tumor weights (left) and resected tumors (right) from PBM-R-bearing FVB mice treated for 2 weeks with vehicle control, olaparib (50 mg / kg, i.p., daily) + anti-PD-1 (200 ug / mouse, i.p., every 3 days), MSA-2 (25 mg / kg, i.p., 3 times per week), or MSA-2 + olaparib + anti-PD-1. Two-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. [Figure 15A] Figure 1 shows the generation of a PARPi-resistant ovarian cancer mouse model by chronic treatment of PBM tumor-bearing mice (PBM-R, PBM-refractory tumors after olaparib treatment) with olaparib showing the characterization of tumor-promoting macrophages in Brca1-deficient ovarian tumors that have acquired secondary resistance to PARP inhibition. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ****P<0.0001. [Figure 15B]Characterization of tumor-promoting macrophages in Brca1-deficient ovarian tumors that have acquired secondary resistance to PARP inhibition. Tumor growth curves of PBM-tumor-bearing mice treated with olaparib and vehicle control. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ****P<0.0001. [Figure 15C] Characterization of tumor-promoting macrophages in Brca1-deficient ovarian tumors that have acquired secondary resistance to PARP inhibition. Measurement of IC50 values ​​for PARPi naive PBM cells and tumor cells isolated from PBM-R tumor-bearing mice (each line originates from a PBM-R tumor-bearing mouse) are shown. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ****P<0.0001. [Figure 15D] Characterization of tumor-promoting macrophages in Brca1-deficient ovarian tumors that have acquired secondary resistance to PARP inhibition. PBM and PBM-R cells were treated with 1 μM olaparib or vehicle control for 24 hours and stained with anti-phospho-H2A.X (Ser139) antibody (γ-H2AX) for flow cytometry analysis (n=3). Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ****P<0.0001. [Figure 15E] Characterization of tumor-promoting macrophages in Brca1-deficient ovarian tumors that have acquired secondary resistance to PARP inhibition. Tumor burden and representative bioluminescence imaging analysis of vehicle control or olaparib-treated PBM or in vitro sensitive PBM-R tumor-bearing mice are shown. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ****P<0.0001. [Figure 16] 1 shows copy number polymorphisms of DNA repair pathway genes detected in in vitro PARPi-resistant PBM-R lines. [Figure 17A]Figure 1 shows that M2-like macrophages are increased in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of tumor-infiltrating tumor-promoting TAMs (M2-like TAMs) in PBM and PBM-R tumor-bearing mice. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 17B] Figure 1 shows that M2-like macrophages are increased in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of total and M2-like TAMs in ascites of PBM and PBM-R tumor-bearing mice. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 17C] Figure 17D shows that M2-like macrophages are increased in PARPi-resistant Brca1-deficient ovarian cancer. A diagram of the workflow in Figure 17D is shown. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 17D] Figure 1 shows that M2-like macrophages are increased in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of bone marrow-derived macrophages (BMDM) cultured for 3 days in 50% complete medium and 50% PBM-CM or PBM-R-CM. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 17E] Figure 17 shows that M2-like macrophages are increased in PARPi-resistant Brca1-deficient ovarian cancer. A diagram of the workflow in Figure 17F is shown. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 17F] Figure 1 shows that M2-like macrophages are increased in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of bone marrow cells (BMC) cultured for 5 days in 50% complete medium and 50% ascites supernatant. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 18] Flow cytometry analysis of tumor-infiltrating CD45+, CD11b+, MDSC, CD4+, CD8+ and Treg cells in PBM and PBM-R tumors. [Figure 19A] Figure 1. Activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. GSEA analysis of RNA sequencing data from PBM and PBM-R tumors is shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19B] Figure 1. Activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of STAT3 (Y705) phosphorylation levels in PBM and PBM-R tumor cells. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19C] We show that activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Representative images of immunohistochemistry (IHC) staining of p-STAT3 (Y705) in PBM and PBM-R tumors are shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19D]Figure 1 shows that activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of p-STAT3 in PBM tumor cells treated with the indicated concentrations of olaparib or vehicle control. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19E] Figure 1. Activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of BMDMs cultured in CM from PBM with or without olaparib treatment. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19F] Figure 2 shows that activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Analysis of cytokines in the medium of PBM treated with olaparib or vehicle control is shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19G] Figure 2 shows that activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Analysis of mouse BMDMs cultured for 3 days with or without 50% PBM-R-CM in the presence or absence of the indicated neutralizing antibodies is shown (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19H] Figure 1. Activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of BMDMs cultured in CM from PBM-R with or without STAT3 knockdown is shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19I]Figure 1 shows that activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Tumor burden in mice implanted with control or STAT3 shRNA-expressing PBM-R tumor cells and treated with olaparib or vehicle control. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19J] We show that activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of the proportion of total TAMs and M1-like and M2-like macrophages in olaparib-treated PBM-R tumors expressing control or STAT3 shRNA in Figure 19I. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 19K] Activation of STAT3 signaling is required for M2-like macrophage polarization in PARPi-resistant Brca1-deficient ovarian cancer. Flow cytometry analysis of CD4+ and CD8+ effector T cells in olaparib-treated PBM-R tumors expressing control or STAT3 shRNA in Figure 19I. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 20A] Figure 2 shows that PARP inhibition upregulates STAT3 signaling and renders ovarian cancer PARPi resistant in Brca1-deficient ovarian cancer.GSEA analysis of the STAT3 signaling pathway in PBM and PBM-R tumors shows *P<0.05, **P<0.01, ***P<0.001. [Figure 20B] Figure 1 shows that PARP inhibition upregulates STAT3 signaling and renders Brca1-deficient ovarian cancer resistant to PARPi. Western blot analysis of total and phosphorylated STAT3 in PBM cells treated with the indicated concentrations of olaparib or vehicle control for 24 hours. *P<0.05, **P<0.01, ***P<0.001. [Figure 20C]Figure 1 shows that PARP inhibition upregulates STAT3 signaling and renders ovarian cancer PARPi resistant in Brca1-deficient ovarian cancer. Western blot analysis of total and phosphorylated Stat3 in PBM and PBM-R cells stably expressing control shRNA or shRNA targeting Stat3. *P<0.05, **P<0.01, ***P<0.001. [Figure 20D] Figure 2 shows that PARP inhibition upregulates STAT3 signaling and renders ovarian cancer PARPi resistant in Brca1-deficient ovarian cancer. Evaluation of IC50 values ​​in control and Stat3-silenced PBM-R cells after olaparib treatment is shown. *P<0.05, **P<0.01, ***P<0.001. [Figure 20E] Figure 1. PARP inhibition upregulates STAT3 signaling and renders ovarian cancer PARPi resistant in Brca1-deficient ovarian cancer. Analysis of tumor-infiltrating M1- and M2-like macrophages in Stat3-silenced PBM-R tumors treated with olaparib or vehicle control. *P<0.05, **P<0.01, ***P<0.001. [Figure 20F] Figure 2 shows that PARP inhibition upregulates STAT3 signaling and renders ovarian cancer PARPi resistant in Brca1-deficient ovarian cancer. Analysis of tumor-infiltrating CD4+ and CD8+ cells in Stat3-silenced PBM-R tumors treated with olaparib or vehicle control. *P<0.05, **P<0.01, ***P<0.001. [Figure 21A] We show that STING agonism reprograms bone marrow cells in a STING-dependent manner in vitro and in vivo. A diagram of the workflow is shown in Fig. 21B and Fig. 21H. Bone marrow-derived macrophages (BMDMs) were cultured with or without STING agonist (ADU-S100 or MSA-2) treatment in 50% PBM-R-CM or control medium for 48 hours. ns: no significant difference. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21B]STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Flow cytometry analysis of macrophage phenotype in Figure 21A. ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21C] STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Diagram of workflow in Figure 21D. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21D] Figure 1 shows that STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Heatmap of differentially expressed genes in myeloid cells (CD45+CD11b+) taken from ascites of PBM-R tumor-bearing mice with the indicated treatments. ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21E] We show that STING agonism reprograms bone marrow cells in a STING-dependent manner in vitro and in vivo. GSEA analysis showing upregulation of the STING signaling pathway in bone marrow cells in ascites of MSA-2-treated PBM-R tumor-bearing mice (Fig. 21F-H). Flow cytometry analysis of TAMs (Fig. 21F), myeloid DCs (Fig. 21G, left), MHC-I+DCs (Fig. 21G, right), and p-TBK-1+DCs (Fig. 21H) in ascites of PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and olaparib+MSA-2 for 24 hours. ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21F] Figure 1. STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Flow cytometry analysis of TAMs in ascites from PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and olaparib + MSA-2 for 24 hours. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21G] We show that STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Flow cytometry analysis of myeloid DCs (left) and MHC-I+ DCs (right) in the ascites of PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and olaparib + MSA-2 for 24 hours. ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Fig. 21H] Figure 1. STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Flow cytometry analysis of p-TBK-1+DCs in the ascites of PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and olaparib + MSA-2 for 24 hours. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21I] Figure 1 shows that STING agonism reprograms bone marrow cells in a STING-dependent manner in vitro and in vivo. Analysis of M1 / M2 ratios in WT and STING- / - BMDMs cultured with CM from ID8-Brca1- / - treated with olaparib or vehicle control (n=3). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21J] STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. A diagram of the workflow in Figure 21K is shown. ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21K] STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Analysis of macrophages (M1 / M2) in WT and STING- / - mice injected with ID8-Brca1- / - tumor cells and treated with the indicated drugs for 24 h. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21L]Shows that STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Shown are tumor burdens in PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and the combination of olaparib and MSA-2 for 14 days (n=7). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21M] STING agonism reprograms myeloid cells in a STING-dependent manner in vitro and in vivo. Flow cytometry analysis of mouse BMDMs cultured for 3 days with CM from control or olaparib-treated ID8-Brca1+ / + and ID8-Brca1- / - cells in the presence or absence of 5 μg / ml MSA-2. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 21N] Figure 1 shows that STING agonism reprograms bone marrow cells in a STING-dependent manner in vitro and in vivo. Flow cytometry analysis of human BMDMs cultured for 3 days with CM from control or olaparib-treated UWB1.289 or UWB1.289+BRCA1 cells in the presence or absence of 5 μg / ml MSA-2. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 22A] Figure 2 shows that STING agonism regulates myeloid cells and sensitizes PBM-R tumors to PARP inhibition.Flow cytometry analysis of bone marrow cells isolated from ascites is shown. [Figure 22B] Figure 2 shows that STING agonism regulates myeloid cells and sensitizes PBM-R tumors to PARP inhibition. Volcano plots showing the significance and magnitude of changes in gene expression in mouse dendritic cells treated with a STING agonist (DMXAA) and an inhibitor of TBK-1 (BX795). [Figure 22C]Figure 2 shows that STING agonism regulates myeloid cells and sensitizes PBM-R tumors to PARP inhibition.Top ranked and upregulated Gene Ontology (GO) terms in myeloid cells treated with MSA-2 or the combination of MSA-2 and olaparib are shown. [Figure 22D] Figure 1 shows that STING agonism regulates myeloid cells and sensitizes PBM-R tumors to PARP inhibition. Analysis of total DCs and p-TBK-1+ myeloid DCs in WT and STING- / - mice injected with ID8-Brca1- / - tumor cells and treated with the indicated drugs for 24 h. *P<0.05. [Figure 23A] Figure 1 shows that STING agonism modulates the TME and resensitizes PBM-R tumors to PARP inhibition. Tumor burden in PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and the combination of olaparib and MSA-2 for 14 days (n=7). Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 23B] Figure 23 shows that STING agonism modulates the TME and resensitizes PBM-R tumors to PARP inhibition. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice, as depicted in Figure 23A: TAM. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 23C] Figure 23 shows that STING agonism modulates the TME and resensitizes PBM-R tumors to PARP inhibition. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice: M1 / M2, as depicted in Figure 23A. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 23D]Figure 23 shows that STING agonism modulates the TME and resensitizes PBM-R tumors to PARP inhibition. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice, as depicted in Figure 23A: CD86+ (left) and p-TBK-1+ (right) in cDCs (CD11c+MHC-II+). Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 23E] We show that STING agonism modulates the TME and resensitizes PBM-R tumors to PARP inhibition. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice, as depicted in Figure 23A: MHC-I+ (left) and p-TBK-1+ in myeloid DCs (CD11b+MHC-II+). Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 23F] Figure 23 shows that STING agonism modulates the TME and resensitizes PBM-R tumors to PARP inhibition. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice: total CD4+ T cells and TNFα+ CD4+ T cells, as depicted in Figure 23A. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 23G] Figure 23 shows that STING agonism modulates the TME and resensitizes PBM-R tumors to PARP inhibition. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice: total CD8+ T cells and TNFα+CD8+ T cells, as depicted in Figure 23A. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 24A]Figure 1 shows that STING agonism in combination with olaparib increases tumor-infiltrating anti-tumor immune cells in PBM-R tumor-bearing mice. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and the combination of olaparib and MSA-2 for 14 days (n=7). CD45+ is indicated. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 24B] Figure 1 shows that STING agonism in combination with olaparib increases tumor-infiltrating anti-tumor immune cells in PBM-R tumor-bearing mice. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and the combination of olaparib and MSA-2 for 14 days (n=7). CD11b+ is indicated. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns, not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 24C] Figure 1 shows that STING agonism in combination with olaparib increases tumor-infiltrating anti-tumor immune cells in PBM-R tumor-bearing mice. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and the combination of olaparib and MSA-2 for 14 days (n=7). cDCs (CD11c+MHC-II+) are shown. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns, not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 24D]STING agonism in combination with olaparib increases tumor-infiltrating anti-tumor immune cells in PBM-R tumor-bearing mice. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and the combination of olaparib and MSA-2 for 14 days (n=7). Myeloid DCs (CD11b+MHC-II+) are indicated. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 24E] Figure 1 shows that STING agonism in combination with olaparib increases tumor-infiltrating anti-tumor immune cells in PBM-R tumor-bearing mice. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and the combination of olaparib and MSA-2 for 14 days (n=7). CD3+ is indicated. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 24F] Figure 1 shows that STING agonism in combination with olaparib increases tumor-infiltrating anti-tumor immune cells in PBM-R tumor-bearing mice. Flow cytometry analysis of tumor-infiltrating immune cells in PBM-R tumor-bearing mice treated with control, olaparib, MSA-2, and the combination of olaparib and MSA-2 for 14 days (n=7). Effector CD4+ and CD8+ T cells are shown. Grey dots: outliers. Two-way analysis of variance (ANOVA). ns: not significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 25A] Figure 25A shows that STING agonist treatment overcomes PARPi resistance in ovarian PDXs. Figure 25A is a diagram of the workflow of (B-E). *P<0.05, **P<0.01, ***P<0.001. [Figure 25B]Figure 1. Overcoming PARPi resistance in ovarian PDXs by STING agonist treatment. Flow cytometry detection of STAT3 (Y705) phosphorylation levels in human ovarian PDXs with or without olaparib treatment. *P<0.05, **P<0.01, ***P<0.001. [Figure 25C] Figure 25 shows that STING agonist treatment overcomes PARPi resistance in ovarian PDXs. Flow cytometry analysis of human BMDMs cultured in CM from PDXs treated with olaparib or control for 3 days as described in Figure 25A. *P<0.05, **P<0.01, ***P<0.001. [Figure 25D] Figure 1 shows overcoming PARPi resistance in ovarian PDXs by STING agonist treatment. Flow cytometry analysis of human BMDMs cultured in CM from control or STAT3 inhibitor (napabucasin) treated PDXs. *P<0.05, **P<0.01, ***P<0.001. [Figure 25E] Figure 1 shows overcoming PARPi resistance in ovarian PDXs by STING agonist treatment. Analysis of human BMDMs cultured in CM from PDXs in the presence or absence of MSA-2 is shown. *P<0.05, **P<0.01, ***P<0.001. [Figure 25F] Figure 25 shows that STING agonist treatment overcomes PARPi resistance in ovarian PDXs. Diagram of workflow in Figure 25G. *P<0.05, **P<0.01, ***P<0.001. [Figure 25G] Figure 2. Overcoming PARPi resistance in ovarian PDXs by STING agonist treatment. Tumor burden in DF86 PDX-bearing mice treated with control, MSA-2, olaparib, and the combination of MSA-2 and olaparib for 2 weeks. *P<0.05, **P<0.01, ***P<0.001. [Fig. 25H] 25G shows that STING agonist treatment overcomes PARPi resistance in ovarian PDXs. Analysis of the indicated immune cells in ascites of DF86 PDX-bearing mice as described in FIG. TAMs (CD11b+; CD68+) are shown. *P<0.05, **P<0.01, ***P<0.001. [Figure 25I] Analysis of the indicated immune cells in the ascites of DF86 PDX-bearing mice as described in Figure 25G. M1 / M2, TAMs were analyzed to identify M1-like (CD80+;CD163-) and M2-like (CD80-;CD163+) polarized phenotypes. *P<0.05, **P<0.01, ***P<0.001. [Figure 25J] Analysis of the indicated immune cells in the ascites of DF86 PDX-bearing mice as described in Figure 25G. CD14+DC (CD14+HLA-DR+) is shown. *P<0.05, **P<0.01, ***P<0.001. [Figure 26A] Shown is a combination treatment of STING agonist and PARPi in ovarian PDXs. Shown is flow cytometry analysis of γ-H2AX in ovarian PDXs treated with olaparib or vehicle control (n=3). *P<0.05, **P<0.01, ***P<0.001. [Figure 26B] Shown are STING agonist and PARPi combination treatments in ovarian PDXs. Shown are tumor burdens in DF86 PDX-bearing mice treated with control, MSA-2, olaparib, and the combination of MSA-2 and olaparib for 2 weeks. *P<0.05, **P<0.01, ***P<0.001. [Figure 26C] Combination treatment of STING agonist and PARPi in ovarian PDX. Analysis of the indicated immune cells in the ascites of DF86 PDX-bearing mice as described in Figure 26B: TAM (CD11b+, CD68+). *P<0.05, **P<0.01, ***P<0.001. [Figure 26D] Combination treatment of STING agonist and PARPi in ovarian PDX. Analysis of the indicated immune cells in the ascites of DF86 PDX-bearing mice as described in Figure 26B: M1 / M2. *P<0.05, **P<0.01, ***P<0.001. [Figure 26E]Combination treatment of STING agonist and PARPi in ovarian PDX. Analysis of the indicated immune cells in the ascites of DF86 PDX-bearing mice as described in Figure 26B: CD14+DC (CD14+HLA-DR+). *P<0.05, **P<0.01, ***P<0.001. [Figure 26F] 1 shows combined STING agonist and PARPi treatment in ovarian PDXs. 1 shows a schematic of TAM polarization. [Figure 27A] Figure 1 shows that osimertinib (AZD9291)-induced adaptive immune activation is necessary for its therapeutic efficacy in vivo. Figure 2 shows CD8+ T cell depletion in FVB mice impaired the in vivo therapeutic efficacy of AZD9291 in a GEM model of EGFR mutant tumors driven by exon 19 deletion / T790M EGFR and loss of Trp53 (referred to as PE) (control: n=6, AZD9291: n=6, AZD9291+CD8 antibody: n=8). Data are presented as mean ± SEM (a and c) or median with quartiles (violin plot, b). ns: not significant. *P<0.05, **P<0.01. [Figure 27B] Figure 1 shows that osimertinib (AZD9291)-induced adaptive immune activation is necessary for its therapeutic efficacy in vivo. Figure 1 shows that AZD9291 treatment induced T cell recruitment and activation in the tumor microenvironment (TME). Each dot represents the result from a single tumor. Data are presented as mean ± standard error (a and c) or median with quartiles (violin plot, b). ns: not significant. *P<0.05, **P<0.01. [Figure 27C] Figure 27C shows that osimertinib (AZD9291)-induced adaptive immune activation is necessary for its therapeutic efficacy in vivo. Figure 27C shows that AZD9291 increased the expression of CCL5 and CXCL10 in cultured tumor cells derived from PE GEMM and in the human EGFR mutant NSCLC cell line PC9GR4. Data are presented as mean ± standard error (a and c) or median with quartiles (violin plot, b). ns: not significant. *P<0.05, **P<0.01. [Figure 27D] We show that osimertinib (AZD9291)-induced adaptive immune activation is necessary for its therapeutic efficacy in vivo. Analysis of clinical data of eight patients with EGFR-mutated advanced NSCLC suggests that the T cell inflammatory signature in tumors is significantly enhanced upon EGFR-TKI (osimertinib or erlotinib) treatment in responders (n=4, progression-free survival [PFS] >8 months) but not in non-responders (n=4, PFS <8 months). Data are presented as mean ± standard error (a and c) or median with quartiles (violin plot, b). ns: not significant. *P<0.05, **P<0.01. [Figure 27E] We show that osimertinib (AZD9291)-induced adaptive immune activation is necessary for its therapeutic efficacy in vivo. We show that elevated T cell inflammation scores upon EGFR-TKI (osimertinib or erlotinib) treatment positively correlate with PFS in patients with EGFR-mutant advanced NSCLC who received EGFR-TKI as first-line treatment. Data are presented as mean ± SEM (a and c) or median with quartiles (violin plot, b). ns: not significant. *P<0.05, **P<0.01. [Figure 28A] Figure 1 shows that an established immunosuppressive TME inhibits the therapeutic efficacy of osimertinib (AZD9291). Figure 2 shows that the therapeutic efficacy of AZD9291 is moderate in large tumors. Data are presented as mean ± standard error (a) or median with quartiles (violin plots, b and c). ns: not significant. *P<0.05, **P<0.01, ****P<0.0001. [Figure 28B]Figure 1 shows that an established immunosuppressive TME inhibits the therapeutic efficacy of osimertinib (AZD9291). Figure 2 shows that flow cytometry analysis revealed that larger tumors (500 mm3) exhibited a more immunosuppressive TME than smaller tumors (100 mm3), as evidenced by decreased T cell and DC infiltration, and increased TAM infiltration and M2 polarization of TAMs. Each dot represents the result from a single tumor. Data are presented as mean ± standard error (a) or median with quartiles (violin plots, b and c). ns: not significant. *P<0.05, **P<0.01, ****P<0.0001. [Figure 28C] Figure 1 shows that an established immunosuppressive TME inhibits the therapeutic efficacy of osimertinib (AZD9291). Figure 2 shows that AZD9291 was unable to induce T cell activation in large tumors (500 mm3). Data are presented as mean ± SEM (a) or median with quartiles (violin plots, b and c). ns: not significant. *P<0.05, **P<0.01, ****P<0.0001. [Figure 29A] We show that tumor-associated macrophages (TAMs) suppress CD8+ T cell activation and impair the therapeutic efficacy of osimertinib (AZD9291) in large established tumors.We present an analysis of clinical data from patients with EGFR-mutated advanced NSCLC, showing that non-responders to TKI (osimertinib or erlotinib) treatment showed a significantly enriched TAM signature prior to TKI treatment compared to responders. [Figure 29B] We show that tumor-associated macrophages (TAMs) suppress CD8+ T cell activation and impair the therapeutic efficacy of osimertinib (AZD9291) in large established tumors. We show that TAM enrichment scores before TKI treatment negatively correlate with T cell inflammation scores upon TKI treatment. [Figure 29C]Figure 1 shows that tumor-associated macrophages (TAMs) suppress CD8+ T cell activation and impair the therapeutic efficacy of osimertinib (AZD9291) in large established tumors. Figure 2 shows that TAMs derived from PE GEMMs significantly suppressed the activation of CD8+ T cells from naive FVB mice in co-culture. Data are presented as median with quartiles (violin plot, c) or mean ± standard error (d). ns: not significant. *P<0.05, ***P<0.001, ****P<0.0001. [Figure 29D] Figure 1 shows that tumor-associated macrophages (TAMs) suppress CD8+ T cell activation and impair the therapeutic efficacy of osimertinib (AZD9291) in large established tumors. Figure 2 shows that TAM depletion with anti-CSF1-R antibody enhanced the therapeutic efficacy of AZD9291 in large PE tumors (control: n=6, AZD9291: n=7, AZD9291+CSF1 antibody: n=5). Data are presented as median with quartiles (violin plot, c) or mean ± standard error (d). ns: not significant. *P<0.05, ***P<0.001, ****P<0.0001. [Figure 30A] Figure 1 shows that systemic delivery of STING agonists in combination with osimertinib induces tumor regression in large established tumors. FVB mice bearing PE tumors were treated with AZD9291, MSA-2, or AZD9291+MSA-2 with or without anti-CD8 antibody (control: n=6, AZD9291: n=8, MSA-2: n=8, AZD9291+MSA-2: n=10, AZD9291+MSA-2+CD8 antibody: n=8). Each dot represents the result from a single tumor. Data are presented as mean ± SEM (a) or median with quartiles (violin plots, b and c). ns: not significant. *P<0.05, **P<0.01, ***P<0.0001, ****P<0.0001. [Figure 30B]Figure 30B shows that systemic delivery of STING agonists in combination with osimertinib induces tumor regression in large established tumors. Figure 30B shows that the combination of AZD9291 and MSA-2, but not single agents, significantly induced immune activation in the TME of PE tumors. Each dot represents the result from a single tumor. Data are presented as mean ± standard error (a) or median with quartiles (violin plots, b and c). ns: not significant. *P<0.05, **P<0.01, ***P<0.0001, ****P<0.0001. [Figure 31A] We show that osimertinib (AZD9291)-induced adaptive immune activation is necessary for its therapeutic efficacy in vivo. We show the generation of GEMMs of EGFR mutant tumors driven by exon 19 deletion / T790M EGFR and loss of Trp53 (referred to as PE). Data are presented as mean ± SEM or median with quartiles (violin plot). ns: not significant. *P<0.05, ****P<0.0001. [Figure 31B] Figure 1 shows that osimertinib (AZD9291)-induced adaptive immune activation is required for its therapeutic efficacy in vivo. Figure 2 shows Western blots showing that EGFR and ERK phosphorylation in PE tumor cells is inhibited by AZD9291, but not erlotinib. Data are presented as mean ± SEM or median with quartiles (violin plot). ns: not significant. *P<0.05, ****P<0.0001. [Figure 31C] Figure 1 shows that osimertinib (AZD9291)-induced adaptive immune activation is necessary for its therapeutic efficacy in vivo. Figure 2 shows the in vivo efficacy of AZD9291 in PE tumor-bearing FVB mice (control: n=4, AZD9291 2.5 mg / mg daily: n=4, AZD9291 10 mg / mg daily: n=6). Data are presented as mean ± SEM or median with quartiles (violin plot). ns: not significant. *P<0.05, ****P<0.0001. [Figure 31D]Figure 1 shows that osimertinib (AZD9291)-induced adaptive immune activation is required for its therapeutic efficacy in vivo. Flow cytometry analysis of CD8+ T cell abundance in PE tumors and tumor-draining lymph nodes (TDLN) after the indicated treatments. Data are presented as mean ± SEM or median with quartiles (violin plot). ns: not significant. *P<0.05, ****P<0.0001. [Figure 32A] Representative plots of flow cytometry are shown in Figure 28B, which shows that an established immunosuppressive TME inhibits the therapeutic efficacy of osimertinib (AZD9291). [Figure 32B] Figure 28C shows that an established immunosuppressive TME inhibits the therapeutic efficacy of osimertinib (AZD9291). [Figure 33A] Figure 1 shows that tumor-associated macrophages (TAMs) suppress CD8+ T cell activation and impair the therapeutic efficacy of osimertinib (AZD9291) in large established tumors. Figure 2 shows that two cohorts of patients with NSCLC (GSK cohort and GSE31210 cohort) were classified into high TAM (above median) and low TAM (below median) groups based on TAM enrichment scores inferred by tumor transcriptome data. Figure 3 shows Kaplan-Meier survival plots showing overall survival (OS) in patients with EGFR mutant and EGFR wild-type NSCLC, respectively, after surgical treatment. Data are presented as mean ± standard error (a) or median with quartiles (violin plot, 33B). ns: not significant. ***P<0.0001. [Figure 33B]Figure 1 shows that tumor-associated macrophages (TAMs) suppress CD8+ T cell activation and impair the therapeutic efficacy of osimertinib (AZD9291) in large established tumors. Figure 2 shows that anti-CSF1-R antibody monotherapy failed to control tumor growth in FVB mice bearing PE tumors. Data are presented as mean ± SEM (a) or median with quartiles (violin plot, 33B). ns: not significant. ***P<0.0001. [Figure 33C] Figure 1 shows that tumor-associated macrophages (TAMs) suppress CD8+ T cell activation and impair the therapeutic efficacy of osimertinib (AZD9291) in large established tumors. Anti-CSF1-R antibody monotherapy significantly reduced TAM abundance but not TAM polarization. Each dot represents the result from a single tumor. Data are presented as mean ± standard error (a) or median with quartiles (violin plot, 33B). ns: not significant. ***P<0.0001. [Figure 34A] Figure 1 shows that systemic delivery of STING agonists in combination with osimertinib induces tumor regression in large established tumors. Figure 2 shows an analysis of cytokine production of CD8+ T cells co-cultured with TAMs. TAMs isolated from treated naive ETP tumors were treated with or without MSA-2 for 1 day. After washing out MSA-2, TAMs were co-cultured with CD8+ T cells isolated from naive FVB mice for 2 days. [Figure 34B] Figure 34 shows that systemic delivery of STING agonists in combination with osimertinib induces tumor regression in large established tumors. Flow cytometry analysis of CD8+ and CD4+ T cells in tumor-draining lymph nodes (TDLN) of FVB mice bearing PE tumors subjected to the indicated treatments. Each dot represents a result from a single tumor. Data are presented as median with quartiles (violin plot, Figure 34B and C). ns: not significant. **P<0.01. [Diagram 35]Figure 1 shows that STING agonists such as MSA-2 enhance the antitumor activity of olaparib in PBM tumor-bearing mice. Tumor burden in PBM tumor-bearing mice treated with the indicated agents was measured by bioluminescence (ROI, area of ​​intensity) 2 weeks after treatment (control: n=6, olaparib: n=7, olaparib+MSA-2: n=4). Ordinary one-way ANOVA. **P<0.01, ***P<0.001. The mouse ovarian tumor model PBM used in this experiment was generated previously (Ding L, Kim HJ, Wang Q, Kearns M, Jiang T, Ohlson CE, Li BB, Xie S, Liu JF, Stover EH, Howitt BE, Bronson RT, Lazo S, Roberts TM, Freeman GJ, Konstantinopoulos PA, Matulonis UA, Zhao JJ. PARP Inhibition Elicits STING-Dependent Antitumor Immunity in Brca1-Deficient Ovarian Cancer. Cell Rep. 2018 Dec 11;25(11):2972-2980.e5.doi:10.1016 / j.celrep.2018.11.054. PMID:30540933; PMCID:PMC6366450.). Approximately 2×105 PBM tumor cells were orthotopically implanted into syngeneic FVB / NJ mice. 10 days after implantation, tumor-bearing mice were divided equally into control and treatment groups according to luminescence intensity. Tumor burden in PBM tumor-bearing mice was measured by bioluminescence (ROI, area of ​​intensity). Olaparib (AZD2281) was administered daily by ip (intraperitoneal) injection at a dose of 50 mg / kg body weight. MSA-2 was prepared by diluting a 50 mg / ml stock in DMSO with PBS (pH 8.0) and administered every other day (3 times a week) by intraperitoneal injection at a dose of 25 mg / kg body weight. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention is based, at least in part, on the discovery that in addition to the inherent synthetic lethality of tumor cells, the immune response elicited by PARP inhibition is also required for an effective response in vivo. Through the provided examples, the primary mechanisms by which BRCA1-deficient breast tumors typically progress through treatment with PARP inhibitors are revealed, in contrast to the much higher success rates observed for BRCA-deficient ovarian cancers. Key findings leading to the underlying discoveries, which are further demonstrated in the Examples, include: (1) BRCA1 mutant mammary tumors are refractory to PARP inhibition in vivo in immune-competent mice; (2) BRCA1 mutant mammary tumor (both mouse and human) cells render tumor-associated macrophages (TAMs) tumor-promoting (M2-like) both in vivo and in vitro, independent of PARP inhibitor treatment; (3) M2-like TAMs directly suppress T cell activation; however, blockade of PD-1 does not add therapeutic benefit to PARP inhibition; (4) M2-like TAMs suppress DNA damage and cell death in tumor cells in response to PARP inhibition, leading to reduced production of cytoplasmic dsDNA and synthetic lethality, thereby suppressing STINs in dendritic cells (DCs) and macrophages. (5) STING agonists reprogram M2-like TAMs into M1-like antitumor macrophages in a STING-dependent manner in macrophages; (6) STING agonists enhance DC activation induced by olaparib upon BP cells in the presence of M2-like TAMs in vitro; (7) STING agonists restore antitumor immunity to the tumor immune microenvironment (TIME) in vivo, thus sensitizing BRCA1-deficient breast tumors to olaparib; (8) STING-deficient / BRCA1-deficient breast tumors do not respond to the combination of intratumorally administered olaparib and STING agonists, but respond effectively to systemic delivery of STING agonists in combination with PARP inhibitors, as do STING-competent tumors.

[0031] Thus, the data presented differ from most other studies suggesting that STING agonists act in tumor cells, but are consistent with pointing out the importance of STING activation in macrophages and DCs.

[0032] Targeting tumor-promoting M2-like TAMs is being actively explored in the field of cancer immunotherapy with various agents, such as small molecule inhibitors or monoclonal antibodies against CSF1 / CSF1R or TGFβ / TGFβR, to deplete or suppress TAMs. Many of these agents have been evaluated in early-stage clinical trials with little success. The examples provided show for the first time that M2-like TAMs suppress olaparib-induced lethal DNA damage and abolish STING activation, and that STING agonists can efficiently reprogram M2-like TAMs into M1-like antitumor macrophages in a STING-dependent manner in macrophages. Thus, STING agonists can not only restore the synthetic lethal response to PARP inhibition, but also reshape TIME to promote an immunogenic antitumor response that synergizes with PARP inhibition in BRCA-mutated breast cancer. In some embodiments, immunotherapy (e.g., blockers / inhibitors of immune checkpoints such as PD-1, PD-L1, PD-L2, CTLA-4, such as blocking antibodies known in the art) may be used in addition to the therapeutic agents and methods described herein.

[0033] In addition, the provided demonstration that activation of STING in immune cells is sufficient to induce antitumor immunity suggests novel therapeutic approaches to treat a significant proportion of patients with cancers that lack tumor cell-intrinsic STING. The translational implications of these findings are therefore highly significant, especially in light of two recent publications in Science reporting the antitumor activity of two non-nucleotidic STING agonists (PMIDs 32820094 and 32820126) that can be administered systemically, as they provide a strong and timely rationale for the combined systemic administration of PARP inhibitors and STING agonists in the treatment of BRCA-deficient breast cancer.

[0034] Thus, the present disclosure provides an important conceptual shift in understanding resistance to PARP inhibitors, which has been explained primarily in terms of restoration of homologous recombination.

[0035] Thus, the present invention provides methods for improving the efficacy of PARP inhibition in subjects with cancer, polarization of tumor-promoting macrophages into anti-tumor macrophages in subjects with cancer, and selection of subjects with cancer for treatment with STING agonists.

[0036] Syngeneic genetically engineered mouse (GEM) models of lung cancer driven by mutant EGFR show that EGFR mutant tumors are highly sensitive to osimertinib in a T cell-dependent manner at early stages of tumor growth, but become resistant as they progress. Thus, the present invention is also based in part on the determination that the presence of immunosuppressive tumor-associated macrophages (TAMs) renders tumors resistant to osimertinib. Depletion of TAMs in these tumors rescues the efficacy of osimertinib. Reprogramming of TAMs with the newly developed STING agonist MSA-2 reactivates antitumor immunity and, when combined with osimertinib, leads to sustained regression of resistant tumors in mice. The results presented herein suggest that an inhibitory tumor immune microenvironment can drive resistance of EGFR mutant tumors to osimertinib. Thus, a new strategy is provided herein to overcome resistance and improve treatment outcomes.

[0037] Accordingly, also provided herein is a method for improving the efficacy of tyrosine kinase inhibitor (TKI) inhibition in a subject having cancer, comprising administering to the subject an effective amount of a STING agonist in combination with an effective amount of a tyrosine kinase inhibitor (TKI).

[0038] In certain embodiments, the method of polarizing tumor-promoting macrophages into anti-tumor macrophages in a subject with cancer comprises administering to the subject an effective amount of a STING agonist.Also provided herein is a method of preventing or reversing drug resistance in a subject with cancer, where the drug resistance is the result of polarizing anti-tumor macrophages into tumor-promoting macrophages, comprising administering to the subject an effective amount of a STING agonist.Also provided herein is a method of improving the efficacy of a DNA synthesis inhibitor in a subject with cancer, where the efficacy of the DNA synthesis inhibitor is improved by administering to the subject an effective amount of a STING agonist in combination with an effective amount of a DNA synthesis inhibitor.

[0039] I. Definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0040] The term "administer" is intended to include modes and routes of administration that allow the agent to perform its intended function. Examples of routes of administration for the treatment of the body that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be a bolus injection or a continuous infusion. Depending on the route of administration, the agent can be coated with or placed in a selected material to protect the agent from natural conditions that may adversely affect its ability to perform its intended function. The agent can be administered alone or in combination with a pharma-ceutically acceptable carrier. The agent can also be administered as a prodrug that is converted to an active form in vivo.

[0041] Unless otherwise specified herein, the terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives contain at least an antigen-binding site. Antibody derivatives may include proteins or chemical moieties conjugated to an antibody.

[0042] In addition, intrabodies are well-known antigen-binding molecules that have the characteristics of antibodies but can be expressed intracellularly to bind and / or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods of adapting antibodies to target (e.g., inhibit) intracellular moieties are well known in the art, such as the use of single-chain antibodies (scFv), modification of immunoglobulin VL domains for hyperstability, modification of antibodies to resist the reducing intracellular environment, generation of fusion proteins to increase intracellular stability and / or modulate intracellular localization. Intracellular antibodies can also be introduced and expressed in one or more cells, tissues, or organs of a multicellular organism, e.g., for prophylactic and / or therapeutic purposes (e.g., as gene therapy) (see at least PCT Publication Nos. 08 / 020079, 94 / 02610, 95 / 22618, and 03 / 014960; U.S. Pat. No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag publs.); Kontermann (2004) Methods 34:163-170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412; Shaki-Loewenstein et al. (see, e.g., et al. (2005) J. Immunol. Meth. 303:19-39).

[0043] The term "antibody" as used herein also includes an "antigen-binding portion" (or simply an "antibody portion") of an antibody. The term "antigen-binding portion" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546), and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (known as single-chain Fvs (scFvs), see, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a particular scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL may also be used to generate Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA techniques. Other forms of single chain antibodies, such as diabodies, are also encompassed.Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing them to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0044] Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion polypeptide formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include the use of streptavidin core regions to generate tetrameric scFv polypeptides (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent biotinylated scFv polypeptides (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab fragments and F(ab')2 fragments, may be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion polypeptides may be obtained using standard recombinant DNA techniques as described herein.

[0045] Antibodies can be polyclonal or monoclonal, xenogeneic, allogeneic, or syngeneic, or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies can also be fully human. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one species of antigen-binding site capable of immunoreacting with a particular epitope of an antigen, and the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically exhibits a single binding affinity for the particular antigen with which it immunoreacts. In addition, antibodies can be "humanized," which includes antibodies made by non-human cells with variable and constant regions modified to more closely resemble antibodies that may be made by human cells. For example, the non-human antibody amino acid sequence is modified to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies encompassed by the invention may, for example, include amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "humanized antibody" also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0046] A "blocking" antibody is one that inhibits or reduces at least one biological activity of the antigen to which it binds. In certain embodiments, the blocking antibodies or fragments thereof described herein substantially or completely inhibit a given biological activity of the antigen. Alternatively, blocking antibodies are referred to herein with the prefix "anti-" in reference to their target (e.g., anti-PARP for antibodies that bind to PARP).

[0047] The term "cancer" or "tumor" or "hyperproliferative" refers to the presence of cells that have properties characteristic of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain distinctive morphological features.

[0048] Cancer cells are often in the form of a tumor, although such cells may exist alone in an animal or may be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-cancerous conditions and malignant cancers. Cancers include, but are not limited to, B cell cancers such as multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases such as alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colon cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematological tissue cancer, and the like. Other non-limiting examples of cancer types amenable to methods encompassed by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, Cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia), and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.In some embodiments, the cancer is of an epithelial nature, including, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers may be characterized in a variety of other ways, including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

[0049] As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are effective in the subject at the same time, which may include a synergistic effect of the two agents). For example, the different therapeutic agents may be administered either simultaneously or sequentially, either in the same formulation or in separate formulations. In certain embodiments, the different therapeutic agents may be administered within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 1 week of each other. Thus, subjects receiving such treatment may benefit from the combined effect of the different therapeutic agents.

[0050] As used herein, the term "DNA synthesis inhibitors" includes, but is not limited to, two types of therapeutic agents used to inhibit DNA synthesis. The first category includes purine and pyrimidine nucleoside analogs that directly inhibit DNA polymerase activity. The second category includes DNA damaging agents, including cisplatin and chlorambucil, that modify the composition and structure of nucleic acid substrates to indirectly inhibit DNA synthesis. Further details regarding DNA synthesis inhibitors can be found in Berdis AJ. Inhibiting DNA Polymerases as a Therapeutic Intervention against Cancer. Front Mol Biosci. 2017 Nov 21; 4: 78. (incorporated herein by reference in its entirety).

[0051] As used herein, epidermal growth factor receptor tyrosine kinase (EGFR-TKI) inhibitors include, but are not limited to, any tyrosine kinase inhibitor that inhibits the activity or reduces the expression level of EGFR peptide, or tyrosine kinase inhibitor that blocks the activity of EGFR peptide or receptor. EGFR is found on the surface of some normal cells and is involved in cell proliferation. It may also be found at high levels in some types of cancer cells. EGFR-TKIs may also be called EGFR inhibitors, epidermal growth factor receptor inhibitors, or epidermal growth factor receptor tyrosine kinase inhibitors. The TK inhibitor (e.g., an EGFR-TK inhibitor) used in any of the methods disclosed herein may be selected from afatinib, dacomitinib, osimertinib (AZD9291), rociletinib (CO-1686), olmutinib (HM61713), nazartinib (EGF816), nacotinib (ASP8273), mavereltinib (PF-0647775), almonertinib, TY-9591, gefitinib, erlotinib, and AC0010.

[0052] As used herein, EGFR activating mutation includes, but is not limited to, any activating mutation that confers sensitivity to EGFR TKI. These mutations include any mutation present in the tyrosine kinase (TK) domain of the EGFR gene. Such mutations include, for example, point mutations, deletion mutations, insertion mutations, missense mutations, or frameshift mutations. Additional exemplary mutations include exon 19 deletion mutations, single point substitution mutation L858R in exon 21, and point mutation T790M.

[0053] Homologous recombination repair (HRR) pathway deficiency (HRD) is involved in tumorigenesis and progression of cancers, including high-grade serous ovarian cancer (HGSOC), and sensitivity to platinum chemotherapy drugs. Homologous recombination (HR) comprises a series of interrelated pathways that function in repairing DNA double-strand breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides important support to DNA replication in the recovery of stalled or broken replication forks, contributing to DNA damage tolerance. As used herein, "HRD cancer" includes any cancer that shows impaired ability of tumor cells to repair DNA double-strand breaks (DSBs) via homologous recombination. Mutations in genes such as RAD51, PALB2, ATM, ATR, CHEK2, RAD51, and FANC can cause HRD cancer. Conversely, a cancer can be a homologous recombination DNA repair competent (HRP) cancer. HRP cancer is any cancer that retains the ability of cancer cells to successfully perform homologous recombination DNA repair (HRR).

[0054] As used herein, PARP inhibitors include, but are not limited to, any agent that inhibits the activity of PARP peptide or reduces its expression level.As disclosed herein, PARP inhibitors also include any agent that blocks PARP enzyme.The PARP family has many essential functions in cellular processes, including the regulation of transcription, apoptosis, and DNA damage response.PARP1 has poly(ADP-ribose) activity, and when activated by DNA damage, it adds branched PAR chains and promotes the recruitment of other repair proteins to promote the repair of DNA single-strand breaks.Exemplary PARP inhibitors include olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, CEP9722, E7016, AG014699, MK4827, BMN-673, iniparib, and 3-aminobenzamide.

[0055] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition, but is at risk of developing it or is susceptible to it.

[0056] As used herein, tyrosine kinase inhibitors (TKIs) include any agent that inhibits the expression or activity of a tyrosine kinase. Kinase inhibitors are either irreversible or reversible. Irreversible kinase inhibitors tend to covalently bind and block the ATP site, resulting in irreversible inhibition. Reversible kinase inhibitors can be further subdivided into four major subtypes based on the identification of the binding pocket as well as the DFG motif. TK inhibitors can be type I, type II, type III, type IV, or type V. Type I inhibitors competitively bind to the ATP binding site of active TK. The positioning of the DFG motif in type I inhibitors has the aspartic acid residue facing the catalytic site of the kinase. Type II inhibitors usually bind to inactive kinases at the ATP binding site. The DFG motif in type II inhibitors protrudes outward from the ATP binding site. Due to the outward rotation of the DFG motif, many type II inhibitors can also utilize regions adjacent to the ATP binding site that would otherwise be inaccessible. Type III inhibitors do not interact with the ATP binding pocket. Type III inhibitors bind exclusively to an allosteric pocket adjacent to the ATP-binding region. Type IV inhibitors bind to an allosteric site distant from the ATP-binding pocket. Type V inhibitors refer to a proposed subset of kinase inhibitors that exhibit multiple binding modes. The TK inhibitor may be a vascular endothelial growth factor receptor (VEGF) TK inhibitor, an epidermal growth factor (EGF) receptor TK inhibitor, a platelet-derived endothelial growth factor receptor (PDGF) TK inhibitor, or the TK inhibitor may be a fibroblast growth factor (FGF) receptor TK inhibitor.

[0057] As used herein, the term "inhibiting" and its grammatical equivalents refer to the reduction, restriction, and / or blocking of a particular action, function, or interaction. A reduction in the level of a given output or parameter can, but need not, mean the absolute absence of the output or parameter. The present invention does not require and is not limited to a method that completely eliminates the output or parameter. A given output or parameter can be determined using methods well known in the art, including, but not limited to, immunohistochemical assays, molecular biological assays, cell biological assays, clinical assays, and biochemical assays discussed herein and in the examples. The antonyms "promoting," "increasing," and their grammatical equivalents refer to an increase in the level of a given output or parameter, which is the opposite of what is described for inhibition or reduction.

[0058] The term "small molecule" is a term of the art and includes molecules less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively contain peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds that may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are non-peptidic small organic compounds. In a further embodiment, the small molecules are not biosynthetic.

[0059] As used herein, the term "nucleotide-based second messenger" refers to a second messenger having a relatively small number (e.g., one, two, or three) of nucleotides or derivatives thereof that transmit signals derived from changes in the environment or intracellular conditions to appropriate cellular responses. It can be cyclic or linear. In one embodiment, the nucleotide-based second messenger is a cyclic dinucleotide, including, but not limited to, a cyclic dipurine (e.g., cyclic di-AMP, cyclic di-GMP, cyclic AMP-GMP), a cyclic pyrimidine (e.g., cyclic di-UMP or cyclic UMP-CMP), or a cyclic purine-pyrimidine hybrid (e.g., cyclic UMP-AMP or cyclic UMP-GMP). In another embodiment, the nucleotide-based second messenger is a cyclic trinucleotide (e.g., cyclic AMP-AMP-GMP). Several bona fide nucleotide signaling pathways, (p)ppGpp, cAMP, cGMP, c-di-AMP, c-di-GMP, and cGAMP, have been characterized with respect to basic pathway modules and phenotypic and physiological outputs (Martin-Rodriguez et al. (2017) Curr Top Med Chem 17:1928-1944). In prokaryotes, cyclic di-GMP has emerged as a critical and ubiquitous second messenger that regulates bacterial lifestyle transitions associated with biofilm formation, virulence, and many other bacterial functions (Pesavento et al. (2009) Curr Opin Microbiol 12:170-176).

[0060] Nucleotide-based second messengers may contain modified nucleotides or non-natural nucleotides. Modified nucleotides may be naturally occurring modified RNA base analogs (Limbach et al. (1994) Nucleic Acids Res 22: 2183-2196; Cantara et al. (2011) Nucleic Acids Res 39: D195-D201; Czerwoniec et al. (2009) Nucleic Acids Res 37: D118-D121; Grosjean et al. (1998) Modification and Editing of RNA. ASM Press, Washington DC.). This includes N 6 -methyladenosine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 2'-O-methyladenosine-5'-triphosphate, 2'-O-methylcytidine-5'-triphosphate, 2'-O-methylguanosine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, inosine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 2'-O-methylpseudouridine-5'-triphosphate, N 1 -Methyladenosine-5'-triphosphate, 2'-O-methyl-5-methyluridine-5'-triphosphate, N 4 -Methylcytidine-5'-triphosphate, N 1 These include, but are not limited to, 5-methylpseudouridine-5'-triphosphate, 5,6-dihydro-5-methyluridine-5'-triphosphate, 5-formylcytidine-5'-triphosphate, 5-hydroxymethylcytidine-5'-triphosphate, 5-hydroxycytidine-5'-triphosphate, 5-hydroxyuridine-5'-triphosphate, 5-methoxyuridine-5'-triphosphate, and 5-carboxymethylester uridine-5'-triphosphate.

[0061] Non-natural nucleotides include 2'-fluoro and 2'-O-methyl NTPs, such as 2'-amino-2'-deoxyadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'-deoxyuridine-5'-triphosphate, 2'-azido-2'-deoxyadenosine-5'-triphosphate, 2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyguanosine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate, 2'-fluoro-2'-deoxyadenosine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine ... 5'-triphosphate, 2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyguanosine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-fluoro-2'-deoxyadenosine-5'-triphosphate, 2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyguanosine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'- Fluoro-2'-deoxyadenosine-5'-triphosphate, 2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyguanosine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate, 2'-O-methyladenosine-5'-triphosphate, 2'-O-methylcytidine-5'-triphosphate, 2'-O-methylguanosine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, pseudouridine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, 2'-amino-2'-deoxycytidine ... thymidine-5'-triphosphate, 2'-amino-2'-deoxyuridine-5'-triphosphate, 2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate, 2'-O-methylpseudouridine-5'-triphosphate, 2'-O-methyl-5-methyluridine-5'-triphosphate, 2'-azido-2'-deoxyadenosine-5'-triphosphate, 2'-amino-2'-deoxyadenosine-5'-triphosphate, 2'-fluoro-thymidine-5'-triphosphate, 2'-azido-2'-deoxyguanosine-5'-triphosphate,N, 4 -methylcytidine-5'-triphosphate, 2'-O-methyladenosine-5'-triphosphate, 2'-O-methylcytidine-5'-triphosphate, 2'-O-methylguanosine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 2'-amino-2'-deoxyadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'-deoxyuridine-5'-triphosphate, aradenosine-5'-triphosphate, araci cytidine-5'-triphosphate, araguanosine-5'-triphosphate, arauridine-5'-triphosphate, 2'-azido-2'-deoxyadenosine-5'-triphosphate, 2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyguanosine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate, 2'-fluoro-2'-deoxyadenosine-5'-triphosphate, 2'-fluoro-2'-deoxycytidine-5' -triphosphate, 2'-fluoro-2'-deoxyguanosine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyladenosine-5'-triphosphate, 2'-O-methylcytidine-5'-triphosphate, 2'-O-methylguanosine-5'-triphosphate, 2'-O-methyluridine-5'-triphosphate, 2'-fluoro-2'-deoxyadenosine-5'-triphosphate, 2'-fluoro These include, but are not limited to, 2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyguanosine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyladenosine-5'-triphosphate, 2'-O-methylcytidine-5'-triphosphate, 2'-O-methylguanosine-5'-triphosphate, and 2'-O-methyluridine-5'-triphosphate.

[0062] As used herein, the term "domain" refers to a functional portion, segment, or region of a protein or polypeptide. An "interaction domain" specifically refers to a portion, segment, or region of a protein, polypeptide, or protein fragment that is involved in the physical affinity of that protein, protein fragment, or isolated domain for another protein, protein fragment, or isolated domain.

[0063] Unless otherwise specified, the term "compound" as used herein includes, but is not limited to, peptides, nucleic acids, carbohydrates, natural product extract libraries, organic molecules (preferentially small organic molecules), inorganic molecules (e.g., including but not limited to chemicals, metals, and organometallic molecules).

[0064] The terms "derivative", "analog", or "variant" as used herein include, but are not limited to, molecules that contain a region that is substantially homologous to a CD-NTase polypeptide, and in various embodiments are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical across the same size amino acid sequence, or compared to aligned sequences where the alignment is performed by computer homology programs known in the art, or whose encoding nucleic acid can hybridize to sequences encoding the component protein under stringent, moderately stringent, or non-stringent conditions. This refers to proteins that are the result of modification of naturally occurring proteins by amino acid substitution, deletion, and addition, respectively, and where the derivative still exhibits (although not necessarily to the same extent) the biological function of the naturally occurring protein. The biological function of such proteins can be examined, for example, by suitable and available in vitro assays, as provided in the present invention.

[0065] The term "functionally active" as used herein refers to a polypeptide (i.e., a fragment or derivative) that, according to the embodiment to which the polypeptide (i.e., fragment or derivative) pertains, has a structural, regulatory, or biochemical function of a protein.

[0066] The term "activity", when used in connection with a protein or molecular complex, means any physiological or biochemical activity exhibited by or associated with a particular protein or molecular complex, including, but not limited to, activity exhibited in biological processes and cellular functions, the ability to interact or bind to another molecule or portion thereof, binding affinity or specificity for a particular molecule, in vitro or in vivo stability (e.g., rate of proteolysis, or, in the case of a molecular complex, the ability to maintain the form of the molecular complex), antigenicity and immunogenicity, enzymatic activity, etc. Such activities may be detected or assayed by any of a variety of suitable methods, as will be apparent to one of skill in the art.

[0067] As used herein, the term "interaction antagonist" refers to a compound that interferes with, blocks, disrupts, or destabilizes a protein-protein or protein-DNA interaction; blocks or interferes with the formation of a molecular complex, or destabilizes, disrupts, or dissociates an existing molecular complex.

[0068] The term "interaction agonist" as used herein means a compound that causes, initiates, propagates, nucleates, or otherwise enhances the formation of a protein-protein or protein-DNA interaction; causes, initiates, propagates, nucleates, or otherwise enhances the formation of a molecular complex; or stabilizes an existing molecular complex.

[0069] The term "STING" or "stimulator of interferon genes", also known as transmembrane protein 173 (TMEM173), refers to a five transmembrane protein that functions as a key regulator of the innate immune response to viral and bacterial infections. STING is a cytoplasmic receptor that senses both exogenous and endogenous cytoplasmic cyclic dinucleotides (CDNs) and activates the TBK1 / IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and proinflammatory cytokine responses. The term "STING" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human STING cDNA and human STING protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI). Human STING isoforms include the longer isoform 1 (NM_198282.3 and NP_938023.1) and the shorter isoform 2 (NM_001301738.1 and NP_001288667.1; which have a shorter 5'UTR and lack an exon in the 3' coding region, resulting in a shorter and different C-terminus compared to variant 1).Nucleic acid and polypeptide sequences of STING orthologues in organisms other than humans are known, e.g., chimpanzee STING (XM_016953921.1 and XP_016809410.1, XM_009449784.2 and XP_009448059.1, XM_001135484.3 and XP_001135484.1), monkey STING (XM_015141010.1 and XP_0149 96496.1), canine STING (XM_022408269.1 and XP_022263977.1, XM_005617260.3 and XP_005617317.1, XM_022408249.1 and XP_022263957.1, XM_005617262.3 and XP_005617319.1, XM_005617258.3 and XP_005617315.1, XM_0224082 53.1 and XP_022263961.1, XM_005617257.3 and XP_005617314.1, XM_022408240.1 and XP_022263948.1, XM_005617259.3 and XP_005617316.1, XM_022408259.1 and XP_022263967.1, XM_022408265.1 and XP_022263973.1), bovine STI These include NG (NM_001046357.2 and NP_001039822.1), mouse STING (NM_001289591.1 and NP_001276520.1, NM_001289592.1 and NP_001276521.1, NM_028261.1 and NP_082537.1), and rat STING (NM_001109122.1 and NP_001102592.1).

[0070] STING agonists have been shown to be useful therapies for treating cancer. Agonists of STING known in the art include, for example, MK-1454, STING agonist-1 (MedChem Express catalog number HY-19711), cyclic di-AMP (c-di-AMP), cyclic di-GMP (c-di-GMP), cGMP-AMP (2'3'cGAMP or 3'3'cGAMP), or cyclic dinucleotides (CDNs) such as 10-carboxymethyl-9-acridanone (CMA) (Ohkuri et al. (2015) Oncoimmunology 4(4):e999523), rationally designed synthetic CDN derivative molecules (Fu et al. (2015) Sci Transl Med. 7(283):283ra52.doi:10.1126 / scitranslmed.aaa4306), and 5,6-dimethyl-xanthenone-4-acetic acid (DMXAA) (Corrales et al. (2015) Sci Transl Med. 7(283):283ra52.doi:10.1126 / scitranslmed.aaa4306). al. (2015) Cell Rep. 11(7):1018-1030). These agonists bind and activate STING, resulting in a strong type I IFN response. Meanwhile, targeting the cGAS-STING pathway with small molecule inhibitors would be beneficial for the treatment of severe debilitating diseases, such as inflammatory and autoimmune diseases, associated with excessive type I IFN production due to aberrant DNA sensing and signaling. STING inhibitors are also known, such as CCCP (MedChem Express, Cat. No. HY-100941) and 2-bromopalmitate (Tao et al. (2016) IUBMB Life. 68(11):858-870). It should be noted that the term may further be used to refer to any combination of the features described herein for STING molecules. For example, any combination of sequence composition, % identity, sequence length, domain structure, functional activity, etc. may be used to describe STING molecules encompassed by the present invention.

[0071] The term "STING pathway" or "cGAS-STING pathway" refers to a STING-regulated innate immune pathway that mediates cytoplasmic DNA-induced signaling events. Cytoplasmic DNA binds and activates cGAS, which catalyzes the synthesis of 2'3'-cGAMP from ATP and GTP. 2'3'-cGAMP binds to the ER adaptor STING and transports it to the ER-Golgi intermediate compartment (ERGIC) and the Golgi apparatus. STING then activates IKK and TBK1. TBK1 phosphorylates STING, which then recruits IRF3 for phosphorylation by TBK1. Phosphorylated IRF3 dimerizes and then enters the nucleus, where it functions with NF-kB to turn on the expression of type I interferons and other immune regulatory molecules. The cGAS-STING pathway not only mediates protective immune defenses against infection by a wide variety of DNA-containing pathogens, but also detects tumor-derived DNA and generates intrinsic antitumor immunity. However, aberrant activation of the cGAS-STING pathway by self-DNA can also lead to autoimmune and inflammatory diseases.

[0072] The term "cGAS" or "cyclic GMP-AMP synthase", also known as Mab-21 domain-containing protein 1, refers to a nucleotidyltransferase that catalyzes the formation of cyclic GMP-AMP (cGAMP) from ATP and GTP (Sun et al. (2013) Science 339:786-791; Krazusch et al. (2013) Cell Rep 3:1362-1368; Civril et al. (2013) Nature 498:332-227; Ablasser et al. (2013) Nature 503:530-534; Kranzusch et al. (2014) Cell 158:1011-1021). cGAS involves both the formation of a 2,5 phosphodiester bond at the GpA step and the formation of a 3,5 phosphodiester bond at the ApG step to generate c[G(2,5)pA(3,5)p] (Tao et al.(2017)J Immunol198:3627-3636, Lee et al.(2017)FEBS Lett.591:954-961). cGAS functions as a major cytoplasmic DNA sensor, and the presence of double-stranded DNA (dsDNA) in the cytoplasm is a danger signal that triggers an immune response (Tao et al.(2017)J Immunol198:3627-3636). cGAS directly binds to DNA in the cytoplasm, causing the activation and synthesis of cGAMP, a second messenger that binds and activates TMEM173 / STING, thereby inducing the production of type I interferons (Tao et al. (2017) J Immunol 198:3627-3636, Wang et al. (2017) Immunity 46:393-404). cGAS has antiviral activity by sensing the presence of dsDNA from DNA viruses in the cytoplasm (Tao et al. (2017) J Immunol 198:3627-3636). cGAS also functions as an innate immune sensor of infection by retroviruses such as HIV-1 by detecting the presence of reverse transcribed DNA in the cytoplasm (Gao et al. (2013) Science 341:903-906).Detection of retroviral reverse transcribed DNA in the cytoplasm may be indirect and mediated through interaction with PQBP1, which directly binds reverse transcribed retroviral DNA (Yoh et al. (2015) Cell 161:1293-1305). cGAS also detects the presence of DNA from bacteria such as M. tuberculosis (Wassermann et al. (2015) Cell Host Microbe 17:799-810). cGAMP can be transferred from the producing cell to neighboring cells via gap junctions, promoting TMEM173 / STING activation and transmitting the immune response to the connecting cells (Ablasser et al. (2013) Nature 503:530-534). cGAMP can also be transferred between cells by packaging within viral particles that contribute to IFN induction in newly infected cells in a cGAS-independent but TMEM173 / STING-dependent manner (Gentili et al. (2015) Science 349:1232-1236). In addition to antiviral activity, cGAS is also involved in the response to cellular stresses such as senescence, DNA damage, or genomic instability (Mackenzie et al. (2017) Nature 548:461-465, Harding et al. (2017) Nature 548:466-470). cGAS functions as a regulator of cellular senescence by binding to cytoplasmic chromatin fragments present in senescent cells, leading to the production of type I interferons via TMEM173 / STING, promoting cellular senescence. cGAS is also involved in the inflammatory response to genomic instability and double-stranded DNA breaks. cGAS acts by localizing to micronuclei that arise from genomic instability (PubMed:28738408; Harding et al. (2017) Nature 548:466-470). Micronuclei are frequently found in cancer cells and consist of chromatin surrounded by its own nuclear membrane.Following the degradation of the micronuclear envelope, a process associated with chromosolysis, MB21D1 / cGAS binds to self-DNA exposed in the cytoplasm, leading to cGAMP synthesis and subsequent activation of TMEM173 / STING and production of type I interferons (Mackenzie et al. (2017) Nature 548:461-465, Harding et al. (2017) Nature 548:466-470). In one embodiment, human cGAS has 522 amino acids with a molecular weight of 58814 Da. cGAS is monomeric in the absence of DNA and when bound to dsDNA (Tao et al. (2017) J Immunol 198:3627-3636). cGAS interacts with PQBP1 (via the WW domain) (Yoh et al. (2015) Cell 161:1293-1305). cGAS also interacts with TRIM14, which stabilizes cGAS / MB21D1 and promotes the production of type I interferons (Chen et al. (2016) Mol Cell 64:105-119). cGAS also interacts with herpesvirus 8 / HHV-8 protein ORF52, which inhibits the enzymatic activity of cGAS.

[0073] The term "cGAS" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human cGAS cDNA and human cGAS protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI). Human cGAS isoforms include the protein (NP_612450.2) encoded by the transcript (NM_138441.2). Nucleic acid and polypeptide sequences of cGAS orthologues in organisms other than humans are known, e.g., chimpanzee cGAS (XM_009451553.3 and XP_009449828.1; and XM_009451552.3 and XP_009449827.1), monkey cGAS (NM_001318175.1 and NP_001305104.1), bovine cGAS (XM_024996918.1 and XP_02485 2686.1, XM_005210662.4 and XP_005210719.2, and XM_002690020.6 and XP_002690066.3), mouse cGAS (NM_173386.5 and NP_775562.2), rat cGAS (XM_006243439.3 and XP_006243501.2), and chicken cGAS (XM_419881.6 and XP_419881.4).

[0074] Suitable anti-cGAS antibodies for detecting cGAS protein are well known in the art, including, for example, antibody TA340293 (Origene), antibodies NBP1-86761 and NBP1-70755 (Novus Biologicals, Littleton, CO), antibodies ab224144 and ab176177 (AbCam, Cambridge, MA), antibody 26-664 (ProSci), etc. In addition, reagents for detecting cGAS are well known. Multiple clinical trials for cGAS are available at the NIH Genetic Testing Registry (GTR®) (e.g., GTR Test ID: GTR000540854.2, Fulgent Clinical Diagnostics Lab, Temple City, CA). Additionally, multiple siRNA, shRNA, CRISPR constructs for reducing expression of cGAS can be found in the listings of commercial products from the companies referenced above, such as, for example, siRNA product number sc-95512 from Santa Cruz Biotechnology, RNAi product SR314484 and TL305813V, and CRISPR product KN212386 from Origene, and multiple CRISPR products from GenScript (Piscataway, NJ). Note that the term may further be used to refer to any combination of the characteristics described herein with respect to a cGAS molecule. For example, any combination of sequence composition, % identity, sequence length, domain structure, functional activity, etc. may be used to describe a cGAS molecule encompassed by the present invention.

[0075] An "antagonist" is one that attenuates, reduces, or inhibits at least one biological activity of at least one protein, such as a receptor. In certain embodiments, an antagonist substantially or completely attenuates or inhibits a given biological activity of at least one protein described herein.

[0076] The term "mode of administration" includes any approach that brings a desired target (e.g., a cell, a subject) into contact with a desired agent (e.g., a therapeutic agent). A route of administration, as used herein, is a specific form of a mode of administration, which specifically encompasses the route by which an agent is administered to a subject or the route by which a biophysical agent contacts a biological material.

[0077] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from cancer. The term "subject" is synonymous with "patient."

[0078] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or the enhancement of desirable physical or mental development and conditions in animals or humans.

[0079] As used herein, the terms "therapeutically effective amount" and "effective amount" refer to an amount of a compound, material, or composition comprising a compound encompassed by the present invention effective to produce some desired therapeutic effect on at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. Toxicity and therapeutic efficacy of a subject compound can be determined, for example, by the LD 50 and ED 50 The LD can be determined by standard pharmaceutical procedures in cell culture or experimental animals to determine the therapeutic index. Compositions that exhibit high therapeutic indices are preferred. In some embodiments, the LD 50 (lethal dose) can be measured and can be reduced, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, for a drug compared to administration of a suitable control drug. 50The IC (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured and can be increased, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, for an agent compared to administration of a suitable control agent. Similarly, the IC 50 (i.e., the concentration that achieves half-maximal cytotoxicity or cytostatic effect on cancer cells) can be measured and can be increased, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, for the agent compared to administration of a suitable control agent.

[0080] II. Subject In some embodiments, the subject is a mammal (e.g., a mouse, a rat, a primate, a non-human mammal, a domestic animal (dog, cat, cow, horse, etc.)), preferably a human. In other embodiments, the subject is an animal model of cancer. In addition, cells (e.g., cells from such a subject), whether in vitro, ex vivo, or in vivo, can be used in accordance with the methods described herein.

[0081] In some embodiments encompassed by the methods of the invention, the subject has not received a treatment, such as a PARP inhibitor. In other embodiments, the subject has received a treatment, such as a PARP inhibitor. In some embodiments encompassed by the methods of the invention, the subject has not received a treatment, such as a TK inhibitor. In some embodiments, the subject has received a treatment, such as a TK inhibitor. In some embodiments encompassed by the methods of the invention, the subject has not received a treatment, such as a DNA synthesis inhibitor. In some embodiments, the subject has received a treatment, such as a DNA synthesis inhibitor. In some embodiments, the subject has a tumor with an M2 enrichment score of greater than 0.27, or any other enrichment score disclosed herein.

[0082] In some embodiments, this mentioned M2 enrichment score, which is also listed in some of the claims, is from TCGA data analysis of cancer types with enrichment scores of M2-like TAMs, which is consistent with the M2 signature data from Pan et al., 2017, Immunity 47, 284-297. The yellow dotted line in the first figure of this article is the average enrichment score (0.27) of the M2 gene signature, and the abbreviations used in this figure are as follows: ACC: adrenocortical carcinoma, BLCA: urothelial carcinoma of the bladder, BRCA: invasive carcinoma of the breast, CESC: squamous cell carcinoma of the cervix and adenocarcinoma of the cervix, COAD: colon adenocarcinoma, DLBC: lymphoid neoplasms diffuse large B-cell lymphoma, GBM: glioblastoma multiforme, HNSC: squamous cell carcinoma of the head and neck, KICH: chromophobe renal carcinoma, KIRC: clear cell carcinoma of the kidney, KIRP: papillary cell carcinoma of the kidney, LAML: acute myeloid leukemia, LGG: brain low-grade glioma, LIHC: hepatocellular carcinoma, LUAD: lung adenocarcinoma, LUSC: lung squamous cell carcinoma, OV: ovarian serous cystadenocarcinoma, PRAD: prostate adenocarcinoma, READ: rectal adenocarcinoma, SKCM: cutaneous melanoma, STAD: gastric adenocarcinoma, THCA: thyroid carcinoma, UCEC: uterine endometrial carcinoma, and UCS: uterine carcinosarcoma.

[0083] Thus, in some embodiments, the subject has lung cancer, such as head and neck squamous cell carcinoma (HNSC), non-small cell lung cancer (NSCLC) or lung squamous cell carcinoma (LUSC), liver cancer, such as hepatocellular carcinoma (HCC), colon cancer, prostate cancer, pancreatic cancer, cutaneous melanoma (SKCM), glioblastoma multiforme (GBM), breast invasive carcinoma (BRCA), lung adenocarcinoma (LUAD), renal clear cell carcinoma (KIRC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), diffuse large B-cell lymphoma (DLBC), gastric adenocarcinoma (STAD), or ovarian cancer, such as high-grade serous ovarian cancer (HGSOC), ovarian cancer, such as high-grade serous ovarian cancer (HGSOC) or homologous recombination (HRP) ovarian cancer, or any homologous recombination (HRP) cancer. The cancer can be any homologous recombination deficient (HRD) cancer (e.g., HRD ovarian cancer). The cancer may be an HRD cancer or tumor that contains a mutation in the RAD51, PALB2, ATM, ATR, CHEK2, RAD51, or FANC gene. The cancer may contain a genetic mutation that upregulates STAT3 signaling and / or converts tumor-associated macrophages into M2-like macrophages (e.g., KRAS G12D The cancer may be any cancer that contains a genetic mutation that polarizes it to a type 2 cancer (such as a cancer with a mutation in the KRAS gene).

[0084] In certain embodiments, the subject has breast cancer that harbors a BRCA mutation (e.g., advanced breast cancer that harbors a germline BRCA1 / 2 mutation). In certain embodiments, the subject has BRCA-naive breast cancer or ovarian cancer.

[0085] In certain embodiments, the subject has a cancer (e.g., lung cancer) that harbors an EGFR mutation. The EGFR mutation can be an activating mutation or any mutation that confers resistance to a TKI.

[0086] In some embodiments, the subject has a cancer that includes a subpopulation of tumors with an M2 enrichment score greater than 0.27 (e.g., even if the cancer itself is one with an M2 enrichment score less than 0.27). In some embodiments, the subject has a cancer that includes a subpopulation of tumors with an M2 enrichment score greater than 0.15 (e.g., 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0. 0.95, 0.96, 0.97, 0.98, 0.99 or higher) (e.g., even if the cancer itself is one of those with an M2 enrichment score of less than 0.15). In some embodiments, the subject has a cancer comprising a tumor that has achieved an M2 enrichment score of greater than 0.27 over the course of treatment (e.g., even if the cancer itself is one that has an M2 enrichment score of less than 0.27).In some embodiments, the subject has an M2 enrichment score greater than 0.15 over the course of treatment (e.g., 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.95, 0.96, 0.97, 0.98, 0.99 or higher) (e.g., even if the cancer itself is one of those having an M2 enrichment score of less than 0.15). In some embodiments, the subject has a defect in activation of STING signaling in intratumoral dendritic cells.

[0087] III. Therapeutic Drugs In some embodiments, the agents used are therapeutic agents that are STING agonists. These agents can, for example, activate STING signaling in macrophages (e.g., those outside the tumor).

[0088] In some embodiments, the STING agonist comprises a modified nucleotide STING agonist. In some embodiments, the STING agonist is selected from DMXAA, MSA-2, SR-717, FAA, CMA, α-mangostin, BNBC, DSDP, diABZI, bicyclic benzamide, and benzothiophene. In some embodiments, any suitable STING agonist (e.g., a STING agonist that is sufficiently suitable for systemic administration, such as a STING agonist that is not a natural ligand of STING) can be used. In general, various further details regarding STING agonists can be found in the literature, for example, Chin et al., Antitumor activity of a systemic STING-activating non-nucleotide cGAMP mimetic, Science 369: 993 (2020), and Pan et al., An orally available non-nucleotide STING agonist with antitumor activity, Science 369: 935 (2020). Data relating to DMXAA can be found in Example 1, and data relating to MSA-2 can be found in Figures 14A-14D.

[0089] In certain embodiments, the STING agonist can be co-administered with another therapeutic agent (e.g., separately or together, at different times or simultaneously). Specifically, the STING agonist can be co-administered with a PARP inhibitor. In some embodiments, the PARP inhibitor is selected from olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, CEP9722, E7016, AG014699, MK4827, BMN-673, iniparib, and 3-aminobenzamide. In some embodiments, co-administering includes administering the STING agonist before the PARP inhibitor. In some embodiments, co-administering includes administering the STING agonist simultaneously with the PARP inhibitor.

[0090] In certain embodiments, the STING agonist can be administered in combination with another therapeutic agent (e.g., separately or together, at different times or simultaneously). Specifically, the STING agonist can be administered in combination with a TKI (e.g., an EGFR-TKI or any other TKI disclosed herein). In some embodiments, the TKI (e.g., an EGFR-TKI or any other TKI disclosed herein) is selected from afatinib, dacomitinib, osimertinib (AZD9291), rociletinib (CO-1686), olmutinib (HM61713), nazartinib (EGF816), nacotinib (ASP8273), mavereltinib (PF-0647775), almonertinib, TY-9591, gefitinib, erlotinib, and AC0010. In some embodiments, the combination administration comprises administering a STING agonist prior to the TKI (e.g., an EGFR-TKI or any other TKI disclosed herein). In some embodiments, the combination administration comprises administering a STING agonist simultaneously with the TKI (e.g., an EGFR-TKI or any other TKI disclosed herein).

[0091] In certain embodiments, the STING agonist can be co-administered with another therapeutic agent (e.g., separately or together, at different times or simultaneously). In particular, the STING agonist can be co-administered with a TK inhibitor. In some embodiments, the co-administering comprises administering the STING agonist before the TK inhibitor. In some embodiments, the co-administering comprises administering the STING agonist simultaneously with the TK inhibitor. The TK inhibitor can be a vascular endothelial growth factor receptor (VEGF) TK inhibitor, an epidermal growth factor (EGF) receptor TK inhibitor, a platelet-derived endothelial growth factor receptor (PDGF) TK inhibitor, or the TK inhibitor can be a fibroblast growth factor (FGF) receptor TK inhibitor. Examples of TK inhibitors include axitinib, dasatinib, erlotinib, imatinib, nilotinib, pazopanib, sorafenib, bosutinib, avapritinib, capmatinib, pemigatinib, ripretinib, selpercatinib, selumetinib, tucatinib, entrectinib, erdafitinib, fedratinib, pexidartinib, tenosynovium, upadacitinib, zanubrutinib, baricitinib, binimetinib, dacomitinib, fostamatinib, gilteritinib, and lapatinib. It may be rotrectinib, lorlatinib, acalabrutinib, brigutinib, midostaurin, neratinib, alectinib, cobimetinib, lenvatinib, osimertinib, ceritinib, nintedanib, afatinib, ibrutinib, trametinib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, tofacitinib, crizotinib, ruxolitinib, vandetanib, pazopanib, lapatinib, gefitinib, or sunitinib.

[0092] In certain embodiments, the STING agonist can be co-administered with another therapeutic agent (e.g., separately or together, at different times or simultaneously). In particular, the STING agonist can be co-administered with a DNA synthesis inhibitor. In some embodiments, co-administering includes administering the STING agonist before the DNA synthesis inhibitor. In some embodiments, co-administering includes administering the STING agonist simultaneously with the DNA synthesis inhibitor. Exemplary DNA synthesis inhibitors include, but are not limited to, gemcitabine, sapacitabine, cytidine analogs, cytarabine, tezacitabine, troxacitabine, DMDC, CNDAC, ECyD, clofarabine, or nucleoside analogs such as decitabine.

[0093] Additional therapeutic agents for combination therapy include radiation therapy, chemotherapy (e.g., paclitaxel, platinum-based drugs (e.g., cisplatin, oxaliplatin), inhibitors of topoisomerase activity such as topoisomerase II (e.g., etoposide), DNA intercalators (e.g., doxorubicin), and / or DNA alkylating agents (e.g., temozolomide)), or DNA damage response (DDR) targeting agents (e.g., ATMi, ATRi, CHK1 / 2i, or Wee1i). Temozolomide is the only FDA-approved first-line therapy for glioblastoma (GMB), an M2-high tumor, but it only provides progression-free survival (PFS) without overall survival (OS) (see, e.g., Fernandes et al. (2017) Current Standards of Care in Glioblastoma Therapy, Chapter 11 of Glioblastoma, De Vleeschouwer S., Ed., Codon Publications (Brisbane, Australia) (2017)).

[0094] IV. Treatment method One aspect encompassed by the present invention relates to methods of treating cancer, for example by improving the efficacy of PARP inhibition in a subject having cancer, which in some embodiments comprises administering to the subject an effective amount of a STING agonist in combination with an effective amount of a PARP inhibitor.

[0095] One aspect encompassed by the present invention relates to methods of treating cancer, for example by improving the efficacy of TK (e.g., EGFR-TKI) inhibition in a subject having cancer, which in some embodiments comprises administering to the subject an effective amount of a STING agonist in combination with an effective amount of a TK (e.g., EGFR-TKI) inhibitor.

[0096] Another aspect encompassed by the present invention relates to methods of treating cancer, for example by improving the effectiveness of DNA synthesis inhibition in a subject having cancer, which in some embodiments comprises administering to the subject an effective amount of a STING agonist in combination with an effective amount of a DNA synthesis inhibitor.

[0097] In some embodiments, administration includes systemic delivery of the STING agonist (e.g., orally, intravenously, or intraperitoneally).

[0098] Aspects encompassed by the present invention relate to a method for polarizing tumor-promoting macrophages into anti-tumor macrophages in a subject with cancer, comprising administering to the subject an effective amount of a STING agonist. In some such aspects, the STING agonist activates STING signaling in macrophages. In some such aspects, the STING agonist does not activate STING signaling in intratumoral dendritic cells. In some embodiments of these aspects, the tumor-promoting macrophages are M2-like. In some embodiments of these aspects, the anti-tumor macrophages are M1-like.

[0099] In some embodiments, the PARP inhibitor is administered at a suitable dosage (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg body weight per day, or any other value or range between these values). A suitable dosage may be administered twice a day, once a day, twice a week, once a week, three times a month, twice a month, or once a month. In some embodiments, the PARP inhibitor is administered multiple times (e.g., at least 2-3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 10 times).

[0100] In some embodiments, the TK inhibitor is administered at a suitable dosage (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / kg body weight per day, or any other value or range between these values). Suitable dosages may be administered twice a day, once a day, twice a week, once a week, three times a month, twice a month, or once a month. In some embodiments, the TK inhibitor is administered multiple times (e.g., at least 2-3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 10 times).

[0101] In some embodiments, the DNA synthesis inhibitor is administered at a suitable dosage (e.g., at least 100 mg / m2 / week, 250 mg / m2 / week, 500 mg / m2 / week, 750 mg / m2 / week, 1,000 mg / m2 / week, 1,500 mg / m2 / week, 1,750 mg / m2 / week, 2,0000 mg / m2 / week, 2,200 mg / m2 / week, or any other value or range between these values). Suitable dosages may be administered twice a day, once a day, twice a week, once a week, three times a month, twice a month, or once a month. In some embodiments, the DNA synthesis inhibitor is administered multiple times (e.g., at least 2-3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 10 times).

[0102] In some embodiments, the STING agonist is administered at a suitable dosage (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mg / kg body weight / week, or any other value or range between these values). A suitable dosage may be administered twice daily, once daily, twice weekly, once weekly, three times monthly, twice monthly, or once monthly. In some embodiments, the STING agonist is administered multiple times (e.g., at least 2-3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 10 times).

[0103] As disclosed herein, STING agonists can be administered in combination with PARP inhibitors.Also, as disclosed herein, STING agonists can be administered in combination with TK inhibitors.

[0104] V. Clinical Efficacy Clinical efficacy can be measured by any method known in the art.For example, the benefit from STING agonist therapy alone or in combination with another agent (e.g., PARP inhibitor, TK inhibitor, or DNA synthesis inhibitor) is related to progression-free survival.As another example, the benefit from STING agonist can be related to tumor volume, which can be measured by suitable method.

[0105] The benefit from using the agents encompassed by the present invention can be determined by measuring the level of cytotoxicity in biological material. The benefit from using the agents encompassed by the present invention can be evaluated by measuring transcriptional profiles, viability curves, microscopic images, biosynthetic activity levels, redox levels, etc. The benefit from using the agents encompassed by the present invention can also be determined by measuring the amount of side effects from STING agonist treatment.

[0106] In some embodiments, the clinical effectiveness of the therapeutic treatments described herein can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the percentage of patients in complete remission (CR), the number of patients in partial remission (PR), and the total number of patients with stable disease (SD) at least 6 months after the end of therapy. A shorthand for this formula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR of a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.

[0107] Further criteria for assessing response to therapy relate to "survival time", including all of the following: survival time to death, also known as overall survival (death can be either from any cause or tumor-related), "recurrence-free survival" (the term recurrence is intended to include both local and distant recurrence). The length of survival time can be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). In addition, criteria for treatment efficacy can be expanded to include response to therapy, survival probability, and recurrence probability.

[0108] For example, to determine the appropriate threshold, a particular STING agonist treatment regimen may be administered to a subject population, and the outcome may be correlated with a biomarker measurement determined before administration of any therapy. The outcome measurement may be a pathological response to the therapy. Alternatively, for subjects after therapy for which biomarker measurements are known, outcome measurements such as overall survival and disease-free survival may be monitored over a period of time. In certain embodiments, the same dose of therapeutic agent, if any, is administered to each subject. In related embodiments, the dose administered is a standard dose known in the art for the agent used in the therapy. The period of time that a subject is monitored may vary. For example, a subject may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.

[0109] VI. Medication Administration To enhance their effects, agents (e.g., STING agonists) encompassed by the present invention are administered to a subject in a biologically compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the protein administered in which the therapeutic effects of the protein outweigh any toxic effects. The term "subject" is intended to include living organisms in which an immune response can be elicited, such as mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Administration of agents described herein can be in any pharmacological form containing a therapeutically active amount of the agent, alone or in combination with a pharma- ceutically acceptable carrier.

[0110] The administration of a therapeutically active amount of the therapeutic composition encompassed by the present invention is defined as an amount effective to achieve the desired result at the required dosage and for the required period of time.For example, the therapeutically active amount of a drug may vary depending on factors such as the individual's medical condition, age, sex, and weight, and the ability of the peptide to induce the desired response in the individual.The administration regimen may be adjusted to provide the optimal therapeutic response.For example, several divided doses may be administered once a day, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0111] The agents encompassed by the present invention can be administered alone or in combination with additional therapies. In combination therapy, the STING agonist encompassed by the present invention and another agent (e.g., a PARP inhibitor, a TK inhibitor, or a DNA synthesis inhibitor) can be delivered to the same or different cells and can be delivered at the same or different times. The agents encompassed by the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions can include one or more agents, or one or more molecules that result in the production of such one or more agents, and a pharma- ceutically acceptable carrier.

[0112] The therapeutic agents described herein may be administered using a mode or route of administration that delivers them to a specific location in the body, or systemically, hi some embodiments, the mode of administration is systemic, such as oral, intravenous, or intraperitoneal.

[0113] The therapeutic agents described herein can be administered in a convenient manner, such as injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound can be coated with a material to protect the compound from the action of enzymes, acids, and other natural conditions that may inactivate the compound. For example, for administration of a drug by a method other than parenteral administration, it may be desirable to coat the drug with a material to prevent its inactivation, or to co-administer the drug with the material.

[0114] The agent is administered to an individual in a suitable carrier, diluent, or adjuvant, co-administered with an enzyme inhibitor, or in a suitable carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinol, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEEP), and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

[0115] As described in detail below, pharmaceutical compositions encompassed by the present invention may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, e.g., by subcutaneous, intramuscular, or intravenous injection as a sterile solution or suspension; (3) topical administration, e.g., as a cream, ointment, or spray applied to the skin; (4) intravaginally or rectally, e.g., as a pessary, cream, or foam; or (5) aerosols, e.g., aqueous aerosols, liposomal preparations, or solid particles containing the compound.

[0116] As used herein, the phrase "pharmacologically acceptable" is used to refer to those agents, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0117] As used herein, the phrase "pharmacologically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a chemical of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Examples of materials which can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; (10) glycerides, such as glycerides, sorbitol ... (10) glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffers, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0118] The term "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of agents that modulate (e.g., inhibit) the expression and / or activity of a biomarker or the expression and / or activity of a complex encompassed by the present invention. These salts can be prepared in situ during the final isolation and purification of the therapeutic agent, or by separately reacting the purified therapeutic agent in its free base form with a suitable organic or inorganic acid and isolating the salt thus produced. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0119] In other cases, agents useful in the methods encompassed by the present invention may contain one or more acidic functional groups and thus can form pharma- ceutically acceptable salts with pharma-ceutically acceptable bases. The term "pharma-ceutically acceptable salts" in these cases refers to the relatively non-toxic inorganic and organic base addition salts of agents that modulate (e.g., inhibit) the expression and / or activity of a biomarker, or the expression and / or activity of a complex. These salts can also be prepared in situ during the final isolation and purification of the therapeutic agent, or by separately reacting the purified therapeutic agent in its free acid form with a suitable base, e.g., a hydroxide, carbonate or bicarbonate of a pharma-ceutically acceptable metal cation, ammonia, or a pharma-ceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).

[0120] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0121] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0122] Formulations useful in the methods encompassed by the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral administration. The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending on the host treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0123] Methods of preparing these formulations or compositions include the step of bringing into association an agent that modulates (e.g., inhibits) expression and / or activity of a biomarker with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the therapeutic agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0124] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powder, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, and the like, each containing a predetermined amount of the therapeutic agent as the active ingredient. The compounds may also be administered as a bolus, electuary, or paste.

[0125] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as refrigerants, glycerol, or glycerol; Mix with starch, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (5) dissolution retarders, such as paraffin, (6) absorption enhancers, such as quaternary ammonium compounds, (7) wetting agents, such as acetyl alcohol and glycerol monostearate, (8) adsorbents, such as kaolin and bentonite clay, (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof, and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain buffering agents. Solid compositions of a similar type may also be used as fillers for soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, and the like.

[0126] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents or dispersing agents. Molded tablets can be made by molding a mixture of powdered peptide or peptidomimetic moistened with an inert liquid diluent in a suitable machine.

[0127] Tablets and other solid dosage forms, such as dragees, capsules, pills and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings known in the art of pharmaceutical formulation. They can also be formulated to provide slow or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain an opacifying agent, and can be of a composition that releases the active ingredient only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, where appropriate, with one or more of the above-mentioned excipients.

[0128] Liquid dosage forms for oral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0129] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0130] Suspensions may contain, in addition to the active agent, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0131] Formulations for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing one or more therapeutic agents with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active agents.

[0132] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0133] Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., inhibits) biomarker expression and / or activity include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and any preservatives, buffers, or propellants which may be required.

[0134] The ointments, pastes, creams and gels may contain, in addition to the therapeutic agent, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0135] Powders and sprays can contain, in addition to the agent that modulates (e.g., inhibits) the expression and / or activity of a biomarker, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0136] Alternatively, the agents disclosed herein can be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension can be used. Ultrasonic nebulizers are preferred because they minimize the exposure of the agent to shear, which can cause the compound to degrade.

[0137] Usually, aqueous aerosols are made by formulating an aqueous solution or suspension of a drug with conventional pharma- ceutically acceptable carriers and stabilizers. Carriers and stabilizers vary according to the requirements of a particular compound, but typically include non-ionic surfactants (Tween®, Pluronic, or polyethylene glycol), innocuous proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0138] Transdermal patches have the added advantage of providing controlled delivery of a therapeutic agent to the body. Such dosage forms can be made by dissolving or dispensing the therapeutic agent in a suitable medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic through the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.

[0139] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the present invention.

[0140] Pharmaceutical compositions encompassed by the invention suitable for parenteral administration comprise one or more therapeutic agents in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders capable of being reconstituted into sterile injectable solutions or dispersions immediately prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0141] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions encompassed by the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, etc. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0142] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of microbial activity can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents in the composition, such as sugars, sodium chloride, etc. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0143] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of the drug. This can be achieved by using a liquid suspension of crystalline or amorphous substances that are poorly water-soluble. In this case, the absorption rate of the drug depends on the dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0144] Injectable depot forms are made by forming microencapsule matrices of the agent that modulates (e.g., inhibits) the expression and / or activity of the biomarker in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0145] When the therapeutic agents encompassed by the present invention are administered to humans and animals as pharmaceuticals, the therapeutic agents may be given as is or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0146] Actual dosage levels of active ingredients in pharmaceutical compositions encompassed by the present invention can be determined by methods encompassed by the present invention to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject.

[0147] The nucleic acid molecule encompassed by the present invention can be inserted into a vector and used as a gene therapy vector. The gene therapy vector can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470), or stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, when the complete gene delivery vector can be produced intact from recombinant cells, for example, in the case of retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0148] In one embodiment, the agent encompassed by the present invention is an antibody. As defined herein, a therapeutically effective amount (i.e., an effective dosage) of an antibody ranges from about 0.001 to 30 mg / kg body weight, preferably about 0.01 to 25 mg / kg body weight, more preferably about 0.1 to 20 mg / kg body weight, even more preferably about 1 to 10 mg / kg, 2 to 9 mg / kg, 3 to 8 mg / kg, 4 to 7 mg / kg, or 5 to 6 mg / kg body weight. One of ordinary skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, may affect the dosage required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of an antibody may include a single treatment or, preferably, may include a series of treatments. In a preferred example, subjects are treated with antibody in the range of about 0.1-20 mg / kg body weight once a week for about 1-10 weeks, preferably 2-8 weeks, more preferably about 3-7 weeks, and even more preferably about 4, 5, or 6 weeks. It will also be understood that the effective dosage of the antibody used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result in diagnostic assay results. EXAMPLES

[0149] Example 1: Overcoming immunosuppression and therapeutic resistance to PARP inhibition in BRCA1-deficient breast cancer by STING agonists PARP inhibitors (PARPi) have dramatically changed the therapeutic landscape for advanced ovarian tumors harboring BRCA mutations. However, the impact of this class of inhibitors in patients with advanced BRCA-mutated breast cancer is relatively modest. Using a syngeneic genetically engineered mouse model of mammary tumors driven by Brca1 deficiency, the experiments provided show that tumor-associated macrophages (TAMs) abolish the efficacy of PARPi both in vivo and in vitro. Mechanistically, BRCA1-deficient mammary tumor cells induce tumor-promoting polarization of TAMs, which in turn suppresses PARPi-induced DNA damage in tumor cells, reducing the production and synthetic lethality of cytoplasmic dsDNA, and thus impairing STING-dependent antitumor immunity. STING agonists reprogram M2-like tumor-educated macrophages (TEMs) to an M1-like antitumor state in a macrophage STING-dependent manner. Systemic administration of STING agonists breaks multiple layers of tumor cell-mediated suppression on macrophages and dendritic cells and synergizes with PARPi. The therapeutic synergy of this combination is mediated by type I IFN responses and CD8+ T cells, but is independent of tumor cell-intrinsic STING. These data demonstrate the importance of targeting innate immune suppression to promote PARPi-mediated engagement of antitumor immunity in breast cancer.

[0150] These results reveal a novel mechanism by which BRCA1-deficient breast tumors exert therapeutic resistance to PARP inhibitors through the induction of an immunosuppressive microenvironment dominated by tumor-promoting macrophages that inhibits both antitumor immune responses and synthetic lethality in tumor cells. Pharmacological activation of the STING pathway in immune cells overcomes resistance and sensitizes tumor cells to PARP inhibition.

[0151] introduction Homologous recombination (HR) deficiency confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), which are utilized therapeutically in both ovarian and breast tumors harboring loss-of-function mutations in HR pathway genes, most commonly BRCA1 and BRCA2 (BRCA1 / 2) (Lord CJ, Ashworth A. Science 2017;355(6330):1152-8). Based on substantial progression-free survival (PFS) benefit, three PARPi have received FDA approval for BRCA-mutated ovarian cancer in both the adjuvant and metastatic settings (Matulonis UA,et al. Ann Oncol 2016;27(6):1013, Moore K,et al. N Engl J Med 2018;379(26):2495-505, Swisher EM,et al. Lancet Oncol 2017;18(1):75-87, Del Campo JM,et al. J Clin Oncol 2019;37(32):2968-73 9). Recently, maintenance treatment with olaparib has been shown to confer unprecedented overall survival benefit to patients with BRCA-mutated recurrent ovarian cancer (Poveda A,et al. Journal of Clinical Oncology 2020;38). However, compared with ovarian cancer, PARPi therapy appears to be less effective in BRCA-mutated breast cancer. Nevertheless, the FDA has approved two PARPi drugs, olaparib and talazoparib, as monotherapy for patients with germline BRCA1 / 2 mutations and HER2-negative advanced breast cancer (Robson M,et al.N Engl J Med 2017;377(6):523-33, Litton JK,et al.N Engl J Med 2018;379(8):753-63).Although these PARPis significantly improved PFS, recent results from the OlympiAD and EMBRACA clinical trials suggest a lack of overall survival benefit for both olaparib and talazoparib in patients with advanced breast cancer harboring germline BRCA1 / 2 mutations (Robson ME, et al. Ann Oncol 2019;30(4):558-66, Litton JK, et al. American Association for Cancer Research (AACR) Virtual Annual Meeting I2020), highlighting the need to understand why BRCA-mutated breast cancer is more refractory to PARPi in order to develop strategies to improve response to PARPi.

[0152] Understanding of the mechanisms underlying the therapeutic efficacy of PARPi is still evolving. Since first being described in 2005 (Bryant HE, et al. Nature 2005;434(7035):913-7;.Farmer H, et al. Nature 2005;434(7035):917-21), PARPi have been shown to exert synthetic lethality in HR-deficient tumor cells through multiple mechanisms, including inhibition of base excision repair (BER), trapping of PARP-DNA complexes, activation of error-prone non-homologous end joining (NHEJ), and interference with PARP1 / POLQ-mediated alternative end joining (alt-EJ) (Konstantinopoulos PA, et al. Cancer Discov 2015;5(11):1137-54; Scott CL, et al. J Clin Oncol 2015;33(12):1397-406). Recently, various experiments have demonstrated that in addition to direct cytotoxicity via synthetic lethality, immune responses induced by PARPi are also required for tumor elimination in vivo (Ding L,et al.Cell Rep 2018;25(11):2972-80; Pantelidou C,et al.Cancer Discov 2019;9(6):722-37;Chabanon RM,et al.J Clin Invest 2019;129(3):1211-28). Experiments using a genetically engineered mouse model (GEMM) of Brca1-deficient ovarian cancer showed that treatment with PARPi led to the release of double-stranded DNA (dsDNA) by tumor cells, which activated stimulator of interferon gene signaling (STING) in intratumoral dendritic cells (DCs), thus inducing a type I interferon (IFN) response and subsequent induction of antitumor CD8+ T cells. Activation of the STING pathway occurs through the production of cyclic dinucleotides by cyclic GMP-AMP synthase (cGAS), which acts as a sensor of cytoplasmic dsDNA in tumor and immune cells (Li T, J Exp Med 2018;215(5):1287-99).PARPi have also been shown to activate tumor cell intrinsic immunity due to their ability to induce DNA double-strand breaks that result in the release of dsDNA fragments from the nucleus (Pantelidou C, et al. Cancer Discov 2019;9(6):722-37, Chabanon RM, et al. J Clin Invest 2019;129(3):1211-28). However, the importance of STING in immune cells and tumor cells in cancer therapy remains unclear.

[0153] In particular, clinical outcomes in breast cancer are strongly influenced by the tumor immune microenvironment (TIME) (Savas P, Loi S. Cancer Cell 2020;37(5):623-4, Salmon H, et al. Nat Rev Cancer 2019;19(4):215-27, Li W, Tanikawa T, et al. Cell Metab 2018;28(1):87-103). Multiple studies have shown that advanced breast tumors often exhibit a pre-existing immunosuppressive TIME, characterized by a significant decrease in tumor-infiltrating lymphocyte (TIL) levels and increased expression of immunosuppressive genes, which correlates with a poor response to chemotherapy and immunotherapy (Li W, et al. Cell Metab 2018;28(1):87-103; Hutchinson KE, et al. Clin Cancer Res 2020;26(3):657-68; Savas P, Loi S. Clin Cancer Res 2020;26(3):526-8). Although there is growing evidence demonstrating the immunomodulatory properties of PARPi in vivo, it is currently unclear whether the already established immunosuppressive TIME in BRCA-mutated breast cancer may affect PARPi efficacy.

[0154] Tumor-associated macrophages (TAMs) constitute one of the most abundant and diverse immune populations that can be found in solid tumors. Although TAM phenotypes and functions are highly plastic and diverse, macrophages can be broadly classified as antitumor (M1 polarized) or tumor-promoting (M2 polarized) based on their ability to suppress or promote tumor growth (DeNardo DG, Ruffell B. Nat Rev Immunol 2019;19(6):369-82). M2 polarized TAMs can exert immunosuppression through multiple mechanisms, including recruitment of immunosuppressive immune cells such as regulatory T cells (Tregs), as well as direct inhibition of immune effector cells such as natural killer (NK) cells and cytotoxic T cells (Cassetta L, Pollard JW. Nat Rev Drug Discov 2018;17(12):887-904). Notably, multiple clinical studies have shown that high TAM infiltration is associated with poor prognosis in most cancer types (Ruffell B, Coussens LM. Cancer Cell 2015;27(4):462-72).

[0155] In this study, the results demonstrate that the response of BRCA1-deficient mammary tumors to PARPi is strongly limited by immunosuppressive TAMs, which not only directly inhibit CD8+ T cells but also suppress PARPi-mediated tumor cell DNA damage, resulting in reduced cytoplasmic dsDNA and synthetic lethality, thereby suppressing STING-dependent activation of antigen-presenting cells (APCs). The addition of exogenous STING agonists redirects TAMs away from a tumor-promoting phenotype, restores the synthetic lethal response to PARPi, and effectively activates APCs. Thus, systemic delivery of STING agonists shows strong therapeutic synergy with PARP inhibition in a Brca1-deficient mouse model of breast cancer, regardless of tumor cell-intrinsic STING expression. This finding reveals a novel approach to reverse the resistance of BRCA1-mutated breast cancer to PARPi therapy.

[0156] result Brca1-deficient mammary tumors show modest response to olaparib in vivo To investigate the response to PARPi in Brca1-deficient breast cancers, we developed syngeneic GEMMs of mammary tumors driven by simultaneous loss of Brca1 and Trp53 (referred to as BP). These BP mammary tumors were generated by intraductal injection of adenovirus expressing Cre recombinase into the mammary ducts of female FVB mice carrying homozygous floxed alleles of both Brca1 and Trp53 (Brca1L / L; Trp53L / L, Figures 1A and 2A). These mice developed mammary tumors with 100% penetrance in approximately 4-7 months (median latency 167 days) (Figure 1B). Histological analysis revealed that these BP tumors were poorly differentiated adenocarcinomas (Figure 2B) similar to advanced BRCA-deficient breast cancers in the clinic (Palacios J, et al. Clin Cancer Res 2003;9(10 Pt1):3606-14). Furthermore, TP53 mutations are frequently found in advanced breast cancers with BRCA1 deficiency (Holstege H, et al. Cancer Res 2009;69(8):3625-33). Thus, this BP tumor model recapitulates both the genetics and pathology of advanced BRCA1-deficient breast tumors in patients.

[0157] Notably, primary tumor cells derived from BP tumors can be cultured in vitro or allogeneically transplanted into the mammary fat pads of syngeneic immune-competent hosts, allowing detailed study of tumor cell-intrinsic activities as well as their interactions with the host immune system and their response to therapeutic interventions. Unfortunately, when FVB mice bearing orthotopic BP tumors were treated with olaparib, the BP tumors initially grew slower than control tumors, but nevertheless progressed throughout treatment and showed comparable growth rates to control tumors at later time points (Figure 1C). Although there was a statistically significant reduction in tumor size compared to control tumors, the effect of olaparib on Brca1-deficient mammary tumors was modest, in contrast to its remarkable effect on a comparable mouse model of high-grade serous ovarian cancer (HGSOC) driven by simultaneous loss of Brca1 and Trp53, as well as the recently developed overexpression of cMyc. Ovarian tumors had a dramatic response to olaparib accompanied by robust activation of antitumor CD8+ T cell responses in TIME, which was essential for the therapeutic efficacy of PARP inhibition.

[0158] BP tumor-associated macrophage 1 mediates immune suppression To investigate how TIME affects the response to PARPi in vivo, we sought to assess the change in BP TIME in response to PARP inhibition. Analysis of TILs revealed that CD8+ T cells and effector CD8+ T cells from the treatment groups were not significantly altered after 7 days of treatment (Figures 2C and 2E). This suggests that olaparib was unable to induce robust T cell activation in BP TIME. Interestingly, after 7 days of treatment with olaparib, the percentage of PD-1+CD8+ T cells increased in BP tumors (Figures 2C and 2E). This indicates increased depletion of CD8+ T cells in tumors over the course of olaparib treatment. However, blocking PD-1 with a monoclonal antibody against mouse PD-1 failed to improve the response of BP tumors to olaparib (Figure 1C). This suggests that BP tumors are protected from T cell-mediated destruction.

[0159] Subsequently, experiments investigated other types of immune cells in BP TIME. Flow cytometry analysis revealed that BP tumors had a large population of tumor-associated macrophages (TAMs, CD45+CD11b+F4 / 80+) that were not significantly affected by olaparib (Figures 2C and 2E). Notably, the proportion of M2-like (MHC-II low CD206+) macrophages was more than 5-fold higher than the proportion of M1-like (MHC-II high CD206-) macrophages in tumors from both the control and olaparib-treated groups (Figures 1D and 2E), suggesting that an immune-suppressive population of TAMs occurred in BP TIME, independent of olaparib treatment.

[0160] Given the striking differences in response to PARPi between BRCA1-null breast and ovarian mouse tumor models, and also considering the higher levels of M2-like macrophages in BP mammary tumors, we hypothesized that exogenous factors might suppress the activation of CD8+ T cells, thereby suppressing the response to therapy in mammary tumors. Therefore, we compared TAMs isolated from mouse breast and ovarian BRCA1-null tumors. Notably, M2 polarization of TAMs was much stronger in BRCA1-null mammary tumors than in BRCA1-deficient ovarian tumors, as evidenced by the M2 / M1 ratio (Figure 1E). This result is consistent with clinical data showing that BRCA1-mutated mammary tumors have a significantly higher enrichment score for the M2 macrophage gene signature than BRCA1-mutated ovarian tumors (Figure 1F) (Pan W, et al. Immunity 2017;47(2):284-97). Together, these data suggest that M2-like macrophages in BRCA1-null mammary tumours may contribute to resistance to PARPi.

[0161] To confirm the immunosuppressive function of TAMs in BRCA1 mutant type 1 mammary tumors, TAMs from BP tumors (14 days after implantation) were sorted and co-cultured with splenic CD8+ T cells isolated from naive mice. Indeed, CD8+ T cells co-cultured with TAMs significantly reduced the production of IFNg, TNFa, and granzyme B, as well as reduced expression of CD25 and reduced levels of effector cells (CD44highCD62Llow) compared to control T cells (Figures 1G and 2D).

[0162] Both mouse and human BRCA1-deficient mammary tumor cells induce M2-like macrophage polarization in vitro. Given the highly M2-like nature of TAMs in BP tumors, experiments next investigated the interaction of macrophages and BP tumor cells in an in vitro system in the presence or absence of Olaparib. A co-culture system of mouse bone marrow-derived macrophages (BMDMs) and primary BP tumor cells was established for 2 days with or without Olaparib (Figure 3A, upper panel). Surprisingly, a dramatic decrease in the M1-like population (from about 40% to about 2%) was simultaneously found to be accompanied by a marked increase in the M2-like macrophage population (from about 0.5% to about 60%) (Figure 3B). Notably, Olaparib alone had little effect on macrophage polarization (Figure 3B). To further investigate this, BMDMs were incubated with conditioned medium (CM) from BP tumor cells (BP-CM) or Olaparib-treated BP cells (BP / OL-CM) (Figure 3A, lower panel). Consistent with the coculture system described above, both BP-CM and BP / OL-CM strongly promoted M2-like polarization, resulting in a substantial induction of the M2 / M1 ratio (Figures 4A and 4B). These data suggest that BP tumor cells can regulate the macrophage phenotype via tumor cell-derived soluble factors.

[0163] We next performed RNA-seq analysis of BMDMs treated with control medium, olaparib, BP-CM, or BP / OL-CM. As shown in Figure 3C, olaparib had no significant direct effect on BMDMs. In contrast, BP-CM strongly upregulated the expression of genes associated with M2-like / tumor-promoting phenotypes (e.g., Ccl2, Vegfa, Arg1, and Mrc1), while downregulated the expression of key M1-like / antitumor genes (e.g., Tnf and Cxcl10). Notably, BMDMs treated with BP / OL-CM had a transcriptional profile similar to that seen with BP-CM (Figure 3C). RT-qPCR further confirmed that the expression levels of M2-like-related genes, including Il6, Il1b, and Cxcl1, were significantly increased in BMDMs treated with BP-CM or BP / OL-CM (Figure 3D). This demonstrates that BP tumor cells contributed significantly to the induction of M2-like macrophages. To assess whether this M2 macrophage polarization induced by mouse BP mammary tumor cells could be recapitulated by human BRCA1 mutant breast cancer cells, human THP-1 macrophages were treated with CM harvested from BRCA1 mutant breast cancer cell lines MDA-MB-436 or HCC1937, and the expression of key genes associated with the M2-like phenotype was assessed. Notably, THP-1 macrophages treated with tumor cell CM significantly upregulated gene expression of IL6, IL1B, and CXCL1, which was not further affected by treating tumor cells with olaparib (Figure 3E). Taken together, the data suggest that both mouse and human BRCA1-deficient mammary tumor cells can "educate" macrophages to become M2-like tumor-promoting macrophages, independent of PARPi. Hereafter, these macrophages co-cultured with tumor cells or incubated with their CM are referred to as tumor cell-educated macrophages (TEM).

[0164] TEM suppresses olaparib-induced DNA damage in BRCA1-deficient breast tumor cells and abolishes STING activation in DCs and tumor cells in vitro. We next investigated how these M2-like TEMs affect the activity of tumor cells (Figure 5A). Because the synthetic lethal response of BRCA1-deficient tumor cells to PARPi is driven by highly deleterious DNA double-strand breaks (DSBs), our experiments first tested how TEMs affect PARPi-induced DSBs in tumor cells. BP tumor cells were incubated with TEM-conditioned medium or naive BMDM-conditioned medium and subsequently treated with Olaparib. Immunofluorescence (IF) analysis using an antibody against double-stranded DNA (dsDNA) revealed that Olaparib induced the accumulation of cytoplasmic dsDNA in BP cells (Figure 5B). TEMs, but not naive BMDMs, abolished the production of cytoplasmic dsDNA in BP cells upon Olaparib treatment (Figure 5B). DNA damage was further assessed by measuring phosphorylation of histone H2AX at serine 139 (γ-H2AX), a surrogate marker and early cellular response to DNA DSBs (30). Notably, BP tumor cells responded to olaparib with a two-fold increase in γ-H2AX (Figure 5C). Consistently, TEM, but not naive BMDM, reduced the upregulation of γ-H2AX in BP cells after olaparib treatment (Figure 5C). Similar results were observed in MDA-MB-436 tumor cells incubated with MDA-MB-436-educated THP-1 macrophages. It was found that the upregulation of γ-H2AX induced by olaparib was significantly inhibited by tumor cell-educated THP-1 macrophages in MDA-MB-436 cells (Figure 5D and Figure 6A). Furthermore, it was found that TEM reduced olaparib-induced tumor cell apoptosis (Annexin V+7-AAD-) by approximately 40%-50% in BP tumor cells or human BRCA1 mutant breast cancer cells (Figure 5E-5F and Figure 6B-6C). Because these TEMs were derived from an in vitro culture system, TAMs were also harvested directly from BP tumors to assess their effect on tumor cells ex vivo. Indeed, TAMs significantly reduced BP cell death in response to olaparib (Figure 5G).These data suggest that BRCA1-deficient breast tumor cells polarize macrophages into M2-like TAMs or TEMs, which in turn suppress the synthetic lethal response of tumor cells to olaparib.

[0165] It was recently reported that dsDNA from BRCA1-deficient ovarian tumor cells treated with PARPi induced STING-dependent activation of DCs, which was a critical step for PARPi to activate antitumor immunity. To test whether this also occurred in BRCA1-deficient mammary tumor cells and to evaluate the effect of TEM on this process, bone marrow-derived DCs (BMDCs) were co-cultured with BP tumor cells pre-incubated with control medium or conditioned medium from naive BMDMs or TEMs, followed by treatment with olaparib and washout (Figure 5H). Consistent with BRCA1-deficient ovarian tumor cells, olaparib-treated BP cells activated the STING pathway in DCs, as indicated by a concomitant increase in the levels of both TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) phosphorylation (p-TBK1+p-IRF3+) (Figure 5I and Figure 6D). In contrast, TEM inhibited the ability of olaparib-treated BP cells, but not naive BMDMs, to activate the STING pathway in DCs (Figure 5I and Figure 6D). Consistently, the upregulation of proinflammatory cytokines (Ifnb, Ccl5, and Cxcl10) found in DCs with an activated STING pathway was blunted when co-cultured with BP cells pre-incubated with TEM (Figure 5J). In parallel, tumor cell-intrinsic immunity induced by PARPi was also evaluated, as reported. BP tumor cells showed a marked upregulation of proinflammatory cytokines (Ifnb, Ccl5, and Cxcl10) in control medium or upon treatment with naive BMDMs, but this upregulation of inflammatory cytokines was found to be significantly reduced by TEM (Figure 6E). Taken together, these results demonstrate that BRCA1-deficient breast tumor cells induce tumor-promoting reprogramming of macrophages, which in turn suppresses DNA damage and synthetic lethality in tumor cells upon PARP inhibition, which suppresses DNA damage and, consequently, reduces cytoplasmic dsDNA in tumor cells and reduces STING pathway activation in both DCs and tumor cells, thus attenuating antitumor immunity.

[0166] STING agonists can reprogram TEMs to an M1-like state in a STING-dependent manner in macrophages. Given the finding that tumor cell-derived dsDNA-mediated STING pathway activation is reduced by tumor cell interaction with macrophages, we hypothesized that exogenous STING agonists could be used to overcome this immune suppression and enhance the efficacy of PARPi.To examine whether the potent murine STING agonist DMXAA (Gao P,et al.Cell 2013;154(4):748-62;Corrales L,et al.Cell Rep 2015;11(7):1018-30)) could inhibit tumor-promoting polarization of macrophages by BP tumor cells, RNA-seq analysis of BMDMs treated with control, olaparib, BP-CM, or BP / OL-CM (shown in Figure 3C) was repeated in the presence of DMXAA (Figure 7A). Transcriptome analysis showed robust upregulated expression of genes related to tumor-promoting M2 polarization, including Arg1, Csf1r, Il1b, Mrc1, Pik3cg, Ptgs1, Stab1, and Tgfb1, in BMDMs cultured in BP-CM or BP / OL-CM compared to controls (Figure 7A). Surprisingly, DMXAA not only suppressed the expression of those tumor-promoting genes, but also strongly stimulated the expression of genes related to antitumor M1 signatures (e.g., Ccl5, Cxcl10, Cd40, Nos2, and Tnf) (Figure 7A). Gene Ontology (GO) analysis revealed that DMXAA significantly increased the "response to virus," "response to interferon gamma," and "type I interferon signaling pathway" signals in these macrophages, while simultaneously decreasing the biological processes of "mitosis" and "organelle division" (Figure 7B and Figure 8A). Consistent with the changes in gene expression, macrophage activation by STING agonism was also reflected in morphological changes. As shown in Figure 8B, BMDMs incubated with BP-CM exhibited elongated or stellate morphology similar to IL4-induced M2-like macrophages.In contrast, addition of DMXAA transformed these cells into a "fried egg" shape (round-shaped cells with a large nucleus centered in the cytoplasm), which resembled the morphology of M1-like macrophages induced by the combination of LPS and IFNg (Young DA, et al,. J Immunol 1990;145(2):607-15). These data suggest that STING agonists can prevent the polarization of BMDMs into M2-like macrophages.

[0167] In our experiments, we next investigated whether STING agonists could reverse M2-like TEMs to M1-like macrophages, as well as the role of the STING pathway in macrophages during STING agonist-mediated macrophage reprogramming. Notably, DMXAA reversed TEMs to M1-like macrophages and significantly increased TEM STING pathway activation (Figure 7C-7D). To determine whether STING in macrophages is required for this reprogramming, we used STING knockout (KO) mice (Sting gt / gt) and co-cultured with BP cells ex vivo. STING- / -1 BMDMs were readily polarized into M2-like TEMs when treated with BP cells (Figure 7E). This suggests that STING is not required for M2-like macrophage polarization. Notably, these STING- / - TEMs did not reverse to M1-like macrophages upon DMXAA treatment (Figure 7E). This suggests that STING is required for M1-like macrophage reprogramming. Furthermore, STING- / - TEMs also failed to increase the expression of the costimulatory molecule CD86 (Figure 7F). We next tested whether STING agonists could reprogram human TEMs by first co-cultured THP-1 macrophages with MDA-MB-436 tumor cells and then treating these TEMs with STING agonists. ADU-S100, a human STING agonist, not only suppressed CD163 (a scavenger receptor with a key role in the activation of tumor-promoting macrophages) (Shiraishi D, et al. Cancer Res 2018;78(12):3255-66, Giurisato E, et al. Proc Natl Acad Sci USA 2018;115(12):E2801-E10), but also significantly increased the expression of CD86 on THP-1 macrophages (Figure 7G). Taken together, the data suggest that STING agonists can reprogram macrophages into M1-like antitumor macrophages (dependent on macrophage STING).

[0168] STING agonists can promote DC activation by BP cells upon olaparib in the presence of TEM in vitro. We next evaluated whether STING agonists could alleviate TEM-mediated suppression of DNA damage in BP cells responding to PARPi, which could lead to increased dsDNA release by BP cells, which in turn could promote DC activation. As shown in Figure 7H, we performed this experiment in parallel with multiple controls and conditions. As expected, BP cells incubated with control medium or treated with naive BMDMs had increased gH2AX upon olaparib treatment, and their co-cultured DCs had activated the STING pathway (Figure 7H-I). Although TEM-treated BP cells did not have increased gH2AX, and their co-cultured DCs did not have activated the STING pathway, pretreatment of TEM with DMXAA restored DNA damage in BP cells in response to olaparib, as well as activation of the STING pathway in co-cultured DCs (Figure 7H-I). Collectively, these data provide a strong rationale for the combination of STING agonists and PARPi for the treatment of BRCA1-deficient mammary tumors.

[0169] STING agonists improve the therapeutic response of orthotopic BP tumors to olaparib in syngeneic immune-competent mice in vivo. To determine whether stimulation of STING can enhance the antitumor activity of PARPi in vivo, a cohort of FVB female mice bearing orthotopic BP tumors was established. When tumor volumes reached approximately 100 mm3, tumor-bearing mice were randomized into four groups and subjected to control, olaparib, DMXAA, or a combination of olaparib and DMXAA. DMXAA was administered to BP tumor-bearing mice via intratumoral (it) injection, as this delivery method has shown promising potential. A relatively low dose of DMXAA (10 mg / kg) was administered once a week for three weeks (a total of three doses). DMXAA and olaparib monotherapy induced moderate tumor growth inhibition, whereas the combination therapy strongly suppressed tumor growth (Figure 9A). Analysis of tumor immune infiltration showed that DMXAA monotherapy strongly upregulated the production of antitumor cytokines (e.g., IFNg, granzyme B, and TNFa) in both CD8+ and CD4+ T cells, which was significantly enhanced by combining with Olaparib therapy (Figure 9B and Figure 9C). Collectively, these results suggest that STING agonists overcome immune suppression and significantly improve the response of Brca1-deficient breast tumors to Olaparib in vivo.

[0170] In parallel, the therapeutic efficacy of PARPi in combination with a colony-stimulating factor 1 receptor (CSF1R) monoclonal antibody against mouse CSF1R was also evaluated in orthotopic allografts of BP tumors. Many CSF1R antagonists are in preclinical and clinical development to combat immune suppression due to their activity in depleting TAMs by blocking survival signaling in macrophages (Cannarile MA, et al. J Immunother Cancer 2017;5(1):53). Anti-CSF1R monotherapy had no significant effect in inhibiting BP tumor growth, but combination treatment with olaparib and anti-CSF1R significantly attenuated tumor growth and improved the efficacy of olaparib (Figure 10A). However, the therapeutic efficacy of the combination of olaparib and anti-CSF1R was significantly lower than that of the combination of olaparib and DMXAA (after 21 days of treatment, T / C%=39.8% [olaparib+aCSF1R] vs. 6% [olaparib+DMXAA]; T / C%=100×[median tumor volume of treatment group] / [median tumor volume of control group]) (FIGS. 9A and 10A). Analysis of tumors treated with anti-CSF1R antibodies indeed revealed a marked decrease in the abundance of TAMs (FIG. 10B), but the levels of activation of intratumoral CD8+ and CD4+ T cells were lower in tumors treated with the combination of olaparib and anti-CSF1R compared to tumors treated with the combination of olaparib and DMXAA (FIGS. 9B-9C and 10C-10D). Collectively, the data indicate that STING agonist-mediated reprogramming of TAMs offers a novel and potentially superior approach to harness TAMs for the treatment of immunosuppressive cancers.

[0171] Systemic delivery of STING agonists sensitizes STING-1 null BP tumors to olaparib in vivo. STING expression is frequently suppressed or lost in most cancers (Konno H, et al. Oncogene 2018;37(15):2037-51). We sought to determine whether STING in tumor cells is required for the therapeutic response to the combination of olaparib and STING agonists. STING-null BP cells were generated via CRISPR / Cas9-mediated gene editing, and the resulting cells were named BP-sgSTING or BP-sg control (Figure 11A). Loss of tumor cell-intrinsic STING did not affect sensitivity to olaparib in vitro (Figure 11B). Similarly, loss of STING in tumor cells, likely due to reliance on dsDNA released from dying, apoptotic tumor cells, did not affect STING pathway activation in DCs cocultured with olaparib-treated BP cells (Figure S12A), and BP-sgSTING tumor cells (with or without olaparib treatment) were still able to strongly promote M2-like polarization of macrophages (Figure S11C). As expected, BP-sgSTING cells failed to upregulate IFNβ, Ccl5, and Cxcl10 in response to olaparib treatment (Figure S11D-F), confirming the loss of STING pathway activation in tumor cells.

[0172] Next, in vivo testing of STING-null BP tumors was performed in response to PARPi in combination with STING agonists. Notably, unlike STING-WT BP tumor responses to DMXAA and its combination with Olaparib (Figure 9A-Figure 9C), BP-sgSTING tumors were completely refractory to DMXAA via intratumoral injection as a single agent or in combination with Olaparib (Figure 12B), consistent with recently reported findings (Sen T, et al. Cancer Discov 2019;9(5):646-61). These data indicate that tumor-intrinsic immunity reduction along with immunosuppression TIME may abolish local activation of antitumor immune responses, thus rendering them resistant to intratumoral administration of PARPi and DMXAA.

[0173] Therefore, experiments were conducted to determine whether systemic administration of DMXAA could overcome such resistance. DMXAA (10 mg / kg) was administered weekly via intraperitoneal (i.p.) injection. Although intraperitoneal injection of DMXAA as a single agent had no significant effect on tumor growth of BP-sg control or BP-sgSTING tumors, the combination of systemic DMXAA administration with olaparib resulted in strong inhibition of tumor growth and significantly extended the survival of both BP-sg control and BP-sgSTING tumor-bearing mice (Figures 11G and 11H). Additional experiments were then performed by treating tumor-bearing mice with a combination of olaparib and systemic DMXAA in the presence or absence of IFNAR1 or CD8 blocking antibodies. As shown in Figures 11I and 11J, the efficacy of the combination therapy was significantly attenuated, but not completely prevented, by IFNAR1 or CD8 neutralization. These results suggest that both innate and adaptive immune functions contribute to the antitumor activity driven by olaparib in combination with STING agonists.1 Collectively, the data provide compelling evidence that the combination of olaparib with systemic delivery of a STING agonist overcomes the resistance of STING-null, BRCA-deficient breast tumors to PARPi.

[0174] Consideration PARPi significantly improved overall survival in patients with BRCA-mutated ovarian cancer, but not in patients with BRCA-mutated breast cancer. Here, we report a previously undefined role for TAMs in orchestrating resistance to PARPi in syngeneic GEMMs of Brca1-deficient mammary tumors. Through a series of studies, the results here demonstrate that BRCA1-deficient mammary tumor cells strongly induce the upregulation of tumor-promoting genes in TAMs through paracrine activation of an M2-like phenotype of macrophages. These TAMs not only suppress CD8+ T cell activation but also significantly reduce DNA damage in PARPi-treated tumor cells, thus attenuating the synthetic lethal response as well as reducing dsDNA-mediated, STING-dependent activation of DCs. Treatment with exogenous STING agonists synergizes with PARPi in reprogramming TAMs, activating DCs, and inducing intratumoral T cell responses to inhibit tumor growth (Figure 13). These are notable findings, as current understanding of PARPi resistance is primarily focused on intratumor mechanisms, including cellular availability of PARPi, restoration of HR or poly ADP-ribosylation (PARylation), and DNA replication fork protection (Noordermeer SM, van Attikum H. Trends Cell Biol 2019;29(10):820-34). These studies provide insight into the role of macrophages in BRCA1-deficient breast cancer therapy and demonstrate a conceptually distinct mechanism of extrinsic immune-mediated resistance of tumor cells to PARPi.

[0175] Consistent with these results, there is growing evidence of TAM-mediated resistance to cytotoxic chemotherapy (Pathria P, et al. Trends Immunol 2019;40(4):310-27). For example, recent studies have reported that TAMs promote chemotherapy resistance by suppressing taxol-induced mitotic arrest (Olson OC, Kim H, Quail DF, Foley EA, Joyce JA. Tumor-Associated Macrophages Suppress the Cytotoxic Activity of Antimitotic Agents. Cell Rep 2017;19(1):101-13 doi10.1016 / j.celrep.2017.03.038) or by inhibiting gemcitabine through pyrimidine release that outpaces drug uptake and metabolism (Halbrook CJ, Pontious C, Kovalenko I, Lapienyte L, Dreyer S, Lee HJ, et al. Macrophage-Released Pyrimidines Inhibit Gemcitabine Therapy in Pancreatic Cancer. Cell Metab 2019;29(6):1390-9 e6). Despite these findings, therapeutic targeting of TAMs remains challenging. Blockade of CSF1R is an effective approach to deplete TAMs that depend on CSF1 / CSF1R signaling for survival (Ries CH, Cannarile MA, Hoves S, Benz J, Wartha K, Runza V, et al. Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy. Cancer Cell 2014;25(6):846-59).However, efforts to reprogram the immunosuppressive TIME with anti-CSF1R therapy have shown limited clinical benefit in advanced solid tumors (Lopez-Yrigoyen M, et al..Macrophage targeting in cancer.Ann NY Acad Sci 2020, Papadopoulos KP, et al.Clin Cancer Res 2017;23(19):5703-10). Several mechanisms may be at work that limit the efficacy of anti-CSF1R therapy. For example, it has been reported that blockade of CSF1R indiscriminately depletes it.

[0176] TAMs contain a subset of proinflammatory macrophages, but not those with proangiogenic characteristics (Zhang L,et al.Cell 2020;181(2):442-59 e29). Furthermore, inhibition of CSF1R has been shown to attract Tregs (Gyori D,et al.JCI Insight 2018;3(11)) or upregulate the expression of granulocyte-specific chemokines in cancer-associated fibroblasts, leading to the recruitment of granulocytic myeloid-derived suppressor cells (MDSCs) (Kumar V,et al.Cancer Cell 2017;32(5):654-68).

[0177] In contrast to TAM depletion strategies, reprogramming TAMs to an antitumor state may be a superior approach to harness the immune system against cancer. Studies have shown that dying tumor cells containing STING agonists such as dsDNA can induce antitumor immunity by activating STING signaling in macrophages (Ahn J,et al. Cancer Cell 2018;33(5):862-73, Zhou Y,et al. Immunity 2020;52(2):357-73). However, this pathway can be silenced by the rapid degradation of tumor-derived DNA during TAM-mediated phagocytic clearance of apoptotic tumor cells (Xu MM,et al. Immunity 2017;47(2):363-73). Here, the results showed that small molecule STING agonists efficiently reprogrammed the phenotype of TAMs from a pro-tumorigenic state to an anti-tumorigenic state, characterized by the induction of type I IFN responses and expression of the costimulatory molecule CD86, which can stimulate T cell cross-priming and induce robust adaptive anti-tumor immunity. Furthermore, while TAMs significantly suppress PARPi-induced synthetic lethal responses in BRCA1-deficient mammary tumor cells, STING agonists reprogrammed these TAMs and inhibited TAM-mediated suppression of synthetic lethality, thus rendering the tumors more sensitive to PARPi therapy. Indeed, these results demonstrated that PARPi in combination with STING agonists had superior anti-tumor efficacy than PARPi in combination with anti-CSF1R in syngeneic GEMMs of Brca1-deficient mammary tumors.

[0178] Although a central role for the STING pathway in PARPi-induced antitumor immunity has been shown, the relative contributions of STING signaling in tumor and immune cells are not fully understood. It has been demonstrated that tumor DNA damage can be sensed by host immune cells, primarily DCs, resulting in STING-dependent IFNβ production and antitumor T cell responses (Corrales L, et al. J Clin Invest 2016;126(7):2404-11; Deng L, et al. Immunity 2014;41(5):843-52; Mender I, et al. Cancer Cell 2020). Consistent with this, the efficacy of PARPi against Brca1-deficient mouse ovarian tumors was found to be significantly reduced in STING-deficient mice. Interestingly, recent studies have reported a role for tumor-intrinsic STING in recruiting CD8+ T cells via stimulation of cytokine production in models of lung and breast cancer. Thus, loss of tumor STING did not affect PARPi cytotoxicity in vitro, but led to resistance to PARPi or other DNA damage response (DDR) inhibition in vivo, which could not be overcome by combining PARPi with PD-1 1 / PD-L1 blockade (Wang Z, et al. J Clin Invest 2019;129(11):4850-62). Herein, we found that loss of tumor cell-intrinsic STING abolished PARPi-induced tumor cell-intrinsic immunity but did not affect tumor cell-mediated polarization of macrophages, leading to complete resistance to PARPi. Loss of tumor cell-intrinsic STING may limit immune activation in TIME due to lack of infiltration of immune cells into STING-deficient tumors (Xiao Y, et al. Clin Cancer Res 2019;25(16):5002-14). Surprisingly, however, systemic administration of a STING agonist overcame the resistance of STING-null tumours to PARPi.This demonstrated that tumor STING does not require systemic administration of STING agonists to enhance host antitumor immunity and synergize with PARPi in priming and mobilizing CD8+ and CD4+ T cells. These findings have important clinical implications because tumor cell-intrinsic STING signaling is frequently suppressed (Xia T, et al. Cell Rep 2016;14(2):282-97; de Queiroz N, et al. Mol Cancer Res 2019;17(4):974-86), and justify further clinical trials of the combination of systemically administered STING agonists with PARPi in STING-lost tumors.

[0179] Given the promising results of many preclinical studies, the majority of efforts to harness the immunomodulatory properties of PARPi have so far focused on the combination of PARPi with immune checkpoint blockers (ICBs), leading to more than 25 clinical trials across different types of HR-deficient cancers, including advanced breast cancer (Takahashi N, et al. Clin Cancer Res 2020;26(11):2452-6). Unexpectedly, however, initial results suggest that the combination may not effectively enhance the objective response rate (ORR) compared to historical cohorts that received PARPi as monotherapy. It is therefore important to better understand the mechanisms that may negate the efficacy of PARP inhibitors. This study shows that TAMs play a key role in influencing the response of BRCA1-deficient breast tumors to PARP inhibition and supports the need to evaluate TIME in early-phase clinical trials. Indeed, accumulation of immunosuppressive macrophages has been frequently observed in T cell-excluded or inactivated TIME in advanced breast cancer (Gruosso T, Gigoux M, Manem VSK, Bertos N, Zuo D, Perlitch I, et al. Spatially distinct tumor immune microenvironments stratify triple-negative breast cancers. J Clin Invest 2019;129(4):1785-800). Importantly, systemic delivery of STING agonists was found to promote activation of antitumor T cells when combined with PARPi. Thus, this combination therapy may prove to be particularly effective against tumors with HR deficiency and high mutational burden but low immune infiltration (e.g., a recently described subtype of advanced breast cancer with downregulated STING signaling and poor immune infiltration).Next generation STING agonists that can be delivered systemically are currently in clinical development (Ramanjulu JM, et al. Nature 2018;564(7736):439-43, Chin EN, et al. Science 2020;369(6506):993-9, Pan BS, et al. Science 2020;369(6506)), findings that systemic delivery of STING agonists that synergize with PARP inhibition1 may inform the design of future clinical therapies.

[0180] Example 2: Method of Example 1 mouse All animal experiments described in this study were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) of DFCI. STING knockout mice (C57BL / 6J-Tmem173gt / J, stock number 017537) were purchased from The Jackson Laboratory. The Brca1loxP / loxP mouse strain was kindly provided by the laboratory of Dr. Jos Jonkers at the Netherlands Cancer Institute. The Trp53loxP / loxP mouse strain was obtained from the National Cancer Institute Mouse Repository. The Brca1loxP / loxP and Trp53loxP / loxP mouse lines were backcrossed onto the FVB / NJ background for more than 10 generations as reported in (Ding L,et al.Cell Rep 2018;25(11):2972-80 e5). To develop a syngeneic genetically engineered mouse model (GEMM) of Brca1-deficient breast cancer, Brca1 mice were further crossed with Trp53 mice. The resulting Brca1;Trp53 mice were intraductally injected with an adenovirus expressing Cre recombinase under the CMV promoter, resulting in the development of mammary tumors driven by the simultaneous loss of Brca1 and Trp53 (referred to as BP).

[0181] cell culture Cells were cultured at 37°C under 5% CO2 in a humidified incubator. MDA-MB-436, HCC1937, and THP-1 human cell lines were obtained from ATCC, tested negative for mycoplasma, and authenticated using short tandem repeat analysis (Promega GenePrint10 system). MDA-MB-436 and HCC1937 breast cancer cells were cultured in RPMI1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gemini) and 100 μg / mL penicillin-streptomycin (Gibco). THP-1 monocytes were cultured in RPMI1640 containing 10% FBS and 0.055 mM 2-mercaptoethanol (Gibco, #21985023). To differentiate THP-1 monocytes into macrophages, cells were treated with 100 nM PMA (Sigma, #P1585) for 48 hours, followed by recovery in PMA-free medium for 24 hours.

[0182] Primary BP tumor cells were obtained from mouse BP breast tumors as described (Palechor-Ceron N, et al. Am J Pathol 2013;183(6):1862-70). Briefly, single cell suspensions obtained from dissociated BP tumors were grown in serum-free F-medium [a 1:3 mixture of Ham's F-12 and DMEM (Gibco) supplemented with 25 ng / mL hydrocortisone (Sigma), 5 μg / mL insulin (Thermo Fisher), 8.5 ng / mL cholera toxin (Sigma), 0.125 ng / mL EGF (Sigma), 100 μg / mL penicillin-streptomycin (Gibco), and 5 μM Rock1 inhibitor Y-27632 (Selleck)]. After tumor cell selection, BP cells were maintained in F-medium supplemented with 10% FBS1.

[0183] Murine macrophages and dendritic cells (DCs) were obtained from the bone marrow (BM) of FVB / NJ mice by modifying the protocol described in (Weischenfeldt J, Porse B. CSH Protoc 2008; Guo X, et al. J Immunol Methods 2016;432:24-9). For the generation of BM-derived macrophages (BMDMs), BM cells were seeded in ultra-low attachment plates (Corning) or Petri dishes (Falcon) and cultured in DMEM supplemented with 10 ng / mL M-CSF (BioLegend, #576404), 10% FBS, and 100 μg / mL penicillin-streptomycin. After 3 days, fresh DMEM containing 10 ng / mL M-CSF was added and the cells were incubated for another 4 days before the adherent cells (BMDMs) were harvested. For differentiation of DCs, BM cells were seeded in tissue culture dishes (Corning) and cultured in RPMI1640 supplemented with 20ng / mL GM-CSF (Stem Cell Technologies, #78017), 10% FBS, and 100μg / mL penicillin-streptomycin. After 3 days, fresh RPMI1640 containing 20ng / mL GM13 CSF was added, and non-adherent cells (DCs) were harvested after another 4 days. For preparation of tumor cell conditioned medium (CM), tumor cells were grown to 60% confluence, washed twice with PBS, and then incubated with fresh DMEM for 2 days. The CM was then harvested, centrifuged, and the supernatant was collected.

[0184] Tumor Growth and Treatment Primary breast tumor cells for orthotopic injection were resuspended in serum-free DMEM (Corning) containing 40% Matrigel. 5 × 105 tumor cells in a total volume of 100 μL were orthotopically injected into the thoracic mammary fat pad of 8-week-old female FVB / NJ mice (Jackson Laboratory). Tumor growth was examined by measuring the maximum longitudinal diameter (length) and maximum transverse diameter (width) with digital calipers, and tumor volume was calculated by using the modified ellipsoid formula (0.52 × length × width2). Tumors were measured 2–3 times per week. All tumor measurements within a single cohort were performed by the same investigator. Tumor-bearing mice were randomized before treatment initiation. Drug treatment was initiated when the mean tumor volume approached 50–100 mm3. Mice were euthanized by CO2 inhalation when tumor volume met the humane endpoint described in the IACUC (diameter 20 mm) protocol or upon severe health deterioration.

[0185] Olaparib (MedChem, #HY-10162) was prepared by diluting a 100 mg / 1 mL stock in DMSO with 10% (2-hydroxypropyl)-β-cyclodextrin (HPCD, MedChem, #101103) in PBS and administered by intraperitoneal (ip) injection immediately after drug preparation at a dose of 50 mg / kg body weight daily. DMXAA (Sigma, #D5817) was reconstituted at 10 mg / mL in 7.5% NaHCO3 (Gibco). For intratumoral injection (it), 250 μg of DMXAA (approximately 10 mg / kg body weight) was administered once a week for three doses. For intraperitoneal injection, DMXAA was administered at a dose of 10 mg / kg body weight once a week and was terminated when tumor size exceeded 600 mm3. Anti-mouse PD-1 antibody (clone 332.8H3, kindly provided by Dr. Gordon Freeman, DFCI) was injected intraperitoneally at a dose of 10 mg / kg every 3 days. Anti-mouse CSF1R antibody (CS7, Eli Lilly) was administered via intraperitoneal cavity at 40 mg / kg every 3 days. For IFNAR1 blockade, anti-mouse IFNAR1 antibody (200 μg / mouse; clone MAR1-5A3; BioXcell) was administered via intraperitoneal cavity 72 hours and 24 hours before the start of combination therapy (olaparib + DMXAA), and every 3 days thereafter. For CD8+ T cell depletion, anti-CD8 antibody (400 μg / mouse; clone YTS169.4, BioXcell) or isotype control (400 μg / mouse; clone LTF-2, BioXcell) was administered intraperitoneally 48 h and 24 h before combination therapy (olaparib + DMXAA) and every 4 days thereafter.

[0186] tissue digestion To obtain single cell suspensions, tumors were excised, minced, and dissociated in collagenase / hyaluronidase buffer [DMEM containing 5% FBS, 10 mM HEPES (Gibco), 100 μg / mL penicillin-streptomycin, 20 μg / mL DNaseI (StemCell), and 1× collagenase / hyaluronidase (StemCell)] for 45 min at 37° C., followed by treatment with ammonium-potassium chloride (ACK) buffer (Lonza) for red blood cell (RBC) lysis and filtering through a 70 μm strainer to remove undigested tumor tissue. Spleens and tumor-draining lymph nodes (TDLN) were mechanically dissociated by passing through a 70 μm strainer using the plunger of a 5 mL syringe, and RBCs were lysed as described above.

[0187] Flow cytometry For flow cytometry analysis of tumor and TDLN samples, single cell suspensions were obtained as described above (Tissue Digestion). Cells were stained with LIVE / DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher) in cold FACS buffer (PBS containing 0.2% BSA and 5 mM EDTA) for 30 min on ice, followed by blocking with anti-CD16 / 32 (BioLegend) for 20 min on ice. Cells were then transfected with IgG4 antibodies against CD45 (clone 30-F11, BioLegend), TCR β chain (clone H57-597, BioLegend), CD3ε (clone 145-2C11, BioLegend), CD4 (clone RM4-5, BioLegend), CD8a (clone 53-6.7, BioLegend), PD-1 (clone 29F.1A12, BioLegend), CD44 (clone IM7, BioLegend), CD62L (clone MEL-14, BioLegend), IFN-γ (clone XMG1.2, BioLegend), TNF-α (clone M The cells were incubated for 30 min on ice in FACS buffer containing antibodies specific for CD11c (clone N418, BioLegend), granzyme B (clone NGZB, eBioscience), CD11c (clone N418, BioLegend), IA / IE (clone M5 / 114.15.2, BioLegend), CD80 (clone 16-10A1, BioLegend), CD86 (clone GL-1, BioLegend), CD103 (clone 2E7, BioLegend), CD11b (M1 / 70, BioLegend), F4 / 80 (clone BM8, BioLegend), phospho-TBK1 (Ser172) (clone D52C2, Cell Signaling Tech.), or phospho-IRF-3 (Ser396) (clone D6O1M, Cell Signaling Tech). For intracellular staining, Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, #00-5523-00) was used for fixation and permeabilization.For cytokine analysis, cells were stimulated with leukocyte activation cocktail containing the protein transport inhibitor brefeldin A (BD Biosciences, #550583) for 4 h at 37°C before staining. Analysis of p-HA2X (Ser139) was performed according to the manufacturer's instructions. Briefly, ice-cold 70% ethanol (Decon) was added dropwise to the cell pellet while vortexing. Cells were then incubated at -20°C for 1 h and washed three times with cold staining buffer. For staining, 5 μL of p-HA2X antibody (clone 2F3, BioLegend) was added to approximately 1 × 106 cells in 100 μL staining buffer and incubated at 4°C for 30 min. For Annexin V and 7-AAD staining, cells were detached with accutase (Sigma, #A6964). Dissociated cells (from media and accutase treatment) were washed twice with cold PBS and then incubated with 5 μL FITC-annexin V (BioLegend, #640906) and 10 μL 7-AAD (BioLegend, #420404) in 100 μL Annexin V binding buffer (BioLegend, #422201) for 15 min at room temperature. Flow cytometry was performed on an LSR Fortessa HTS analyzer (BD Biosciences). All data were analyzed using FlowJo software. Gating strategies are shown in the figures.

[0188] Cell viability assay Tumor cells were seeded in 96-well plates at a density of 5000 cells per well and allowed to attach overnight. Cells were then treated with the indicated drugs at the indicated concentrations for 72 hours. Cell viability was measured using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega, #G9242) according to the manufacturer's instructions. Growth inhibition was calculated by comparing the absorbance at 490 nm of drug-treated wells to that of the untreated control 1, set at 100%. Dose-response curves and IC50 values ​​were generated using nonlinear regression models in GraphPad Prism8.

[0189] Co-culture experiments For in vitro co-culture of CD8+ T cells and tumor-associated macrophages (TAMs), TAMs (7-AAD-CD45+CD11b+F4 / 80+) were isolated from BP tumors 14 days after tumor cell implantation using a FACSAria™ II cell sorter (BD Biosciences), and CD8+ T cells were isolated from the spleens of FVB / NJ mice using a mouse CD8+ T cell isolation kit (StemCell, #19853). In a 96-well plate, 1×105 CD8+ T cells per well were cultured alone or co-cultured with TAMs at a 1:1 ratio for 2 days in RPMI1640 supplemented with 10% FBS, 0.055 mM 2-mercaptoethanol, 2 ng / mL IL-2 (Peprotech), 2.5 ng / mL IL-7 (Peprotech), and 50 ng / mL IL-15 (Peprotech). CD8+ T cells were analyzed by flow cytometry to assess IFNg expression as described above.

[0190] For in vitro coculture of macrophages with tumor cells, 2 × 105 tumor cells per well were cocultured with macrophages at a ratio of 1:1 (BP cells:mouse BMDM) or 1:1.5 (MDA-MB-436 cells:THP-1 macrophages) in 6-well plates containing the indicated drug treatments or DMSO vehicle control for 2 days.

[0191] For in vitro coculture of tumor cells with DCs, 2 × 105 tumor cells per well were seeded in 6-well plates and allowed to attach overnight, followed by incubation with macrophage CM and treatment with olaparib or DMSO vehicle control. After 2 days, tumor cells were washed twice with PBS and cocultured with 2 × 105 mouse BM-derived DCs per well in 1 mL of RPMI1640 supplemented with 20 ng / mL GM-CSF, 10% FBS, and Lipofectamine 3000 (2 μL / mL, Invitrogen). After 24 h, cocultured cells were harvested for flow cytometry, and floating cells (enriched in DCs to approximately 90%) were harvested for mRNA analysis as reported in (15).

[0192] Generation of STING-deficient BP cells CRISPR double nickase plasmids with improved editing specificity (67) were used to generate STING-deficient BP tumor cells. Briefly, BP tumor cells cultured in 6-well plates were transfected with 2 μg / well of STING (Tmem173) double nickase plasmid (Santa Cruz, #SC-428364-NIC) or control double nickase plasmid (Santa Cruz, #SC-1 437281) using Lipofectamine 3000 (Invitrogen). Two days after transfection, cells were passaged into 10 cm dishes and cultured in growth medium containing 3 μg / mL puromycin (Thermo Fisher) for selection. Puromycin-resistant cells were then used for isolation of single clones. After assessment of STING expression by Western blot, all of the clones with STING depletion were combined, expanded, and used to generate tumors via allografting into syngeneic FVB hosts as described above.

[0193] RNA extraction and reverse transcription quantitative PCR (RT-qPCR) Total RNA was extracted using RNeasy® Plus Mini Kit (QIAGEN, #74134). iScript Reverse Transcription Supermix (Bio-Rad, #1708841) was used for first strand cDNA synthesis using 1 μg of total RNA.Real-time PCR was performed using SYBR Select Master Mix (Thermo Fisher, #4472908) and gene-specific primers (mouse Il6, forward 5'-TAGTCCTTCCTACCCCAATTTCC-3', reverse 5'-TTGGTCCTTAGCCACTCCTTC-3'; mouse Il1b, forward 5'-GCAACTGTTCCTGAACTCAACT-3', reverse 5'-ATCTTTTGGGGTCCGTCAACT-3'; mouse Cxcl1, forward 5'-CCGAAGTCATAGCCACACTC AA-3', reverse 5'-GCAGTCTGTCTTCTTTCTCCGTTAC-3'; mouse Ifnb, forward 5'-TCCGAGCAGAGATCTTCAGGAA-3', reverse 5'-TGCAACCACCACTCATTCTGAG-3'; mouse Ccl5, forward 5'-GCTGCTTTGCCTACCTCTCC-3', reverse 5'-TCGAGTGACAAACACGACTGC-3'; mouse Cxcl10, forward 5'-CCAAGTG CTGCCGTCATTTTC-3', reverse 5'-GGCTCGCAGGGATGATTTCAA-3'; mouse Actb, forward 5'-CGGTTCCGATGCCCTGAGGCTCTT-3', reverse 5'-CGTCACACTTCATGATGGAATTGA-3'; human IL6, forward 5'-ACTCACCTCTTCAGAACGAATTG-3', reverse 5'-CCATCTTTGGAAGGTTCAGGTTG-3'; human IL1B, forward Analysis was performed using the following primers: human CXCL1, forward 5'-ATGATGGCTTATTACAGTGGCAA-3', reverse 5'-GTCGGAGATTCGTAGCTGGA-3'; human CXCL1, forward 5'-AAGTGTGAACGTGAAGTCC-3', reverse 5'-GGATTTGTCACTGTTCAGCA-3'; human GAPDH, forward 5'-CTCTGCTCCTCCTGTTCGAC-3', reverse 5'-TTAAAAGCAGCCCTGGTGAC-3'. Relative mRNA levels were calculated using the ΔΔCT method. Mouse Actb and human GAPDH were used as endogenous controls for mouse and human samples, respectively.

[0194] Cytoplasmic double-stranded 1DNA (dsDNA) staining and imaging BRCA1-deficient tumor cells were cultured on glass coverslips in 6-well plates. Cells were treated with 5 μM olaparib or DMSO vehicle control in the presence or absence of macrophage-derived CM for 2 days. After treatment, cells were fixed and stained for cytoplasmic dsDNA as described in (Bakhoum SF, et al. Nature 2018;553(7689):467-72). Briefly, cells were first fixed with 4% paraformaldehyde for 10 min. To stain cytoplasmic dsDNA, selective plasma membrane permeabilization was performed by incubating fixed cells with 0.02% saponin (Sigma) in PBS for 5 min. The cells were then blocked with 2.5% normal goat serum in PBS for 30 min and stained overnight at 4 °C with anti-dsDNA antibody (1:200 dilution, Thermo Fisher, #MAB1293MI) in PBS containing 1% BSA, followed by staining with Alexa Fluor 594 (Thermo Fisher, #A-11032), a goat anti-mouse IgG (H+L) highly cross-adsorbing secondary antibody. The cells were mounted with ProLong Diamond Antifade Mountant containing DAPI (Thermo Fisher, #P36966). The staining was imaged using a Leica SP5X laser scanning confocal microscope. The fluorescence intensity of cytoplasmic dsDNA was analyzed using ImageJ / Fiji software as described in Pantelidou C, et al. Cancer Discov 2019;9(6):722-37.

[0195] Immunoblotting Cells were pelleted and lysed using ice-cold RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher). Protein concentration was determined using the DC protein assay (Bio-Rad). Equal amounts of protein extracts (40-60 μg) were loaded and separated by SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk (Bio-Rad) in TBS + 0.05% Tween® 20 for 45 min at room temperature, followed by incubation with primary antibodies overnight at 4°C, then with fluorescently labeled anti-mouse IgG (Rockland Immunochemicals, #RL610-145-002) or anti-rabbit IgG (Molecular Probes, #A-21109) for 1 h at room temperature. Western blots were visualized with an Odyssey® scanner (LI-COR).

[0196] IFNβ ELISA Tumor cells were treated with olaparib or DMSO vehicle control for 2 days. To evaluate the production of IFNβ by tumor cells, cell culture supernatants were collected and centrifuged at 1,500×g for 10 min at 4° C. to remove floating cells and debris. IFNβ was then detected via a Mouse IFNβ ELISA Kit (Thermo Fisher, #424001) according to the manufacturer's instructions.

[0197] Cancer Genome Atlas (TCGA) analysis RNA-seq data were obtained from GEO DataSets (GEO:GSE62944), where TCGA RNA-seq data of 24 cancer types were reprocessed by aligning FASTQ files downloaded from Cancer Genomics Hub to compare gene expression across cancer types (Rahman M, et al. Bioinformatics (Oxford, England) 2015;31(22):3666-72). BRCA1 mutation information was obtained for patients in the TCGA cohort according to a recent study (Riaz N, et al. Nature communications 2017;8(1):857). M2 TAM immunosuppressive gene signature was derived as described in (Pan W, et al. Immunity 2017;47(2):284-97). Enrichment scores of M2 signature were generated by single sample gene set enrichment analysis (ssGSEA) as implemented in the GSVA R package.

[0198] Transcriptome analysis Total RNA was isolated by RNeasy®Plus mini kit (QIAGEN) and sequenced on the Ion Torrent platform (Thermo Fisher) using an Ion AmpliSeq custom panel targeting 4,604 mouse genes most relevant to the study, as described in (15,71). Data were analyzed using the Torrent Suite and AmpliSeqRNA Analysis Plugin (Thermo Fisher) to generate read counts per gene. Differential gene expression was then examined using the DESeq2 package (Love MI, Huber W, Anders S. Genome Biol 2014;15(12):550) in the R software environment. Genes with Log2(fold change)>1 and P<0.001 were considered as differentially expressed genes (DEGs). Volcano plots showing the significance and magnitude of log2(fold change) of these DEGs were generated by the ggplot2 package in R. Gene Ontology (GO) analysis of DEGs was performed using the topGO package in R. For GSEA, genes were first ranked according to log2 (fold change) and then analyzed using the GSEAPreranked tool with the MSigDB v7.1 HALLMARK gene set and the "classical" method (Subramanian A, et al. Proc Natl Acad Sci USA 2005;102(43):15545-50). Heatmaps showing changes in gene expression were generated using the heatmap.3 package in R.

[0199] statistical analysis Statistical analysis was performed using Prism8 (GraphPad Software Inc.) as described in the legend of each figure. For analysis of tumor growth, two-way ANOVA with Tukey's multiple comparison test was used. For survival analysis, the log-rank Mantel-Cox test was used. For other analyses, unpaired two-tailed Student's t test (for normally distributed data) and Mann-Whitney nonparametric test (for skewed data departing from normality) were used to compare two conditions. One-way ANOVA with Tukey's multiple comparison test (for normally distributed data) and Kruskal-Wallis nonparametric test (for skewed data) were used to compare three or more means. Differences with P<0.05 were considered statistically significant.

[0200] Data availability All data generated during this study will be available in the manuscript. Transcriptomic data supporting the findings will be deposited prior to publication.

[0201] Example 3: Novel treatment for ovarian cancer PARP inhibitors (PARPi) have demonstrated strong therapeutic efficacy in ovarian cancer treatment. Acquired resistance to PARPi is a major problem in the clinic, and therapeutic approaches that can overcome secondary resistance to PARP inhibition are urgently needed. By using PARPi-resistant mouse and PDX models of ovarian cancer, a novel mechanism underlying secondary resistance to PARP inhibition mediated by tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) has been identified. Mechanistically, PARP inhibition activates the STAT3 signaling pathway in tumor cells and promotes tumor-promoting polarization of TAMs in the TME of ovarian cancer. STING agonists reprogram myeloid cells in the TME of ovarian tumors by repolarizing TAMs and increasing myeloid DCs in a STING-dependent manner. Furthermore, STING agonism was shown to overcome acquired immunosuppressive TME-induced secondary resistance to PARPi in mouse models and human PDX. The data presented herein elucidate a novel mechanism of PARPi resistance and provide a new therapeutic strategy to overcome acquired therapeutic resistance to PARP inhibition in ovarian cancer.

[0202] introduction Homologous recombination deficiency (HRD), particularly mutations and dysregulation of BRCA1 and BRCA2, is frequently found in various human malignancies, including ovarian, breast, pancreatic, and prostate cancer. Based on the concept of synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency, PARP inhibitors (PARPi) have been developed for the treatment of BRCA-deficient tumors (Chan et al., 2021; Farmer et al., 2005). An increasing number of PARPi have received FDA approval for their promising therapeutic efficacy in clinical practice, especially in ovarian cancer (Abida et al., 2020 J Clin Oncol 38, 3763-3772; de Bono et al., 2020, New England Journal of Medicine 382, ​​2091-2102; Gong et al., 2021 Oncology (Williston Park) 35, 119-125; Gonzalez-Martin et al., 2019 New England Journal of Medicine 381, 2391-2402; Ledermann et al., 2012 New England Journal of Medicine 366, 1382-1392; Mullard, 2017 PARP inhibitors plough on. Nat Rev Drug Discov 16, 229; Robson et al., 2017 New England Journal of Medicine 377, 523-533). In particular, recent studies have shown that maintenance with PARP inhibitors improves progression-free survival (PFS) in all subsets of patients with platinum-sensitive, recurrent, high-grade ovarian cancer, with the greatest benefit in patients with BRCA1 / 2 mutations (Lee et al., 2021 A meta-analysis. Cancer 127, 2432-2441; Mirza et al., 2016; New England Journal of Medicine 375, 2154-2164).In addition to synthetic lethality, PARPi has been reported to induce potent antitumor immune responses, which can be further enhanced by immune checkpoint blockade (Ding et al., 2018 Cell Rep25, 2972-2980.e2975.; Pantelidou et al., 2019 Cancer Discov9, 722-737.; Sen et al., 2019 Cancer Discov9, 646-661). These preclinical findings are further supported by recent results from clinical trials of PARPi and ICB (Domchek et al., 2020 Lancet Oncol21, 1155-1164; Konstantinopoulos et al., 2019 JAMA Oncol5, 1141-1149; Lee et al., 2018 Annals of Oncology29, viii334.). Although PARPi has changed the landscape of ovarian cancer treatment, resistance to PARPi, including primary and secondary, has become a major problem in the clinic, and appropriate management of patients with PARPi-resistant tumors is an urgent concern. Several mechanisms underlying resistance to PARP inhibition, including restoration of homologous recombination, reduced PARP trapping, cell cycle dysregulation, and enhanced drug efflux, have been identified from both clinical and preclinical studies (D'Andrea, 2018 Mechanisms of PARP inhibitor sensitivity and resistance. DNA Repair (Amst) 71, 172-176; Pantelidou et al., 2019 Cancer Discov 9, 722-737; Pettitt et al., 2020 Cancer Discovery 10, 1475). BRCA1 / 2 reversion mutations have been found as the main molecular mechanism of HR restoration and PARPi resistance in ovarian cancer. Of note, recent studies have reported that approximately 20–50% of recurrent ovarian cancer cases acquire BRCA1 / 2 reversion mutations ( Lin et al., 2019 ; Norquist et al., 2011 ).Although most studies have focused on tumor cell-intrinsic resistance to PARPi, some recent findings suggest the role of the tumor microenvironment or the host immune system in PARPi resistance. For example, PARPi has been found to induce immunosuppression through upregulation of PD-L1 expression or enhancement of both antitumor and protumor properties of macrophages in breast cancer (Jiao et al., 2017 Clinical Cancer Research 23, 3711; Mehta et al., 2021 Nature Cancer 2, 66-82). It remains unclear whether the host immune system also plays a role in PARPi resistance in ovarian cancer.

[0203] Using a preclinical mouse model of secondary resistance to PARP inhibition, we uncovered a novel mechanism of PAPRi resistance mediated by tumor cell-intrinsic STAT3 signaling and TAMs in BRCA1-deficient ovarian tumors. We further demonstrated that STING agonism efficiently reprograms myeloid cells in the TME and overcomes TME-dependent secondary resistance to PARP inhibition in both mouse and human ovarian cancer models, thus providing a new therapeutic option for some ovarian cancer patients with resistance to PARP inhibition.

[0204] BRCA1-deficient ovarian tumors developed secondary resistance to PARP inhibition A syngeneic genetically engineered mouse model of ovarian cancer driven by simultaneous loss of Brca1 and Trp53 and overexpression of c-Myc, termed PBM, which recapitulates the high-grade serous carcinoma of human ovarian cancer, was generated (Ding et al., 2018 Cell Rep25, 2972-2980.e2975). These PBM tumors initially had a robust response to PARP inhibition, but eventually most of the tumors progressed with olaparib treatment (termed PBM-R, Figures 15A and 15B) (Ding et al., 2018 Cell Rep25, 2972-2980.e2975). To investigate the mechanisms underlying this secondary resistance to PARPi in the BRCA1-deficient ovarian tumor model, 12 PBM-R tumors were harvested and their primary cell lines were established in culture (Figure 15B). Strikingly, 11 of 12 PBM-R tumor lines were sensitive to PARPi in vitro and IC 50 The values ​​are comparable to PBM tumor cells (Figure 15C). Only PBM-R3 tumor cells were truly resistant to Olaparib, with an IC50 of 4.19 μM, 7.48-fold higher than 0.56 μM. Consistently, PBM-R tumor cells showed significantly increased DNA damage in response to Olaparib, as measured by phosphorylation of histone H2AX at Seine139 (γ-H2AX), an early cellular response marker to DNA DSBs, whereas PBM-R3 cells showed only modest DNA damage signaling (Figure 1D). Whole exome sequencing (WES) analysis revealed that PBM-R3 tumor cells had copy number alterations (specifically, copy number gains) in multiple genes involved in DNA repair pathways that were not detected in PBM-R1 and -R2 (Figure 16). It has been shown that reduced DNA damage and / or increased DNA repair, i.e., BRCA reversion and copy number gains in DNA repair genes, are associated with resistance to PAPRi (Figure 16). The majority of secondary resistant PBM-R tumors responded to olaparib in vitro, implicating tumor-extrinsic resistance mechanisms, and we further confirmed that these PBM-R tumors were indeed resistant to PARPi in vivo when orthotopically reimplanted into syngeneic host mice (Figure S15E).

[0205] Tumor-promoting macrophages enriched in the TME of PARPi-resistant ovarian tumors To explore the extracellular mechanisms underlying PAPRi resistance in Brca1-deficient ovarian tumors, we assessed immune cells in the TME of PBM and PBM-R tumors by flow cytometry analysis. The data showed that total CD11b + Although the populations of TAMs and MSDCs were similar, tumor-promoting (M2-like, MHC-II) expression was significantly higher in PBM-R tumors compared to PBM tumors. 低 CD206 + ) macrophages were significantly increased between PBM and PBM-R tumors (Figure 17A and Figures 18A-18C). +、 CD8 +、 No significant changes in TAM and Treg cells were observed (Figures S2D-S2F). Because ascites is an important specific microenvironment that contributes to ovarian tumor growth, we further analyzed immune cells in ascites and found that both TAM and M2-like macrophages were significantly increased in PBM-R tumor-bearing mice compared to PBM-tumor-bearing mice (Figure S17B). These results indicate that PBM-R tumors may have acquired the ability to promote tumor-promoting macrophage polarization.

[0206] To evaluate the effect of PBM and PBM-R tumor cells on macrophage polarization, bone marrow-derived macrophages (BMDMs) were incubated with conditioned medium (CM) from PBM (PBM-CM) or PBM-R (PBM-R-CM). PBM-R-CM was able to promote M2-like polarization of TAMs ex vivo, and showed a much stronger effect on M2-like TAM polarization (Figures 17C and 17D). Bone marrow cells in ascites (BMCs) were further cultured in supernatants collected from PBM or PBM-R tumor-bearing mice. As a result, PBM-R ascites supernatants promoted the proliferation and differentiation of myeloid cells (CD11b +) and TAM differentiation, as well as promoting M2-like polarization ex vivo (Figures 17E and 17F). These data suggest that PBM-R tumor cells can strongly promote M2-like macrophage polarization in the TME of tumor-bearing mice, both in an ex vivo coculture system and in vivo.

[0207] PARP inhibition upregulates the STAT3 signaling pathway in tumor cells, which in turn promotes the polarization of tumor-promoting macrophages. To investigate the mechanism underlying the ability of PBM-R cells to induce M2-like macrophage polarization, we performed RNA-seq analysis of PBM and PBM-R tumors, which contain more than 95% cancer cells. GSEA analysis revealed that the STAT3 signaling pathway was significantly activated in PBM-R tumors when compared to PBM tumors (Figure 19A and Figure 20A). Flow cytometry analysis also showed that PBM-R tumor cells had higher levels of phosphorylated STAT3 (Y705) than PBM tumor cells (Figure 19B). This observation was further confirmed by immunohistochemical analysis of p-STAT3 in PBM and PBM-R tumors (Figure 19C).

[0208] We next investigated whether PAPRi could directly induce STAT3 phosphorylation in tumor cells. Treatment of PAPRi-naive PBM tumor cells with olaparib, followed by flow cytometry and Western blot analysis, revealed that the phosphorylation level of STAT3 in cultured PBM cells increased in a dose-dependent manner (Figures 19D and 20B). Notably, conditioned medium (CM) collected from olaparib-treated PBM cells (PBM-OLA-CM) showed a stronger effect on M2-like TAM polarization of BMDMs than PBM-CM (Figure 19E). Cytokine array analysis showed that several STAT3-regulated chemokines (CCL2, CX3CL1, CCL20, and CXCL1) were significantly increased in the CM of olaparib-treated PBM cells (Figure 19F) (Ikeda et al., 2016 Oncotarget7, 13563-13574; Liu et al., 2013 PLOS ONE 8, e75804; Shen et al., 2018 Neurochem Res43, 556-565; Yang et al., 2016 Cancer Research76, 4124-4135). Blockade of these chemokines partially abolished the effect of olaparib-treated PBM cells on promoting tumor-promoting macrophage polarization (Figure 19G). These data suggest that activation of STAT3 in PARPi-induced tumor cells may contribute to the M2-like polarization of macrophages.

[0209] To further evaluate whether activation of STAT3 in tumor cells is necessary for M2-like macrophage polarization in PARPi-resistant BRCA1-deficient ovarian cancer, STAT3 was silenced by introduction of two individual lentivirus-mediated shRNAs targeting different regions of the STAT3 gene in PBM-R tumor cells (Figure 20C). Knockdown of STAT3 in PBM-R cells reduced the M2 / M1 ratio induced by CM from PBM-R tumor cells (PBM-R CM) (Figure 19H). Notably, knockdown of STAT3 in PBM-R cells did not affect the tumor cell response to olaparib treatment in vitro (Figure 20D), but PBM-R-shStat3 tumors became sensitive to olaparib treatment in vivo. Flow cytometry analysis revealed that the antitumor activity and immunomodulatory properties induced by olaparib treatment were enhanced by an increase in the M1 / M2 ratio and a decrease in M2-like TAMs, as well as an increase in the M2 / M1 ratio and a decrease in the M2-like TAMs in total T cells and effector CD4 + and CD8 + STAT3 activation in PBM-R tumors was shown to be restored in PBM-R-shStat3 tumors, as reflected by an increase in T cells (Figures 19J and 19K, Figures 20E and 20F). These data suggest that STAT3 activation in PBM-R tumors is involved in inducing M2-like TAM polarization in the TME.

[0210] STING agonists reprogram TAMs and activate myeloid DCs in the TME of PBM-R tumors Increasing evidence indicates that activation of STING signaling can remodel the TME by antagonizing MDSC proliferation and reprogramming immunosuppressive macrophages into immune-activating subtypes (Downey et al., 2014 PLOS ONE 9, e99988; Jassar et al., 2005 Cancer Res65, 11752-11761; Jing et al., 2019; Zhang et al., 2019 Therapy of Cancer7, 115). We tested whether STING agonists could alter the PARPi-induced immunosuppressive TME to overcome the resistance of PBM-R tumors to PARPi. We first tested the effect of STING agonists on macrophage polarization in vitro. BMDMs generated from FVB / NJ mice were incubated with PBM-R-CM and treated with vehicle control or STING agonists MSA-2 or ADU-S100 (Figure 21A). As expected, PBM-R-CM decreased the M1 / M2 ratio (Figure 21B). Addition of either MSA-2 or ADU-S100 significantly increased the M1 / M2 ratio (Figure 21B), suggesting that STING agonists shifted macrophages to an M1-like state.

[0211] To further explore this in vivo, PBM-R tumor cells were injected intraperitoneally into FVB / NJ mice to induce an immunosuppressive TME in the ascites. PBM-R tumor-bearing mice were then challenged with olaparib and MSA-2, either as single agents or in combination, for 24 hours (Figure 21C). + Using a positive selection kit, bone marrow cells (CD45 + CD11b +) were isolated and the purity of the isolated bone marrow cells was confirmed by flow cytometry analysis (Figure 22A). Transcriptome analysis of the isolated bone marrow cells was performed and it was found that MSA2 treatment not only inhibited the expression of genes related to tumor-induced M2 polarization (Fn1, Plxdc2, Tgfb2, Ltbp1, Alox15, etc.) but also strongly upregulated the expression of M1-like / anti-tumor-induced genes (Ccl5, Ly6a, Ly6i, Il18bp, Ifi44, etc.) compared to the control group (Figure 21D).

[0212] To derive a characteristic gene expression signature of cGAS-STING pathway activation, in vitro cultured mouse DCs were treated with DMSO control, DMXAA, or DMXAA in combination with STING pathway inhibitor BX795. RNA sequencing analysis was performed to identify a gene expression signature containing 94 upregulated genes and 7 downregulated genes for cGAS-STING pathway activation in DCs upon STING agonist treatment (Figure 22B). By using the cGAS-STING pathway signature generated in DCs, the data further revealed that bone marrow cells from MSA-2-treated PBM-R tumors had a significantly higher enrichment score of cGAS-STING pathway gene signature than control mice. This indicates that MSA-2 activated the cGAS-STING signaling pathway in bone marrow cells (Figure 21D). Interestingly, MSA-2 also strongly stimulated the expression of genes related to antigen processing and presentation (e.g., Flg2, Tap1, Psmb8, Cts, Psmb9, Tap2, and B2m) (Figure 21D). Gene Ontology (GO) analysis revealed that MSA-2 significantly increased myeloid leukocyte-mediated immunity, neutrophil activation, response to viruses, type I interferon signaling pathway, and T cell activation signal (Figure 22C). Flow cytometry analysis revealed that MSA-2 treatment not only reduced the population of TAMs (Figure 21F), but also increased the expression of CD11b + CD11c +It was further shown that STING agonist-induced TAM repolarization increased the population of myeloid DCs and enhanced class I antigen presentation in this subset of DCs (Figure 21G). Further analysis confirmed that the STING pathway was indeed activated in activated myeloid DCs (Figure 21H). These data suggest that STING agonism reprograms myeloid cells by promoting the repolarization of M2-like TAMs to an M1-like state and enhancing class I antigen presentation of myeloid DCs in the TME of BRCA1-deficient ovarian tumor-bearing mice. To further investigate whether the STING pathway is required for STING agonist-induced TAM repolarization, we used STING knockout mice (STING - / - BMDMs generated from wild-type mice (WT BMDMs) and syngeneic BRCA1-deficient cell lines (ID8-Brca1 BMDMs) were cultured with or without olaparib treatment. - / - ) (Figure 21I). These results show that MSA-2 cells were cultured in CM from control or olaparib-treated ID8-Brca1 - / - Reverses M2-like TAM polarization in WT BMDMs induced by normal but not STING cells - / - Furthermore, in vivo data showed that MSA-2 successfully activated myeloid DCs and repolarized M2-like TAMs to an M1-like state in WT mice, but not in BMDMs (Figure 21I). - / - We further demonstrated that this was not the case in mice (Figures 21J and 4K, and Figure 22D). These data demonstrated that STING agonism reprograms bone marrow cells in a STING-dependent manner.

[0213] STING agonism overcomes immunosuppressive TME-induced secondary resistance to PARPi in a mouse model of Brca1-deficient ovarian cancer Because STING agonism can reprogram immunosuppressive myeloid cells to an antitumor state, we next investigated whether STING agonism could overcome acquired TME-dependent PARPi resistance in a BRCA1-deficient mouse model of ovarian cancer. PBM-R tumor cells were orthotopically injected into FVB / NJ mice. Tumor-bearing mice were randomized into four groups and subjected to control, olaparib, MSA-2, or combination treatment. While MSA-2 or olaparib alone had little or no effect on inhibiting tumor growth, the combination of MSA-2 and olaparib treatment significantly suppressed PBM-R tumor growth when compared to the control group, with an inhibitory effect of 60-70% (Figure 23A). Analysis of tumor-infiltrating immune cells in each group of mice revealed a dramatic decrease in the proportion of TAMs in mice treated with MSA-2, along with an increase in the M1 / M2 ratio (Figures 23B and 23C). On the other hand, treatment with MSA-2 or olaparib alone activated the STING pathway and promoted tumor invasion (CD86 + ) conventional DCs (cDCs), and the combination of MSA-2 with olaparib further enhanced this effect (Figures 23D and 24C). Similarly, MSA2, in combination with olaparib, activated the STING pathway in intratumoral myeloid DCs and enhanced their antigen presentation in PBM-R tumor-bearing mice (Figures 23E and 24D). Furthermore, the combination of MSA-2 and olaparib treatment increased tumor-infiltrating CD4 + and CD8 + The population of CD4 and cytokine production were significantly increased (Fig. 23F and 23G, Fig. 24E). + and CD8 + Effector T cells (CD44 高 CD62L 低 ) significantly accumulated in the combination treatment group (Figure 24F). Collectively, these data indicate that STING agonists can overcome immunosuppressive TME-induced resistance to PARPi in BRCA1-deficient ovarian tumors by reprogramming immunosuppressive myeloid cells.

[0214] STING agonism resensitized PARPi-resistant ovarian cancer PDXs to PARP inhibition Ovarian patient-derived xenograft (PDX) models were previously generated by transplantation of cancer cells from patients' ascites into immunodeficient mice (Liu et al., 2017 Clinical cancer research: an official journal of the American Association for Cancer Research 23, 1263-1273.). PARPi-resistant ovarian PDXs, DF86 and DF101 (BRCA1-deficient), DF118 and DF149 (no BRCA1 deficiency), were harvested from ascites of NSG mice, cultured in vitro for 24 h, and then treated with 5 μM olaparib for 24 h. Increased phosphorylation levels of STAT3 were detected in three out of four PDX cells after olaparib treatment (Figures 25A and 25B). In parallel, human BMDMs were cultured in CM harvested from ovarian cancer PDXs with or without olaparib treatment. Consistent with previous findings in mouse and human ovarian cancer cell lines, CM from most ovarian PDXs significantly promoted M2-like macrophage polarization, and activation of the STAT3 signaling pathway induced by olaparib treatment further enhanced M2-like polarization induced by ovarian PDXs (Figure 25C). Inhibition of the STAT3 signaling pathway with the STAT3 inhibitor napabucasin partially reduced M2-like macrophage polarization induced by CM from DF86 and DF118 (Figure 25D). Addition of MSA-2 in the co-culture system reversed M2-like macrophage polarization induced by ovarian PDXs (Figure 25E). To evaluate whether STING agonism could reprogram myeloid cells and overcome immunosuppression in ovarian PDX models in vivo, DF86 cells, which are relatively sensitive to PARPi treatment in vitro (Figure 26A), were mixed with primary human bone marrow monocytes (BMMs) and injected into NSG mice. Approximately 3 weeks after injection, when a highly immunosuppressive TME had formed, DF86 tumor cell-bearing mice were grouped and treated with vehicle control, olaparib, MSA-2, or a combination of olaparib and MSA-2 (Figure 25F).MSA-2 treatment significantly reduced tumor burden (reflected by fold change in intensity of luciferase signal) in DF86 tumor-bearing and NSG mice compared with control or olaparib-treated mice (Figure 25G). Flow cytometry analysis of human immune cells in ascites revealed that mice in the MSA-2 treatment group had a decreased percentage of TAMs and an increased M1 / M2 ratio (Figures 25H and 25I, Figure 26B). In particular, CD14. + Myeloid DC (CD14 + HLA-DR + ) was significantly increased in the STING agonist-treated group (Figure 25J). We also evaluated the effect of MSA-2 in the DF101 PDX model, which was more resistant to olaparib in vitro compared to DF86 (Figure 26A). The results showed that DF101 tumor cell-bearing mice were less responsive to MSA-2 treatment alone or in combination with olaparib compared to DF86 PDX tumors, but MSA-2 significantly reduced the proportion of TAMs and reprogrammed M2-like TAMs to M1-like in the ascites of DF101 tumor cell-bearing mice (Figures 26C-E).

[0215] Example 4: Method of Example 3 Generation of PARPi-resistant ovarian cancer mouse models A Brca1-deficient ovarian cancer mouse model - PBM (Trp53- / -; Brca1- / -; c-Myc) was previously developed in FVB / NJ mice (Ding et al.,2018 Cell Rep25,2972-2980.e2975.). PBM tumor cells were orthotopically implanted into syngeneic FVB / NJ mice and treated with vehicle control or olaparib (AZD2281) at a dose of 50 mg / kg body weight, 6 days per week, by intraperitoneal injection. PBM tumors initially responded well but relapsed with olaparib treatment after long-term treatment. Tumor cells from refractory tumor-bearing mice (one cell line from each treated mouse) (designated PBM-R) were cultured in MOT medium (DMEM / F12, 0.6% FBS, 10 ng / ml EGF, 1 μg / ml hydrocortisone, 1 ng / ml cholera toxin, 100 μg / ml penicillin-streptomycin, 5 μM Y27632) for further evaluation.

[0216] Cell lines and PDX models UWB1.289 and UWB1.289+BRCA1 were purchased from ATCC and cultured in epithelial complete growth medium (50% ATCC formulated RPMI-1640 medium, 50% MEGM medium, and 3% fetal bovine serum) as previously described (Ding et al., 2018, Cell Rep25, 2972-2980.e2975). + / + and ID8-Brca - / -The cells were previously generated by CRISPR-Cas9 technology. Ovarian cancer patient-derived tumor xenografts (PDXs) were established at Dana-Farber Cancer Institute by intraperitoneally implanting tumor cells isolated from the patient's ascites into irradiated nude mice (Liu et al., 2017; Clinical cancer research: an official journal of the American Association for Cancer Research 23, 1263-1273). The established PDX models were maintained by intraperitoneal implantation in NOD / SCID IL2Rg null mice (NSG, The Jackson Laboratory). Ovarian PDX cells could be cultured for about 3-4 days in epithelial complete growth medium for in vitro experiments.

[0217] Determination of IC50 values ​​in tumor cells Tumor cells were seeded in 96-well plates at a density of 2000-3000 cells / well and allowed to adhere overnight. Cells were then exposed to appropriate concentrations of therapeutic agents (or vehicle control) with continuous exposure for 72 hours. Growth inhibition was measured by the CellTiter-Glo® 2.0 cell viability assay from Promega, according to manufacturer's instructions. IC50 values ​​were calculated using a nonlinear regression model (log inhibitor vs. normalized response variable slope) in Graphpad Prism9.

[0218] Tumor Growth and Treatment PBM and PBM-R tumor cells were orthotopically implanted into syngeneic FVB / NJ mice to generate tumors for drug evaluation. Tumor-bearing mice were equally divided into control and treatment groups according to luminescence intensity as previously described (Ding et al., 2018; Cell Rep25, 2972-2980.e2975). Olaparib (AZD2281) was administered daily by intraperitoneal injection at a dose of 50 mg / kg body weight. Anti-PD-1 antibody (clone, 332.8H3) was diluted in PBS (250 μg / 100 μl / mouse) and injected intraperitoneally every 3 days. MSA-2 was prepared by diluting a 50 mg / ml stock in DMSO with PBS (pH 8.0) and administered every other day by intraperitoneal injection at a dose of 25 mg / kg body weight (3 times a week, 2 weeks on, followed by 1 week off). Endpoints were determined by tumor burden and ascites.

[0219] In PDX in vivo experiments, approximately 3 × 10 6 PDX tumor cells were cultured at 3 × 10 in serum-free DMEM / F12 medium containing 50% Matrigel (Cat. No. 70001, STEMCELL Technology). 6 The PDXs were mixed with human bone marrow mononuclear cells and intraperitoneally implanted into NOD / SCID IL2Rg null mice (NSG, The Jackson Laboratory). Approximately 3 weeks after injection, the PDX-bearing mice were divided equally into 4 groups according to luminescence intensity and treated with vehicle control, olaparib, MSA-2 and MSA-2 combination using the same dosing and schedule as described above. After 3 weeks of treatment, ascites was collected for analysis.

[0220] Flow cytometry analysis Tumors were cut and digested in collagenase buffer as previously described (Ding et al., 2018 Cell Rep25, 2972-2980.e2975). Single cell suspensions of tumors and ascites were obtained by filtration through a 70 μm strainer and treated with 1× eBioscience RBC lysis buffer (Thermo Fisher) before staining. Single cell suspensions were incubated with LIVE / DEAD Fixable Aqua Dead Cell Stain (Life Technologies, Cat. No. L34965) for 30 min and then blocked with anti-CD16 / 32 (Biolegend, clone 93) for 20 min on ice. Samples were then incubated with the appropriate antibodies for 30 min on ice. For staining of intracellular markers, Foxp3 staining buffer set (eBioscience, Cat. No. 00-5523-00) was applied. For intracellular cytokine analysis, cells were stimulated with leukocyte activation cocktail (BD Biosciences, catalog no. 550583) for 4–6 h at 37°C prior to FACS staining. The following antibodies were used in this study (antibodies were purchased from BioLegend unless otherwise indicated): CD45 (clone 30-F11), CD3ε (clone 145-2C11), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD44 (clone IM7), CD62L (MEL-14), CD25 (PC61), IFNγ (clone XMG1.2), TNFα (clone MP6-XT22), CD11b (clone M1 / 7), and CD45 (clone 145-2C11). 0), CD11c (clone BM8), F4 / 80 (clone BM8), Ly-6C (clone HK1.4), Ly-6G (clone 1A8), MHC-II (clone M5 / 114.15.2), CD80 (clone 16-10A1), CD86 (clone GL-1), MHC-I (clone KH114), FoxP3 (clone FJK-16s; eBioscience), Phospho-IRF-3 (Ser396) (clone D6O1M, Cell signaling technology), and Phospho-TBK1 / NAK (Ser172) (clone D52C2, Cell signaling technology).The following human antibodies were used in this study: cell surface markers include CD45 (clone HI30), CD11b (clone M1 / 70), CD11c (clone Bu15), CD80 (clone 2D10), CD86 (clone IT2.2), CD14 (clone 63D3), CD15 (clone 30-F11), HLA-DR (clone L243), and HLA-A,B,C (clone W6 / 32). Intracellular markers include CD163 (clone GHI / 61), CD68 (clone Y1 / 82A), and CD206 (clone 15-2). Flow cytometry was performed on an LSRII (BD Biosciences) or Fortessa HTS (BD Biosciences) at the DFCI Flow Cytometry Core. All data were analyzed using FlowJo software.

[0221] Analysis of p-STAT3 and γ-H2AX (p-HA2X-Ser139) was performed according to the two-step protocol of Intracellular Phosphorylated Signaling Proteins (Thermo Fisher). Briefly, cells were incubated with LIVE / DEAD Fixable Aqua Dead Cell Stain for 30 min. After washing, cells were suspended in 100 μl of PBS and then fixed by adding an equal volume of IC Fixation Buffer (catalog no. 00-822-49, Thermo Fisher) directly to the cells and incubated at room temperature for 20 min, followed by fixation with ice-cold 90% methanol in PBS for 30 min. Fixed cells were blocked and stained with p-STAT3 or γ-H2AX antibodies for flow cytometry analysis as described above.

[0222] Cytokine array analysis PBM tumor cells were cultured in 6-well plates for 24 hours and treated with olaparib or vehicle control. After 24 hours of treatment, the drug was removed and the cells were cultured in fresh medium for another 48 hours. Cell culture supernatants were obtained by centrifugation at 1,500g for 5 minutes at 4°C according to the manufacturer's instructions, and then all debris and cells were removed and subjected to cytokine array analysis (ARY028, R&D system). Briefly, cell culture supernatants were mixed with a cocktail of biotinylated detection antibodies and then incubated with mouse cytokine arrays. The arrays were then incubated with streptavidin-horseradish peroxidase, followed by detection by chemiluminescence. Array images were analyzed using Image J software.

[0223] Lentivirus-mediated knockdown of Stat3 in PBM-R tumor cells Control shRNA and Stat3 shRNA plasmids (sh-Stat3-1: TRCN0000071456 and sh-Stat3-2: TRCN0000071453) were obtained from Sigma-Aldrich. Stat3 shRNA and control shRNA plasmids were co-transfected with pCMV-delta8.9 and pVSVG in a ratio of 2:2:1 with PEI (1 μg / μl) (4:1 to DNA) into HEK293T cells. 24 hours after transfection, the medium was replaced and the viral supernatant was harvested 48 hours later by filtering through a 0.45 μm filter. PBM-R cells were cultured in 6-well plates and infected with Stat3-shRNA lentiviral particles, and puromycin (3 μg / mL) was added to the culture for selection. Puromycin-resistant cells were selected and expanded. Western blot analysis was performed to evaluate the silencing effect of lentiviral Stat3 in PBM-R tumor cells.

[0224] Western blot analysis Tumor cells were harvested and lysed in ice-cold RIPA buffer supplemented with protease phosphatase inhibitor cocktail (Thermo Fisher). Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher). Approximately 50 μg of protein extract was loaded and separated by SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF) membrane. After blocking with 5% nonfat milk (Bio-Rad) in PBST (PBS + 0.2% Tween® 20) for 1 h, the membrane was incubated with primary antibodies overnight at 4°C. Fluorescently labeled anti-mouse IgG (Rockland Immunochemicals, #RL610-145-002) or anti-rabbit IgG (Molecular Probes, #A-21109) was used as secondary antibodies, and western blots were visualized with an Odyssey scanner (LI-COR).

[0225] Co-culture experiments For ex vivo culture of bone marrow cells in ascites supernatant, bone marrow cells were isolated from FVB / NJ mice and cultured in conditioned medium containing 50% ascites supernatant and 50% complete DMEM medium (90% DMEM and 10% FBS) supplemented with 100 μg / ml penicillin-streptomycin. Cells were incubated in conditioned medium for 6 days and the medium was changed on day 3. Cellular components were analyzed by flow cytometric analysis.

[0226] For ex vivo culture of mouse macrophages, bone marrow cells were isolated from FVB / NJ mice and cultured in DMEM containing 10% FBS, 55 μM 2-mercaptoethanol, and 20 ng / ml M-CSF. On day 7, BMDMs (bone marrow derived macrophages) were harvested and further cultured for 72 hours in 2.0 ml of the above control medium (90% DMEM, 10% FBS, 55 μM 2-mercaptoethanol, 5 ng / ml M-CSF) or medium containing 50% tumor cell conditioned medium, and then analyzed. For tumor cell conditioned medium, approximately 3 × 10 5Tumor cells were cultured in 6-well plates for 24 hours and then treated with DMSO or Olaparib. After 24 hours of incubation with DMSO or Olaparib, tumor cells were washed twice with PBS and cultured in DMEM containing 10% FBS for 48 hours. Tumor cell conditioned medium was collected by centrifugation at 1,500g for 5 minutes at 4°C to remove all debris and cells. For cytokine blockade experiments, monoclonal antibodies specific for each cytokine were added to the tumor cell conditioned medium and then applied to the macrophage cultures.

[0227] For ex vivo culture of human macrophages, human BMDMs were generated from bone marrow mononuclear cells (BMMs) obtained from STEMCELL Technologies (catalog no. 70001). Briefly, BMMs were cultured in DMEM containing 10% FBS, 55 μM 2-mercaptoethanol (catalog no. 21985023, Thermo Fisher), and 50 ng / ml M-CSF for 5-7 days, with medium changes every 3 days to obtain mature macrophages (BMDMs). BMDMs were further cultured in medium with or without the addition of 50% conditioned medium obtained from human ovarian cancer cell lines for 72 hours. Flow cytometry analysis was performed to analyze the macrophage phenotype for both mouse and human BMDMs.

[0228] Transcriptome analysis Transcriptome analysis of tumor samples: Total RNA was isolated from bulk tumors by RNeasy Plus mini kit (QIAGEN) and sequenced on the Ion Torrent platform (Thermo Fisher) using an Ion AmpliSeq custom panel targeting 4,604 mouse genes. Data were analyzed using the Torrent Suite and AmpliSeq RNA Analysis plugin (Thermo Fisher) to generate read counts per gene. Differential gene expression analysis was performed using DESeq2 with default parameters to obtain log2 fold changes (MAP) and adjusted p-values ​​(Benjamini-Hochberg procedure). Genes were ranked by log2 fold changes (MAP) and GSEA was performed using the GSEA prerank tool.

[0229] Transcriptome analysis of bone marrow cells: Approximately 1 × 10 6 PBM-R tumor cells were injected intraperitoneally into FVB / NJ mice. Approximately 2-3 weeks after injection, the mice were grouped and treated with control, olaparib, MSA-2, and a combination of MSA-2 and MSA-2 for 24 hours. After treatment, bone marrow cells (CD45 + CD11b + ) was isolated from the ascites of each mouse (n=3 per group). Total RNA was isolated and sequenced on the Ion Torrent platform (Thermo Fisher) as described above using the Ion AmpliSeq transcriptomic mouse gene expression panel. Gene Ontology (GO) analysis of DEGs was performed using the topGO package in R. GSEA analysis was performed as described above. Heatmaps showing changes in gene expression were generated using the heatmap.3 package in R.

[0230] Quantification and statistical analysis Statistical analysis was performed using Prism9 (Graphpad Software Inc.). Two conditions were compared using unpaired two-tailed Student's t-test for normally distributed data and Mann-Whitney nonparametric test for skewed data that deviated from normality. Three or more means were compared using one-way analysis of variance with Bonferroni post-hoc test for normally distributed data and Kruskal-Wallis nonparametric test for skewed data. Differences with P<0.05 were considered statistically significant.

[0231] Example 5: Targeting the tumor immune microenvironment to overcome resistance in lung cancer Osimertinib (AZD9291) is a third-generation EGFR tyrosine kinase inhibitor (TKI) for patients with non-small cell lung cancer (NSCLC) with EGFR activating mutations or acquired T790M mutations resistant to previous generation EGFR-TKIs. The emergence of resistance to osimertinib is inevitable, and overcoming such resistance remains a key challenge in the clinic. As provided herein, the inventors have used a syngeneic genetically engineered mouse (GEM) model of lung cancer driven by mutant EGFR to show that EGFR mutant tumors are highly sensitive to osimertinib in the early stages of tumor growth in a T cell-dependent manner, but become resistant as they progress. Furthermore, it has been shown that the presence of immunosuppressive tumor-associated macrophages (TAMs) renders tumors re...

Claims

1. A combination of drugs for use in a method for improving the efficacy of PARP inhibition in a subject having cancer, comprising (i) an effective amount of a STING agonist, and (ii) an effective amount of a PARP inhibitor, an effective amount of a TK inhibitor, and / or an effective amount of a DNA synthesis inhibitor. (i) the method comprises systemic delivery of the STING; (ii) the method comprises oral, intravenous, or intraperitoneal delivery; (iii) the STING agonist is a modified nucleotide STING agonist; and / or (iv) The pharmaceutical combination for use according to claim 1, wherein the STING agonist is selected from DMXAA, MSA-2, SR-717, FAA, CMA, α-mangostin, BNBC, DSDP, diABZI, bicyclic benzamides, and benzothiophenes.

3. (i) The PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, CEP9722, E7016, AG014699, MK4827, BMN-673, iniparib, and 3-aminobenzamide; (ii) the TKI inhibitor is an EGFR-TKI inhibitor and is selected from the group consisting of afatinib, dacomitinib, osimertinib, rociletinib (CO-1686), olmutinib (HM61713), nazartinib (EGF816), nacotinib (ASP8273), mavereltinib (PF-0647775), almonertinib, TY-9591, gefitinib, erlotinib, and AC0010; and / or (iii) 2. The pharmaceutical combination for use according to claim 1, wherein the DNA synthesis inhibitor is gemcitabine, sapacitabine, a cytidine analogue, cytarabine, tezacitabine, troxacitabine, DMDC, CNDAC, ECyD, clofarabine, or decitabine.

4. the method comprises administration of the STING agonist prior to the PARP inhibitor, the TK inhibitor, and / or the DNA synthesis inhibitor; or The pharmaceutical combination for use according to claim 1, wherein the method comprises administration of the STING agonist simultaneously with the PARP inhibitor, the TK inhibitor, and / or the DNA synthesis inhibitor.

5. The cancer is (i) includes tumors with an M2 enrichment score greater than 0.27; (ii) tumors with a TAM M2 / M1 ratio greater than 1; (iii) head and neck squamous cell carcinoma (HNSC), lung cancer, non-small cell lung cancer (NSCLC), lung squamous cell carcinoma (LUSC), liver cancer, hepatocellular carcinoma (HCC), colon cancer, prostate cancer, pancreatic cancer, cutaneous melanoma (SKCM), glioblastoma multiforme (GBM), breast invasive carcinoma (BRCA), lung adenocarcinoma (LUAD), renal clear cell carcinoma (KIRC), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), diffuse large B-cell lymphoma (DLBC), gastric adenocarcinoma (STAD), ovarian cancer, high-grade serous ovarian cancer (HGSOC), homologous recombination prognostic (HRP) ovarian cancer, or homologous recombination deficient (HRD) ovarian cancer; (iv) breast cancer harboring a BRCA mutation or advanced breast cancer harboring a germline BRCA1 / 2 mutation; (v) lung cancer containing an EGFR mutation or a non-small cell lung cancer containing an EGFR activating mutation; (vi) comprises a subpopulation of tumors with an M2 enrichment score greater than 0.27; (vii) comprises tumors that have achieved an M2 enrichment score of greater than 0.27 over the course of treatment; and / or (viii) A pharmaceutical combination for use as described in claim 1, wherein the tumor cell-specific STING is defective. (i) the subject is a rodent, primate, human, or animal model of cancer, and optionally, the subject is a human; (ii) the subject has a defect in activation of STING signaling in tumor cells; (iii) the method comprises administration of a PARP inhibitor at a dosage of 50 mg / kg body weight per day, administration of an EGFR-TKI inhibitor at a dosage of 5-50 mg / kg body weight per day, or administration of a DNA synthesis inhibitor at 2,000 mg / m2 per week; (iv) the method comprises administration of a STING agonist at a dosage of 10 mg / kg body weight per week; (v) the method comprises 2 to 3 administrations of a STING agonist; and / or (vi) A pharmaceutical combination for use according to any one of claims 1 to 5, wherein the method comprises an additional therapy.

7. The additional therapy is (i) Includes radiation therapy; (ii) chemotherapy, optionally comprising paclitaxel, platinum-based drugs, cisplatin, oxaliplatin, inhibitors of topoisomerase, etoposide, DNA intercalators, doxorubicin, DNA alkylating agents, or temozolomide; and / or (iii) a DNA damage response (DDR) targeting agent, optionally wherein the DDR targeting agent comprises ATMi, ATRi, CHK1 / 2i, or Wee1i.

8. A composition for use in a method for polarizing tumor-promoting macrophages into anti-tumor macrophages in a subject with cancer, the composition comprising an effective amount of a STING agonist.

9. A composition for use in a method for preventing or reversing drug resistance in a subject having cancer, said composition comprising an effective amount of a STING agonist, and said drug resistance is the result of polarization of anti-tumor macrophages into tumor-promoting macrophages.

10. (i) the STING agonist activates STING signaling in macrophages; (ii) the intratumoral STING agonist, optionally tumor cell cytoplasmic dsDNA / cGAMP or intratumorally delivered STING agonist, does not activate STING signaling in intratumoral dendritic cells; (iii) the subject has a defect in the STING signaling pathway in tumor cells; (iv) the tumor-promoting macrophages are M2-like; (v) the anti-tumor macrophages are M1-like; (vi) the method comprises systemic delivery of the composition; (vii) the method comprises oral, intravenous, or intraperitoneal delivery of the composition; (viii) the STING agonist is a modified nucleotide STING agonist; and / or (ix) The composition of claim 8 or claim 9, wherein the STING agonist is selected from DMXAA, MSA-2, SR-717, FAA, CMA, α-mangostin, BNBC, DSDP, diABZI, bicyclic benzamides, and benzothiophenes.

11. (i) the cancer comprises a tumor having an M2 enrichment score greater than 0.27; (ii) the cancer comprises head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), non-small cell lung cancer (NSCLC), liver cancer such as hepatocellular carcinoma (HCC), colon cancer, prostate cancer, pancreatic cancer, cutaneous melanoma (SKCM), glioblastoma multiforme (GBM), breast invasive carcinoma (BRCA), lung adenocarcinoma (LUAD), renal clear cell carcinoma (KIRC), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), diffuse large B-cell lymphoma (DLBC), gastric adenocarcinoma (STAD), or ovarian cancer such as high-grade serous ovarian cancer (HGSOC), ovarian cancer such as high-grade serous ovarian cancer (HGSOC), homologous recombination prostate cancer (HRP), or homologous recombination deficient (HRD) ovarian cancer; (iii) the cancer comprises a BRCA mutated breast cancer; (iv) the cancer comprises advanced breast cancer harboring a germline BRCA1 / 2 mutation; (v) the cancer comprises a lung cancer harboring an EGFR mutation, e.g., an activating EGFR mutation or a T790M mutation; (vi) the cancer comprises a non-small cell lung cancer harboring an EGFR mutation, e.g., an EGFR activating mutation or a T790M mutation; (vii) the drug resistance is resistance to PARP inhibition or resistance to EGFR-TK inhibition; (viii) the cancer comprises a subpopulation of tumors with an M2 enrichment score greater than 0.27; (ix) the cancer comprises a tumor that has achieved an M2 enrichment score greater than 0.27 over the course of treatment; (x) the subject is a rodent, primate, human, or animal model of cancer, and optionally, the subject is a human; (xi) the subject has a defect in the STING signaling pathway in tumor cells, such that an intratumoral STING agonist, optionally a dsDNA / cGAMP released from tumor cells or an intratumoral delivered STING agonist, is unable to activate intratumoral dendritic cells and macrophages; (xii) administration of the STING agonist is at a dosage of 10 mg / kg body weight per week; (xiii) the composition is administered two to three times; and / or (xiv) The composition of claim 8 or 9, wherein the method comprises an additional therapy.

12. The additional therapy is (i) comprises a PARP inhibitor, optionally wherein said PARP inhibitor is selected from the group consisting of olaparib, rucaparib, niraparib, talazoparib, veliparib, pamiparib, CEP9722, E7016, AG014699, MK4827, BMN-673, iniparib, and 3-aminobenzamide; (ii) comprising a TK inhibitor, optionally wherein the TK inhibitor is an EGFR-TK inhibitor selected from the group consisting of afatinib, dacomitinib, osimertinib, rociletinib (CO-1686), olmutinib (HM61713), nazartinib (EGF816), nacotinib (ASP8273), mavereltinib (PF-0647775), almonertinib, TY-9591, gefitinib, erlotinib, and AC0010; (iii) includes radiation therapy; (iv) chemotherapy, optionally comprising paclitaxel, platinum-based drugs, cisplatin, oxaliplatin, inhibitors of topoisomerase, etoposide, DNA intercalators, doxorubicin, DNA alkylating agents, or temozolomide; and / or 12. The composition of claim 11, wherein (v) the composition comprises a DNA damage response (DDR) targeting agent, and optionally the DDR targeting agent comprises ATMi, ATRi, CHK1 / 2i, or Wee1i.

13. A method for obtaining an M2 enrichment score for a tumor derived from a subject as an indicator for selecting a subject having cancer for treatment with a STING agonist, the method comprising: detecting the M2 enrichment score for a tumor derived from the subject; and selecting the subject if the M2 enrichment score is higher than 0.

27.

14. (i) the cancer comprises head and neck squamous cell carcinoma (HNSC), lung squamous cell carcinoma (LUSC), non-small cell lung cancer (NSCLC), liver cancer, hepatocellular carcinoma (HCC), colon cancer, prostate cancer, pancreatic cancer, cutaneous melanoma (SKCM), glioblastoma multiforme (GBM), breast invasive carcinoma (BRCA), lung adenocarcinoma (LUAD), renal clear cell carcinoma (KIRC), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), diffuse large B-cell lymphoma (DLBC), gastric adenocarcinoma (STAD), ovarian cancer, high-grade serous ovarian cancer (HGSOC), homologous recombination proliferative (HRP) ovarian cancer, or homologous recombination deficient ovarian cancer; (ii) the cancer comprises a BRCA mutated breast cancer; (iii) the cancer comprises advanced breast cancer harboring a germline BRCA1 / 2 mutation; (iv) the cancer comprises a lung cancer harboring an EGFR mutation, optionally an activating EGFR mutation or a T790M mutation; (v) the cancer comprises a non-small cell lung cancer harboring an EGFR mutation, optionally an activating EGFR mutation or a T790M mutation; (vi) the subject is a rodent, primate, human, or animal model of cancer, optionally wherein the subject is a human; and / or (vii) The method of claim 13, wherein the subject has a defect in activation of STING signaling in tumor cells.

15. A combination of drugs for use in treating a subject with advanced breast cancer harboring a germline BRCA1 / 2 mutation, the combination comprising (i) a STING agonist and (ii) a PARP inhibitor, wherein the cancer comprises a tumor with an M2 enrichment score greater than 0.27, and optionally wherein the STING agonist is for administration at about 10 mg / kg body weight and the PARP inhibitor is for administration at about 50 mg / kg body weight.

16. 1. A combination of drugs for use in treating a subject with non-small cell lung cancer harboring a germline EGFR mutation, said combination comprising (i) a STING agonist, and (ii) an EGFR-TK inhibitor, and said cancer comprising a tumor with an M2 enrichment score greater than 0.

27.

17. Use of a combination of drugs comprising (i) an effective amount of a STING agonist, and (ii) an effective amount of a PARP inhibitor, an effective amount of a TK inhibitor, and / or an effective amount of a DNA synthesis inhibitor, for the manufacture of a pharmaceutical agent for improving the efficacy of PARP inhibition in a subject having cancer.

18. Use of an effective amount of a STING agonist for the manufacture of a pharmaceutical agent for polarizing tumor-promoting macrophages into anti-tumor macrophages in a subject with cancer.

19. Use of an effective amount of a STING agonist for the manufacture of a pharmaceutical agent for preventing or reversing drug resistance in a subject having cancer, wherein the drug resistance is the result of polarization of anti-tumor macrophages into tumor-promoting macrophages.

20. Use of a combination of drugs comprising (i) a STING agonist and (ii) a PARP inhibitor for the manufacture of a medicament for treating a subject having advanced breast cancer harboring a germline BRCA1 / 2 mutation, wherein the cancer comprises a tumor having an M2 enrichment score greater than 0.27, and optionally, the STING agonist is for administration at about 10 mg / kg body weight and the PARP inhibitor is for administration at about 50 mg / kg body weight.

21. Use of a combination of drugs comprising (i) a STING agonist, and (ii) an EGFR-TK inhibitor, for the manufacture of a medicament for treating a subject with non-small cell lung cancer harboring a germline EGFR mutation, wherein the cancer comprises a tumor having an M2 enrichment score greater than 0.27.

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