Combination therapy for treating glioblastoma

Abstract

The present disclosure provides methods for the treatment of cancer characterized by rapid tumor cell proliferation and strong invasion to surrounding tissues using a synergistic combination of Compound 1, or a pharmaceutically acceptable salt thereof, with an anti-cancer therapy, the anti-cancer treatment is such as at least one kinase in RTK-PI3K-mTOR, at least one kinase in the MAPK signaling pathway, at least one PARP1 inhibitor or radiation of a therapeutic dose. The method results in tumor reduction significantly beyond the cumulative effect of the individual composition. # imgabs0 #

Description

[0001] Priority application

[0002] This application claims priority to U.S. patent application serial number 63 / 431,234, filed on December 8, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Government support statement

[0004] This invention was made with government support under Grant No. R01NS118513 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Cell migration is a fundamental step in cancer metastasis, and the mechanism of cell movement depends on the activity of the actomyosin network in cells. The actin cytoskeleton regulates cell polarity and adhesion, promoting the transformation of cells to malignant phenotypes (including invasion of adjacent tissues and metastasis). The main cellular actin-binding molecular motor protein, non-muscle myosin II, regulates and mechanically changes the microenvironment during cancer cell migration and tumor proliferation. Members of the myosin superfamily can act as enhancers or inhibitors of tumor progression, where the inactivation or upregulation of myosin is associated with malignant phenotypes, cancer cell migration and metastasis. Interestingly, knockdown of NMII leads to changes in gene expression associated with epithelial-to-mesenchymal transition (EMT), angiogenesis and aging (Halder D. et al., Nonmuscle myosin IIA and IIB differentially modulate migration and alter gene expression inprimary mouse tumorigenic cells. Molecular Biology of the Cell, 2019. 30 (12): 1463-1476). In summary, targeting non-muscle myosin II in cancer represents an important strategy for treating various cancers by reducing proliferation and metastasis (Naydenov NG, et al. Myosin Motors: Novel Regulators and Therapeutic Targets in Colorectal Cancer. Cancers. 2021. 13(4): 741; ZK. Xia, et al., Nonmuscle myosin IIa is associated with poor prognosis of esophagealsquamous cancer, Diseases of the Esophagus. 2012. (25)(5): 427–-436; Li Y., et al. Myosins as fundamental components during tumorigenesis: diverse and indispensable. Oncotarget. 2016; 7: 46785-46812).

[0006] Glioblastoma (GBM) is the most common and lethal primary glial tumor and resists successful treatments for other solid malignancies (Puduvalli, VK et al., Chemotherapy of High-Grade Astrocytomas in Adults. Prog Neurol Surg, 2018. 31: p. 116-144; Verhaak, RG, et al., Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell, 2010. 17(1): p. 98-110; Yuan, J., et al., Single-cell transcriptome analysis of lineage diversity in high-grade glioma. Genome Med, 2018. 10(1): p. 57; Neftel, C., et al., An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma. Cell, 2019. 178(4): p. 835-849e21; Wang, LB, et al., Proteogenomic and metabolomic characterization of human glioblastoma. Cancer Cell, 2021. 39(4): p. 509-528e20). Glioblastoma is an aggressive and malignant brain tumor that accounts for nearly half of all malignant brain tumors and originates from glial cells in the brain (NORD Rare Disease Report, Glioblastoma, last updated October 30, 2023, available at https: / / rarediseases.org / rare-diseases / glioblastoma-multiforme / ).Glioblastoma is the most common primary malignant brain cancer in adults, with an overall survival rate of ≤5% to 7.2% within 5 years after diagnosis (Batash, RN, et al., Glioblastoma Multiforme, Diagnosis and Treatment; Recent Literature Review. Curr Med Chemm_2017.24(27):3002-3009; Chien, LNet al., Comparative Brain and Central Nervous System Tumor Incidence and Survival between the United States and Taiwan(China) Based on Population-Based Registry. Front Public Health,2016.4:151; Ding, ZZ, et al., Leukemia-Associated Rho Guanine Nucleotide Exchange Factor and Ras Homolog Family Member C Play a Role in Glioblastoma Cell Invasion and Resistance. Am J Pathol,2020.190(10):2165-2176; Wu, W., et ... al., Glioblastoma multiforme (GBM): An overview of current therapies and mechanisms of resistance. PharmacolRes, 2021.171:105780; Ostrom, QT, et al., CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2014-2018. Neuro Oncol, 2021.23 (12Suppl 2):iii1-iii105).The standard of care includes maximum surgical resection, followed by radiation and temozolomide (TMZ) (alkylating chemotherapeutic agent) (Rahman, MI, et al., Selective Vulnerability of Senescent Glioblastoma Cells to BCL-XL Inhibition. Mol Cancer Res, 2022.20 (6): 938-948), and sometimes tumor-treating field (TTF). However, due to the high recurrence rate and aggressiveness of GBM, this care can only extend survival after initial diagnosis to about one year. Therefore, despite decades of research, no cure has been determined, and GBM remains an ultimately fatal disease.

[0007] Many factors contribute to GBM treatment failure. These include limited ability to resect the tumor due to extensive invasion of single tumor cells into adjacent normal brain, significant radioresistance of these invasive tumor cells, and the fact that more than half of all GBM patients do not respond to TMZ due to their genetic and epigenetic status (IDH wild-type and MGMT unmethylated) (Ding et al., 2020; Kamson, DO, et al., The Role of Temozolomide in Patients With Newly Diagnosed Wild-Type IDH, Unmethylated MGMTp Glioblastoma During the COVID-19 Pandemic. JAMA Oncol. 2021; 7(5): 675-–676). In addition, oncogenic kinases in GBM are often redundant, allowing cells to bypass the blockade by activating or upregulating another kinase, thereby overcoming the blockade of one kinase (Ivkovic, S., et al., Direct inhibition of myosin II effectively blocks gliomainvasion in the presence of multiple motogens. Mol Biol Cell, 2012. 23 (4): p. 533-42). In addition, GBM is an immunosuppressive tumor that evades immune responses through multiple mechanisms that constitute an obstacle to effective tumor vaccines (Frederico, SC, et al., Making a Cold Tumor Hot: The Role of Vaccines in the Treatment of Glioblastoma. Front Oncol, 2021. 11: p. 672508). In addition, although the blood-brain barrier (BBB) is leaky in certain areas of GBM, invasive tumor cells can exist in the normal brain and are surrounded by an intact BBB that protects the tumor cells from CNS non-permeable treatments. GBM tumors contain a subpopulation of cells with stem-like characteristics that resist DNA-damaging therapy, leading to inevitable relapse and death (Bao, S., et al., Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature, 2006. 444(7120): p. 756-60).Furthermore, two defining phenotypes of GBM are invasion and proliferation, also known as Go and Grow: GBM cells only perform one of these two. However, blocking one stimulates the other.

[0008] The prognosis for patients with recurrent high-grade glioma is extremely poor—if untreated, GBM is often fatal within three months of diagnosis—and there have been no new drugs for these patients since bevacizumab was approved in 2008. Survival rates for patients with GBM have not improved significantly across demographics over the past three decades (Tamimi, AF and Juweid, M., Epidemiology and Outcome of Glioblastoma. In: De Vleeschouwer S, editor. Glioblastoma [Internet]. Brisbane (AU): Codon Publications; 2017 Sep 27. Chapter 8. PMID: 29251870).

[0009] According to the National Brain Tumor Society, more than 14,490 people are expected to receive a GBM diagnosis in 2023. In the United States, the incidence of GBM is reported to be as high as 3.3 per 100,000 people, with a duration of 1.2 years, indicating that there are currently approximately 13,264 GBM patients in the United States, well below the 200,000 case threshold required for epidemiological criteria to define an orphan disease.

[0010] Patients with GBM experience two types of symptoms: systemic and localized (NORD Glioblastoma Report). Localized symptoms depend on the location and size of the tumor; for example, if the tumor is located in a functional area of the brain, the patient may have more problems speaking or understanding language (NORD Glioblastoma Report). However, according to the NORD Glioblastoma Report and AANS.org, common symptoms include headaches, seizures, nausea / vomiting, confusion, memory loss, muscle weakness, visual changes, blurred or distorted vision, speech defects, cognitive changes, and a decline in normal function.

[0011] Individuals suspected of having GBM typically undergo a comprehensive physical and neurological examination. The neurological examination assesses the patient's sensory and muscle response (NORD Glioblastoma Report). Contrast-enhanced magnetic resonance imaging (MRI) is often used to identify GBM; however, a tissue biopsy is required to make any definitive diagnosis (NORD Glioblastoma Report). GBM can be located anywhere in the brain and does not often spread outside the brain (NORD Glioblastoma Report). GBM tends to invade older individuals (45 to 70 years old) and rarely occurs in children (NORD Glioblastoma Report). The average age of diagnosis is 64 years old, and the rate is slightly higher in men than in women (NORD Glioblastoma Report). Caucasians have the highest GBM diagnosis rate compared to other racial groups (such as African Americans, Asians, and Native Americans) (NORD Glioblastoma Report).

[0012] The World Health Organization (WHO) divides GBM into four major categories: glioblastoma isocitrate dehydrogenase (IDH)-mutant, glioblastoma IDH-wild type, glioblastoma NOS (not otherwise specified), and not-elsewhere-classified (NEC) glioblastoma, as further detailed in Table 1 (Grochans, SA, et al., Epidemiology of Glioblastoma Multiforme-Literature Review. Cancers (Basel), 2022. 14(10):2412).

[0013] Table 1: WHO classification of GBM into four main categories

[0014]

[0015]

[0016] WHO = World Health Organization; IDH = isocitrate dehydrogenase; NOS = not otherwise specified; NEC = not classified

[0017] The Karnofsky performance status (KPS) score is used to determine functional ability, with a score of 100 indicating that the patient can perform all tasks normally with mild signs of illness, while a score of 50 indicates that the patient requires considerable assistance in daily activities (NORD Glioblastoma Report). O-6-methylguanine DNA methyltransferase (MGMT) is an enzyme responsible for DNA repair, and patients with GBM often undergo methylation profiling to determine MGMT status because it can affect the effectiveness of chemotherapy (NORD Glioblastoma Report). Individuals with a normal form (methylated or partially methylated) of MGMT tend to perform better during and after chemotherapy (NORD Glioblastoma Report). Furthermore, individuals with both wild-type IDH and unmethylated MGMT showed no benefit from TMZ, which affects quality of life and has immunosuppressive effects (Kamson and Grossman, 2021; Torre, MPY, et al., The predictive value of partial MGMT promoter methylation for IDH-wild-type glioblastoma patients, Neuro-Oncology Practice, 2023. 10(2): 126-–131). As a result, a shift away from TMZ treatment was observed in this group, which constitutes more than half of all GBM cases.

[0018] As mentioned above, standard treatment for primary GBM includes maximal safe surgical resection followed by radiation and TMZ (alkylating chemotherapeutic agent) (Rahman et al. 2022); treatment methods may also include TTF, which is alternating electric field therapy, to prevent cancer cell proliferation (NORD Glioblastoma Report). Bevacizumab and Gliadel wafers are FDA-approved chemotherapeutic agents for the treatment of GBM, but have shown limited success (NORD Glioblastoma Report).

[0019] GBM is one of the most complex, lethal, and difficult-to-treat cancers (National Brain Tumor Society). The significant unmet medical need for GBM treatment is underscored by a 5-year survival rate of less than approximately 5% (Batash et al. 2017; Chien et al., 2016; Ostrom et al., 2021), with no significant improvement in survival rates for decades (Tamimi and Juweid, 2017; National Brain Tumor Society). The National Brain Tumor Society estimates that GBM kills more than 10,000 people in the United States each year.

[0020] The median survival time for patients with GBM who receive a combination of surgery, chemotherapy, and radiation is 14.6 months (NORD Glioblastoma Report). Individuals with GBM IDH mutant protein have a higher overall survival rate than individuals with GBM IDH wild-type protein (NORD Glioblastoma Report).

[0021] The nonmuscle myosin II (NMII) family of molecular motors is an irreplaceable component of the mechanism that drives the invasive phenotype of tumor cell metastasis. They also play an important role in driving mitosis of tumor proliferation (Picariello, HS, et al. Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner. Proc Natl Acad Sci USA, 2019. 116 (31): 15550-15559; Ivkovic, S., et al. Direct Inhibition of myosin II effectively blocks glioma invasion in the presence of multiple mitogens. Mol Biol Cell, 2012. 23 (4): 533-42; Beadle, C., et al. The role of myosin II in glioma invasion of the brain. Mol Biol Cell, 2008. 19 (8): 3357-68). For example, in GBM, inhibition of NMIIA and / or NMIIB effectively blocks GBM dispersal, regardless of the activity of upstream and invasion-stimulating receptor tyrosine kinases (RTKs). Cytokinesis is the final stage of mitosis, which is blocked by deleting NMIIA and IIB together, and this has been shown to significantly prolong survival in genetically engineered mouse models (GEMMs) of GBM. Specifically, extensive genetic interventions have shown that simultaneous disruption of two non-muscle myosin II (NMII) molecular motors (NMIIA and IIB) meets the criteria for blocking GBM invasion and proliferation.In addition, NMII plays other non-canonical roles that are particularly relevant to cancer biology, including 1) regulation of ERK and SRC signaling and integrin and RTK function (Picariello, HS, et al. Myosin IIA suppresses glioblastoma development in a mechanically sensitive manner. Proc Natl Acad Sci USA, 2019. 116(31): 15550-15559; Rai, V., et al. Myosin IIA Heavy Chain Phosphorylation Mediates Adhesion Maturation and Protrusion in Three Dimensions. J Biol Chem, 2017. 292(8): 3099-3111; Morin, NA, et al. Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration. J Exp Med, 2008. 205: 195-205); 2) Regulation of p53 function in squamous cell carcinoma (Schramek, D. et al. Direct in vivo RNAiscreen unveils myosinIIa as a tumor suppressor of squamous cell carcinomas, Science, 2014. 343(6168): 309-13); and 3) Control of resistance to MEK / ERK inhibitors and control of mitochondrial fission and reactive oxygen species formation in melanoma (Orgaz, JLet al. Myosin II Reactivation and Cytoskeletal Remodeling as a Hallmark and a Vulnerability in Melanoma Therapy Resistance. Cancer Cell, 2020. 37, 85-103).

[0022] However, the translational potential of this research is limited by the lack of clinically safe CNS-penetrant small molecule inhibitors of NMII. To this end, compound 1, (S)-3a-hydroxy-6-methyl-1-(2-methylquinolin-6-yl)-1,2,3,3a-tetrahydro-4H-pyrrolo[2,3-b]quinolin-4-one:

[0023]

[0024] A well-tolerated, highly brain-penetrating dual small-molecule inhibitor of NMIIA and IIB was identified (WO 2019 / 241469, incorporated by reference in its entirety), which is needed for effective GBM treatment. Summary of the Invention

[0025] To overcome these challenges, the present disclosure provides methods for treating cancer in subjects with cancer. In some embodiments, the cancer is characterized by rapid tumor cell proliferation and aggressive invasion of surrounding tissues. The methods comprise administering to the subject a combination of Compound 1 or a pharmaceutically acceptable salt thereof and at least one anticancer treatment.

[0026]

[0027] As another embodiment, provided is a combination of Compound 1, or a pharmaceutically acceptable salt thereof, and at least one anti-cancer treatment for use in treating cancer characterized by rapid tumor cell proliferation and aggressive invasion of surrounding tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A and Figure 1B Dose response curves of mouse and human primary GBM cells to compound 1. Mouse proneural and mesenchymal GBM cell lines showed similar sensitivity to compound 1, with EC 50 Values ranged from 4 to 6 μM ( Figure 1A The corresponding dose response curves for 8 primary human GBM lines showed similar potency and efficacy for compound 1 ( Figure 1B ).

[0029] Figure 2A and Figure 2B The dose-response surface generated by MuSyC showed a synergistic interaction between compound 1 and sunitinib ( Figure 2A The synergistic dose range is indicated by the darker shading in the lower right corner of the face. The dose responses were fitted with the MuSyc algorithm, defining synergy in terms of potency (β) and valence (log(α12) and log(α21)). Figure 2B ).

[0030] Figure 3Kaplan Meier survival curves for Trp53(- / -) GEMMs treated with Compound 1, sunitinib, or the combination. Mice were treated with vehicle (-XXX), Compound 1 (----), sunitinib (XXXXXX), or the combination of Compound 1 and sunitinib (-) until onset of disease or 85 days after retroviral injection, whichever occurred first. Compound 1 or sunitinib vs. vehicle = p < 0.0001, log-rank test, with a 32% improvement in median survival; the combination of sunitinib and Compound 1 more than doubled median survival, with 35% of mice tumor-free at 100 days.

[0031] Figure 4A and Figure 4B Trp53-deficient (Trp53(- / -)) mouse GBM cells respond to 5μM compound 1 and sunitinib ( Figure 4A ) or saracatinib ( Figure 4B ) in vitro dose response (EC 50 Curve ) shows the synergistic effect between compound 1 and RTK inhibitors.

[0032] Figure 5 In vivo synergistic effect of paxalisib + compound 1. Genetically engineered mice harboring a floxed p53(- / -) allele were injected with PDGF-IRES-cre retrovirus and treated with vehicle, paxalisib, compound 1, or a combination of compound 1 and paxalisib until disease progression. The differences between paxalisib or compound 1 and vehicle, as well as between the combination and single-agent compound 1 or paxalisib, were highly significant (p < 0.0001, log-rank test).

[0033] Figure 6A and Figure 6B . Human PDX ( Figure 6A ) or p53-deficient GEMMs ( Figure 6B ) model was treated with daily doses of vehicle, compound 1, radiation therapy, or a combination of compound 1 and radiation therapy, followed by survival follow-up. The differences in median survival between vehicle, compound 1, radiation, and the combination were significant by log-rank test.

[0034] Figure 7A and Figure 7B Effects of compound 1 alone and in combination with everolimus in pancreatic adenocarcinoma cells (PANC-1; Figure 7A ) The Combination Index (CI) of Compound 1 and Everolimus in PANC-1 shows the synergistic effect of the combination ( Figure 7B; Fa = action level function).

[0035] Figure 8A and Figure 8B Effects of compound 1 alone and in combination with sunitinib in pancreatic adenocarcinoma cells (PANC-1; Figure 8A ) The combination index (CI) plot of compound 1 and sunitinib in PANC-1 shows the synergistic effect of the combination ( Figure 8B ; Fa = action level function).

[0036] Figure 9A and Figure 9B Effects of compound 1 alone and in combination with olaparib in pancreatic adenocarcinoma cells (PANC-1; Figure 9A ) The combination index (CI) plot of compound 1 and olaparib in PANC-1 showed the synergistic effect of the combination ( Figure 9B ; Fa = action level function).

[0037] Figure 10A and Figure 10B Effects of compound 1 alone and in combination with sunitinib in pancreatic adenocarcinoma cells (MIAPaCa-2; Figure 10A ) The combination index (CI) plot of compound 1 and sunitinib in PANC-1 shows the synergistic effect of the combination ( Figure 10B ; Fa = action level function).

[0038] Figure 11A and Figure 11B Effects of compound 1 alone and in combination with olaparib in pancreatic adenocarcinoma cells (MIAPaCa-2; Figure 11A ) The combination index (CI) plot of compound 1 and olaparib in PANC-1 showed the synergistic effect of the combination ( Figure 11B ; Fa = action level function).

[0039] Figure 12A and Figure 12B Effects of compound 1 alone and in combination with everolimus in TNBC cells (MDA-MB-468; Figure 12A ) Combination index (CI) plot of compound 1 and everolimus in MDA-MB-468 cells showed synergistic effect of the combination ( Figure 12B ; Fa = action level function). DETAILED DESCRIPTION

[0040] The present disclosure is based on the unexpected synergistic effect found in the combination of Compound 1 and at least one anticancer therapy. The combination can significantly prolong the survival of cancer patients beyond the sum of the survival achieved by Compound 1 and the anticancer therapy alone. As demonstrated throughout the various exemplary embodiments herein, the synergistic effect found in the combination is reflected in patient survival even after cessation of treatment and tumor elimination.

[0041] Because NMII drives cancer cell growth, pro-invasive signaling, and DNA damage response, NMII is an attractive but relatively unexplored target for treating aggressive tumors such as GBM. The present disclosure is based in part on the inhibition of two defining components of such tumors, invasion and proliferation, respectively, of which GBM is an exemplary phenotype (Ivkovic, S., et al. (2012); Dhruv, HD, et al., Reciprocal activation of transcription factors underlies the dichotomy between proliferation and invasion of glioma cells. PLoS One, 2013. 8 (8): p.e72134; Picariello, HS, et al (2019)). Inhibiting invasion stimulates proliferation, and vice versa (Dhruv, HD, et al (2013)), and therefore the most effective GBM treatment should block both. Established studies have shown that the NMII family of molecular motors plays an indispensable role in cell motility that drives invasion, mitosis, and proliferation (Picariello, HS, et al. (2019)). While targeting either NMIIA or IIB is sufficient to block GBM invasion, targeting both blocks GBM proliferation optimally (Ivkovic, S., et al. (2012); Picariello, HS, et al. (2019)), meaning that targeting both NMIIA and IIB reduces proliferation and invasion regardless of how many oncogenic signaling pathways are activated because NMII represents a point where these pathways converge (Ivkovic, S., et al. (2012); Picariello, HS, et al. (2019); Kenchappa, RS, et al., Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma. iScience, 2020. 23(12): p. 101802; Beadle, C., et al. (2008)). However, little attention has been paid to therapeutically targeting NMII given the view that such NMII inhibitors would be toxic. However, as shown herein, Compound 1, a CNS-penetrant small molecule inhibitor of both NMIIA and NMIIB, reaches therapeutic concentrations in the mouse brain and is well tolerated.

[0042] definition

[0043] As used herein, and unless otherwise clear from the context or indicated to the contrary, the term "compound" is inclusive, ie, it covers a compound or a pharmaceutically acceptable salt thereof.

[0044] Unless the context clearly dictates otherwise, nouns modified by a quantifier include one or more.

[0045] Some compounds described herein may exist in a variety of isomeric forms (including configurations, geometric isomers, and conformational isomers, including, for example, cis or trans conformations). Compounds may also exist in one or more tautomeric forms (including both single tautomers and mixtures of tautomers). The term "isomer" is intended to encompass all isomeric forms of the compounds of the present disclosure, including tautomeric forms of the compounds. The compounds of the present disclosure may also exist in open-chain or cyclized forms. In some cases, one or more of the cyclized forms may be produced by dehydration. The specific composition of the open-chain and cyclized forms may depend on how the compounds are separated, stored, or administered. For example, a compound may exist primarily in an open-chain form under acidic conditions, but cyclize under neutral conditions. All forms are included in the present disclosure.

[0046] Some compounds described herein may have an asymmetric center and therefore exist in different enantiomers and diastereoisomers. Compounds as described herein may be in the form of optical isomers or diastereoisomers. Therefore, the present disclosure encompasses compounds and their uses in the form of optical isomers, diastereoisomers, and mixtures thereof (including racemic mixtures) of the compounds as described herein. The optical isomers of the compounds in the present disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology, or by chemically separating stereoisomers using optically active resolving agents.

[0047] Unless otherwise indicated, the term "stereoisomer" means one stereoisomer of a compound that is substantially free of other stereoisomers of the compound. Thus, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound, or greater than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of other stereoisomers of the compound. The stereoisomers described above can be viewed as compositions comprising both stereoisomers present in their respective weight percentages as described herein.

[0048] If there is a discrepancy between the structure shown and the name given to that structure, the structure shown controls. Additionally, if the stereochemistry of a structure or portion of a structure is not indicated by, for example, bold or dashed lines, that structure or portion of that structure should be interpreted as encompassing all stereoisomers thereof. However, in some cases, where there is more than one chiral center, the structure and name may be indicated as a single enantiomer to aid in describing the relative stereochemistry. One skilled in the art of organic synthesis will know whether a compound is prepared as a single enantiomer by the process used to prepare it.

[0049] In various embodiments, the term "synergistic effect" may refer to a survival benefit resulting from a combination of Compound 1 and an anticancer treatment that is greater than the sum of the survival benefits resulting from the individual Compound 1 and anticancer treatments. In some embodiments, synergy is assessed in terms of the benefit it produces for a particular subject. In other embodiments, synergy is assessed by measuring the survival benefit in a population of subjects and then calculating the average or mean survival benefit for the population. The population of subjects can be selected by common diagnosis of a particular cancer (e.g., GBM), cancer stage, prognosis, and combinations thereof.

[0050] In this disclosure, a "pharmaceutically acceptable salt" is a pharmaceutically acceptable organic or inorganic acid or base salt of a compound described herein. Representative pharmaceutically acceptable salts include, for example, alkali metal salts, alkaline earth metal salts, ammonium salts, water-soluble and water-insoluble salts such as acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glucoheptonate, gluconate, glutamate, glycolyllarsanilate, hexafluorophosphate, hexylresorcinol salt, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothiocyanate (i Pharmaceutically acceptable salts include, but are not limited to, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate In this case, the pharmaceutically acceptable salt may have multiple counterions.Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0051] The term "treat" and variations thereof refer to the improvement or eradication of a disease or symptoms associated with a disease. In various embodiments, the term refers to minimizing the spread or worsening of a disease resulting from the administration of one or more prophylactic or therapeutic compounds described herein to a patient suffering from such a disease.

[0052] The term "prevent," "prevent," and variations thereof refer to preventing the onset, recurrence, or spread of a disease in a patient as a result of the administration of one or more prophylactic or therapeutic compounds described herein.

[0053] The term "effective amount" refers to an amount of a compound or other active ingredient as described herein that is sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease, or to delay or minimize symptoms associated with a disease. Additionally, a therapeutically effective amount with respect to a compound as described herein means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. When used in combination with a compound as described herein, the term can encompass an amount that improves overall therapy, alleviates or avoids symptoms or causes of a disease, or enhances the therapeutic efficacy of another therapeutic agent or acts synergistically with another therapeutic agent.

[0054] "Patient" or "subject" includes animals, such as humans, cows, horses, sheep, lambs, pigs, cats, dogs, mice, rats, rabbits, or guinea pigs. According to some embodiments, the animal is a mammal, such as non-primates and primates (e.g., monkeys and humans). In one embodiment, the patient is a human, such as a human infant, child, adolescent, or adult. In this disclosure, the terms "patient" and "subject" are used interchangeably.

[0055] How to use

[0056] In some embodiments, the present disclosure provides a method for treating cancer in a subject suffering from cancer. Cancer is a cancer characterized by rapid tumor proliferation and aggressive invasion of surrounding tissues. In some embodiments, cancer can be defined as a cancer in which NMIIA or NMIIB is expressed at a higher level relative to healthy cells (i.e., non-cancerous cells, such as those adjacent to cancer). See, for example, P.Zhou et al., Oncogene 38 (2019) 5500-5515. In some embodiments, the NMIIA or NMIIB expression level that meets this standard is typically elevated, such as about twice as high or more than the level observed in healthy tissue. In other embodiments, regardless of the expression level of NMIIA or NMIIB, the activity of constitutively activated NMIIA or NMIIB in cancer can be increased or decreased relative to healthy tissue, such as by post-translational modification (PTM) including abnormal phosphorylation and mutation. See Nevitt, C. et al., N-terminal acetylation and methylation differentially affect the function of MYL9. Biochem J 2018;475(20):3201-–3219.

[0057] In various embodiments, cancers treatable by the methods described herein include glioblastoma (GBM), pancreatic adenocarcinoma, triple negative breast cancer (TNBC), bile duct cancer, head and neck squamous cell carcinoma, testicular germ cell tumors, hepatocellular carcinoma, gastric adenocarcinoma, renal chromophobe cell carcinoma, esophageal cancer, brain low-grade glioma, ovarian serous cystadenocarcinoma, thyroid cancer, bladder urothelial carcinoma, skin melanoma, renal clear cell carcinoma, prostate adenocarcinoma, cervical squamous cell carcinoma, adrenal cortical carcinoma, lung adenocarcinoma, rectal adenocarcinoma, lung squamous cell carcinoma, invasive breast cancer, colon adenocarcinoma, uterine corpus endometrial cancer, pheochromocytoma and paraganglioma, renal papillary cell carcinoma, uterine carcinosarcoma, acute myeloid leukemia, lymphoid neoplasms diffuse large B-cell lymphoma, mesothelioma, oropharyngeal squamous cell carcinoma, sarcoma, thymoma, and uveal melanoma.

[0058] In one embodiment, the cancer is GBM. In another embodiment, the cancer is TNBC. In yet another embodiment, the cancer is pancreatic adenocarcinoma. In another embodiment, the cancer is carcinoma or adenocarcinoma. In another embodiment, the adenocarcinoma is lung adenocarcinoma. In another embodiment, the cancer is squamous cell carcinoma of the lung or invasive breast carcinoma.

[0059] The method comprises administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, in combination with at least one anti-cancer therapy.

[0060]

[0061] In various embodiments, Compound 1 is administered as a pharmaceutically acceptable salt. In an exemplary embodiment, the pharmaceutically acceptable salt is the monohydrochloride salt.

[0062] In some embodiments, the anti-cancer treatment is selected from:

[0063] (a) at least one inhibitor of at least one kinase in the RTK-PI3K-mTOR signaling pathway, or a pharmaceutically acceptable salt thereof,

[0064] (b) at least one inhibitor of at least one kinase in the mitogen-activated protein kinase (MAPK) signaling pathway, or a pharmaceutically acceptable salt thereof,

[0065] (c) inhibitors of poly(ADP-ribose) polymerase 1 (PARP1), and

[0066] (d) Therapeutic doses of radiation.

[0067] Inhibitors of kinases in the RTK-PI3K-mTOR signaling pathway

[0068] In some embodiments, the anticancer treatment is at least one inhibitor of at least one kinase in the RTK-PI3K-mTOR signaling pathway, or a pharmaceutically acceptable salt thereof. In one embodiment, the kinase in the RTK-PI3K-mTOR signaling pathway is a receptor tyrosine kinase (RTK), and the inhibitor is an RTK inhibitor. In various other embodiments, Compound 1 is administered in combination with one or more RTK inhibitors disclosed generally and specifically herein.

[0069] According to the understanding of the art, RTK inhibitors refer to substances that block or otherwise inhibit the enzymatic activity of receptor tyrosine kinases or the activity of multiple receptor tyrosine kinases, such as small molecules. In multiple embodiments, RTK inhibitors inhibit two or more receptor tyrosine kinases, three or more receptor tyrosine kinases or four or more receptor tyrosine kinases. In some embodiments, more than one RTK inhibitor is administered to the subject. Therefore, for example, multiple RTK inhibitors are selected to inhibit multiple receptor tyrosine kinases. In some exemplary embodiments, the RTK inhibitors suitable for methods described herein are RTK inhibitors that exhibit at least some brain permeability, CNS permeability or both brain permeability and CNS permeability.

[0070] In various embodiments, the RTK inhibitor is an RTK inhibitor that targets one or more receptor tyrosine kinases from at least one of the following recognized kinase classes:

[0071] RTK class I (EGF receptor family; ErbB family);

[0072] RTK class II (insulin receptor family);

[0073] RTK class III (PDGF and c-KIT receptor family);

[0074] RTK class IV (VEGF receptor family);

[0075] RTK class V (FGF receptor family);

[0076] RTK class VI (CCK receptor family);

[0077] RTK class VII (NGF receptor family);

[0078] RTK class VIII (HGF receptor family);

[0079] RTK class IX (Eph receptor family);

[0080] RTK class X (AXL receptor family);

[0081] RTK class XI (TIE receptor family);

[0082] RTK class XII (RYK receptor family);

[0083] RTK class XIII (DDR receptor family);

[0084] RTK class XIV (RET receptor family);

[0085] RTK class XV (ROS receptor family);

[0086] RTK class XVI (LTK receptor family);

[0087] RTK class XVII (ROR receptor family);

[0088] RTK class XVIII (MuSK receptor family); and

[0089] RTK class XIX (LMR receptors).

[0090] Many RTK inhibitors are known in the art and are suitable for use in the methods described herein. Thus, in various embodiments, Compound 1 is administered in combination with one or more exemplary RTK inhibitors selected from the group consisting of fasudil (eril), sirolimus (rapamune), imatinib, gefitinib, erlotinib, sorafenib, sunitinib, sacatinib, dasatinib, lapatinib, nilotinib, temsirolimus, and temsirolimus. s), everolimus, pazopanib, ruxolitinib, vandetanib, vemurafenib, crizotinib, icotinib, axitinib, tofacitinib, bosutinib, cabozantinib, ponatinib, regorafenib, afatinib, dabrafenib ib), trametinib, ibrutinib, nintedanib, idelalisib, alectinib, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, bosutinib, dacomitinib, neratinib, osimertinib, gilteritinib, midostaurin midostaurin, erdafitinib, larotrectinib, axitinib, carbozantinib, lenvatinib, encorafenib, acalabrutinib, binimetinib, cobimetinib, abemaciclib, palbociclib and ribociclib, and their salts and / or hydrates.RTK inhibitors can inhibit one or more of RTK classes I to XIX. The selection of RTK inhibitors can be guided by genetic profiling of glioblastoma. Alternatively, the selection of RTK inhibitors can be guided by the expected side effects in the patient. In one embodiment, the RTK inhibitor is sunitinib or a salt thereof.

[0091] In other embodiments, the kinase in the RTK-PI3K-mTOR signaling pathway is phosphoinositide 3-kinase (PI3K), and the inhibitor is a PI3K inhibitor. In various embodiments, Compound 1 is administered in combination with one or more specific examples of PI3K inhibitors suitable for use in the methods described herein, including idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, leniolisib, buparlisib, dactolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatol ... isib), linperlisib, nemiralisib, pictilisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, tenalisib, voxtalisib, AMG319, AZD8186, GSK2636771, SF1126, LY294002, acalisib, omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, hibiscone C, IC87114, PI-103, Wortmannin, pharmaceutically acceptable salts thereof, and combinations thereof. In a specific embodiment, the PI3K inhibitor is paxalisib.

[0092] In other embodiments, the kinase in the RTK-PI3K-mTOR signaling pathway is a mechanistic target of rapamycin (mechanistic target of rapamycin, mTOR) kinase, and the inhibitor is an mTOR inhibitor. In multiple embodiments, compound 1 is administered in combination with one or more specific examples of an mTOR inhibitor, and the mTOR inhibitor includes rapamycin (rapamycin), temsirolimus, everolimus, ridaforolimus (ridaforolimus), sirolimus (sirolimus), ridaforolimus, umirolimus (umirolimus), zotarolimus (zotarolimus), torin-1, torin-2, vistusertib (vistusertib), its pharmaceutically acceptable salts and combinations thereof. In one embodiment, the mTOR inhibitor is everolimus.

[0093] In various embodiments, the anticancer treatment is at least one inhibitor of at least one kinase in the MAPK signaling pathway, or a pharmaceutically acceptable salt thereof. In some embodiments, the kinase in the MAPK signaling pathway is a Src kinase, and the inhibitor is a Src inhibitor. In various embodiments, Compound 1 is administered in combination with one or more exemplary Src inhibitors, including KX2-391, bosutinib, saracatinib, dasatinib, PP1, PP2, pharmaceutically acceptable salts thereof, and combinations thereof. In one embodiment, the Src inhibitor is saracatinib.

[0094] In other embodiments, the kinase in the MAPK signaling pathway is mitogen-activated protein kinase (MEK), and the inhibitor is a MEK inhibitor. In various embodiments, Compound 1 is administered in combination with one or more specific examples of MEK inhibitors, including bimetinib, cobimetinib, selumetinib, trametinib, pharmaceutically acceptable salts thereof, and combinations thereof.

[0095] In other embodiments, the kinase in the MAPK signaling pathway is an extracellular signal-regulated kinase (ERK), and the inhibitor is an ERK inhibitor. In multiple embodiments, Compound 1 is administered in combination with one or more specific examples of an ERK inhibitor, including ravoxertinib, MK-8353, ulixertinib, temuterkib, KO-947, CC-90003, ONC201, tizaterkib, SCH772984, pluripotin, VX-lle, DEL-22379, FR 180204, ERK5-IN-1, pharmaceutically acceptable salts thereof, and combinations thereof.

[0096] In various embodiments, the anticancer treatment is a PARP1 inhibitor. In various embodiments, Compound 1 is administered in combination with one or more specific examples of PARP1 inhibitors, including rucaparib, iniparib, olaparib, veliparib, niraparib, talazoparib, CEP-9722, and E7016. In one illustrative embodiment, the PARP1 inhibitor is olaparib.

[0097] In various embodiments, anticancer therapy can be selected based on established standards of care for a particular cancer. Thus, for example, in embodiments where the cancer is pancreatic cancer, the anticancer therapy combined with Compound 1 can be selected from paclitaxel, fluorouracil (5fu), capecitabine, gemcitabine hydrochloride, irinotecan hydrochloride liposomes, mitomycin, everolimus, erlotinib, olaparib, and sunitinib. In other embodiments, where the cancer is pancreatic cancer, the anticancer therapy combined with Compound 1 can be selected from everolimus, sunitinib, and olaparib.

[0098] In other embodiments, wherein the cancer is TNBC, the anticancer treatment combined with Compound 1 can be selected from fluorouracil (5FU); anthracyclines such as doxorubicin; alkylating agents such as cyclophosphamide; taxanes such as taxol and taxotere; paclitaxel, decetaxel, doxorubicin, epirubicin; ADC sacituzumab; PARP inhibitors such as olaparib and talazoparib; growth factor inhibitors such as lapatinib, gefitinib and cetuximab; mTor inhibitors such as rapamycin; Abl / Src inhibitors such as dasatinib; anti-androgen receptor agonists such as sirolimus. receptor (AR) treatment, such as bicalutamide and enzalutamide; immune checkpoint inhibitors targeting PDL1 or PD1, such as atezolizumab and pembrolizumab; and platinum-based chemotherapy, such as cisplatin. In other embodiments, where the cancer is TNBC, the anticancer treatment in combination with Compound 1 can be selected from everolimus and olaparib.

[0099] In other embodiments, wherein the cancer is GBM, the anti-cancer treatment combined with Compound 1 may be selected from paxalisib and sunitinib.

[0100] In various embodiments, the anti-cancer treatment is a therapeutic dose of radiation. According to established practice in the art, and as used herein, the term "radiation" refers to radiation therapy that entails the use of directed X-rays or subatomic particles, which are primarily used in cancer management in both therapeutic and palliative settings. In various embodiments, the radiation is administered externally or internally. In one embodiment, the radiation is administered as external beam radiation, in which a radiation source external to the patient emits energy that is focused and shaped to a target of interest. In another embodiment, the radiation occurs in brachytherapy, in which a naturally occurring radioactive source is placed that decays over time and produces a high dose of radiation at a focal area.

[0101] In some embodiments, the ionizing radiation is in the form of photons. In other embodiments, where it is desired to increase the radiation dose close to the skin, the ionizing radiation is in the form of electrons. In other embodiments, the radiation is in the form of particles (including protons, carbon ions, and neutrons). One skilled in the art will determine how and where the radiation dose is deposited in tissues according to sound medical practice and can use knowledge of these patterns to limit the dose to normal structures, thereby improving the therapeutic window.

[0102] Compared with normal cells that quickly repair double-strand breaks, the damaged DNA repair mechanism in cancer cells makes cancer cells susceptible to radiation. Therefore, in one embodiment, the total radiation dose can be applied by a fractionated approach, that is, the total radiation dose is divided into multiple daily treatments: DNA damage in normal cells is repaired between treatments, while damage to cancer cells accumulates over time, causing cancer cells to die preferentially. Both dose / fraction and total dose affect the response of tumors and normal tissues. Generally speaking, the lower the daily dose of radiation, the less likely it is to cause toxicity. Therefore, according to reasonable treatment practice, technicians reach a balance between daily doses that are low enough to avoid damaging normal tissues but high enough to cause cancer cell death. In some exemplary embodiments, the therapeutic dose of radiation can be 180 to 200 cGy / day.

[0103] Application

[0104] Anticancer therapy and compound 1 include combination therapies for treating subjects with cancers described herein. In some embodiments, anticancer therapy and compound 1 are administered to the subject on the same day. In some embodiments, compound 1 is administered at a certain time of day, such as in the morning, followed by administration of anticancer therapy at another certain time of day, such as in the afternoon, or vice versa. In other embodiments, compound 1 and anticancer therapy are administered within a prescribed time interval (e.g., one hour) of each other. In other embodiments, for example, when the anticancer therapy is a chemotherapeutic agent administered by iv route, IP route, PO route, or subcutaneous route, the anticancer therapy is administered simultaneously with compound 1. In other embodiments, compound 1 and anticancer therapy are administered by different routes. For example, in one embodiment, compound 1 is administered by subcutaneous route. In other embodiments, anticancer therapy and compound 1 are administered sequentially, typically with an interval of 15 to 60 minutes between administrations to monitor the reaction. Other embodiments are more specific combinations of compound 1 and anticancer therapy, combined with any embodiment described herein for administration, administration of a combination to a subject, and treatment endpoints. Thus, in various embodiments, combinations include Compound 1 and tamoxifen, Compound 1 and olaparib, Compound 1 and everolimus, Compound 1 and sunitinib, and Compound 1 and paxalisib.

[0105] Many dosing regimens provide variations on the combination therapy. For example, in some embodiments, each of the anticancer therapy and Compound 1, including specific combinations thereof described herein, is administered to a subject once daily for 4 days a week, 5 days a week, or 6 days a week. The anticancer therapy and Compound 1 can be administered on the same day, every other day, and combinations thereof. In some embodiments, the anticancer therapy and Compound 1 are each administered to a subject on each of the seven days a week.

[0106] Some embodiments define treatment endpoints, which may vary according to several factors, including the age and overall health of the subject, the presence of comorbidities, the time of cancer diagnosis relative to the start of therapeutic intervention, and the severity of the cancer. In some embodiments, the treatment endpoint is that the survival of the subject substantially exceeds the survival that would otherwise be caused by no treatment or by a single treatment with a separate compound 1 or a single anticancer treatment. All combinations of compound 1 and anticancer treatments are contemplated, including those described in more detail herein. In other embodiments, the treatment endpoint is that the survival of the subject exceeds the survival that would otherwise be expected by the cumulative effect of a single treatment with compound 1 and an anticancer treatment. In one embodiment, the term "subject" as used herein may refer to a specific individual. In another embodiment, the term may refer to a population of individuals for which a mean or average treatment endpoint may be determined.

[0107] In some exemplary embodiments, where the cancer is GBM, resection and radiation as treatment result in an average survival rate of 12 to 18 months for most patients. Those patients with recurrent glioblastoma taking RTK inhibitors (e.g., sunitinib) see little additional survival advantage. Thus, in one embodiment, the addition of compound 1 according to the methods described herein increases the average life expectancy of patients after resection and radiation to at least 15 months, 18 months, or two years.

[0108] In other embodiments, the therapeutic endpoint for treatment with the combination is that the subject survives for about twice or more the duration of survival of a subject administered either the anti-cancer therapy or Compound 1 alone. In other embodiments, the therapeutic endpoint is cancer tumor incidence, subject survival beyond the time of cessation of the combination treatment, or both.

[0109] Other advantages of the present disclosure arise from the unexpected synergistic effects of the combination of Compound 1 and the anticancer therapy used in the methods described herein. Thus, in some embodiments, the dose of the anticancer therapy or Compound 1 in the combination is less than the dose of the anticancer therapy or Compound 1 required to achieve the same therapeutic endpoint when administered alone to a subject. The ability to utilize lower doses of Compound 1 or the anticancer therapy, or both, can minimize or eliminate any toxicity to the subject.

[0110] Pharmaceutical composition

[0111] The present disclosure also provides pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds described herein or pharmaceutically acceptable salts thereof, stereoisomers and / or tautomers mixed with a pharmaceutically acceptable carrier. In some embodiments, according to the approved practice of pharmaceutical compounding, the composition further comprises one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers and / or flavoring agents.

[0112] The pharmaceutical compositions of the present disclosure are formulated, dosed, and applied in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific subject being treated, the subject's clinical condition, the cause of the condition, the site of delivery of the medicament, the method of administration, the timing of administration, and other factors known to medical practitioners.

[0113] The "therapeutically effective amount" of the compound or its pharmaceutically acceptable salt, stereoisomer and / or tautomer administered is determined by such considerations. A therapeutically effective amount may be less than an amount that is toxic to normal cells or the subject as a whole. Generally speaking, a therapeutically effective amount is the minimum amount of Compound 1 necessary to inhibit NMIIA, inhibit NMIIB, exhibit toxicity to cancer cells, and combinations thereof. In various embodiments, the therapeutically effective amount of Compound 1 is an amount of about 0.1 mg / kg to about 15 mg / kg, about 0.5 mg / kg to about 13 mg / kg, about 1 mg / kg to about 11 mg / kg, or about 5 mg / kg to about 10 mg / kg based on the subject's body weight. In other embodiments, the therapeutically effective amount of Compound 1 is about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, or about 15 mg / kg. Thus, in some illustrative embodiments, the combination includes a therapeutically effective amount of Compound 1 as described herein and anticancer therapy (a), anticancer therapy (b), anticancer therapy (c), or anticancer therapy (d). Some embodiments include specific combinations of the anti-cancer therapies described herein and Compound 1, including Compound 1 and tamoxifen, Compound 1 and olaparib, Compound 1 and everolimus, Compound 1 and sunitinib, and Compound 1 and paxalisib.

[0114] A therapeutically effective amount for anticancer therapy is the minimum amount necessary to exert oncology and / or immuno-oncology therapeutic activity, inhibit one or more receptor tyrosine kinases or other kinases described herein, and combinations thereof. In various embodiments, a therapeutically effective amount for anticancer therapy in combination with Compound 1, as generally and specifically described herein, is an amount of about 0.1 to about 200 mg / kg, about 0.5 to about 150 mg / kg, about 1 to about 125 mg / kg, about 2 to about 110 mg / kg, about 5 mg / kg to about 100 mg / kg, about 10 mg / kg to about 80 mg / kg, about 15 mg / kg to about 60 mg / kg, about 20 mg / kg to about 50 mg / kg, or about 30 mg / kg to about 40 mg / kg based on the subject's body weight. In other embodiments, the therapeutically effective amount for anti-cancer treatment is about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, or about 60 mg / kg.

[0115] In some exemplary embodiments, unit dosage forms include intraperitoneal (IP) injection, intravenous (IV) injection, subcutaneous (SQ) injection, and oral (PO) dosage forms. The dosage form may contain about 0.1 mg to about 1000 mg, about 50 mg to about 500 mg, about 25 mg to about 200 mg, or about 10 mg to about 100 mg of Compound 1 or anticancer therapy, or each of Compound 1 or anticancer therapy mixed with each other. In any of the foregoing embodiments, optionally in combination with any other embodiment described herein, the dosage form can be administered once a day, twice a day, or three times a day.

[0116] The compositions of the present disclosure may be administered orally, parenterally, by inhalation or spray, or rectally in dosage unit formulations.The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

[0117] Suitable oral compositions as described herein include, but are not limited to, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs.

[0118] In another aspect, also contemplated are pharmaceutical compositions suitable for a single unit dose comprising a compound of the present disclosure, or a pharmaceutically acceptable stereoisomer, salt, or tautomer thereof, and a pharmaceutically acceptable carrier.

[0119] Compositions of the present disclosure suitable for oral use can be prepared according to any method known in the art for preparing pharmaceutical compositions. For example, liquid formulations of compounds of the present disclosure include one or more agents selected from sweeteners, flavorings, coloring agents, and preservatives to provide pharmaceutically palatable formulations of compounds of the present disclosure.

[0120] For tablet compositions, the compounds of the present disclosure are mixed with non-toxic pharmaceutically acceptable excipients for tablet preparation. Examples of such excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating agents and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or coated using known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained therapeutic effect over a desired time period. For example, a time-delaying agent such as glyceryl monostearate or glyceryl distearate may be used.

[0121] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or medium-chain oils).

[0122] For aqueous suspensions, the compounds of the present disclosure are mixed with excipients suitable for maintaining a stable suspension (e.g., wherein no compound degradation or precipitation occurs). Examples of such excipients include, but are not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth, and gum arabic.

[0123] Oral suspensions may also include a dispersant or wetting agent, such as a naturally occurring phospholipid, such as lecithin, or a condensation product of an alkylene oxide with a fatty acid, such as polyoxyethylene stearate, or a condensation product of an alkylene oxide with a long-chain fatty alcohol, such as heptadecanol, or a condensation product of an alkylene oxide with a partial ester derived from a fatty acid and a hexitol, such as polyoxyethylene sorbitol monooleate, or a condensation product of an alkylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, such as polyethylene sorbitan monooleate. Aqueous suspensions may also include one or more preservatives, such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin.

[0124] Oily suspensions can be formulated by suspending a compound of the present disclosure in a vegetable oil (such as peanut oil or medium chain oil) or in a mineral oil (such as liquid paraffin). Oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol.

[0125] Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0126] Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide a compound of the present disclosure mixed with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweeteners, flavorings, and coloring agents, may also be present.

[0127] The pharmaceutical composition of the present disclosure can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil (e.g., olive oil or peanut oil), or a mineral oil (e.g., liquid paraffin), or a mixture thereof. Suitable emulsifiers can be naturally occurring gums (e.g., gum arabic or tragacanth), naturally occurring phosphatides (e.g., soybean, lecithin), and esters or partial esters (e.g., condensation reaction products of sorbitan monooleate and the partial ester with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate)) derived from fatty acids and hexitol anhydrides. Emulsions can also include sweeteners and flavorings.

[0128] Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain a demulcent, a preservative and flavoring and coloring agents.

[0129] The pharmaceutical composition can be in the form of a sterile injectable, aqueous suspension or oily suspension. The suspension can be prepared according to known techniques using those suitable dispersants or wetting agents and suspending agents mentioned above. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, for example as solutions in 1,3-butanediol. Among acceptable carriers and solvents, water, Ringer's solution and isotonic sodium chloride solution can be used. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids (such as oleic acid) can be used in the preparation of injectables.

[0130] Compositions for parenteral administration are administered in a sterile medium. Depending on the carrier and concentration (concentration of the drug in the preparation) used, parenteral formulations can be suspensions or solutions containing dissolved drugs. Excipients (e.g., local anesthetics, preservatives, and buffers) can also be added to parenteral compositions.

[0131] Example

[0132] The following non-limiting and illustrative examples constitute further embodiments of the present disclosure.

[0133] Example 1: Combined deletion of NMIIA and IIB severely impairs GBM tumorigenesis and prolongs survival in a genetically engineered model (GEMM) of the disease.

[0134] A virally induced mouse model of proneural, IDH wild-type, unmethylated GBM has been described (Lei L, et al., Glioblastoma models reveal the connection between adult glial progenitors and the proneural phenotype. PLoS One. 2011; 6(5): e20041). Injection of a bicistronic retrovirus encoding HA-tagged PDGF and Cre recombinase into the white matter of tumor suppressor (Pten or Trp53) conditional knockout (cKO) mice resulted in infection of glial progenitor cells, recombination of the cKO allele, and malignant transformation with 100% penetrance (Lei L, et al. 2011). Comparison of Kaplan Meier survival curves of Pten-deficient GBM mice with Pten, Myh9 (NMIIA) and Myh10 (NMIIB) cKO mice showed that co-deletion of NMIIA and IIB prevented tumorigenesis and significantly improved long-term survival.

[0135] Example 2: Compound 1 is a CNS penetrant and a well-tolerated dual inhibitor of NMIIA and IIB.

[0136] Blebbistatin is an NMII allosteric inhibitor (Straight, AF, et al., Dissecting temporaland spatial control of cytokinesis with a myosin III inhibitor. Science, 2003. 299 (5613): p. 1743-7), due to its potent inhibition of cardiac myosin II (CMII), poor tolerance is shown at a dose as low as 0.5 mg / kg (IV). In view of the high sequence homology in the residues lining the (line) blebbistatin binding site in NMII and CMII, it is believed that selective targeting of NMII is thorny. However, compound 1 shows high brain penetrability, is stronger than CMII in the selectivity characteristics of NMII, and importantly, is identical in potency for both NMII and IIB (WO 2019 / 241469), which is a potential feature of suppressing invasion and proliferation. Although compound 1 exhibits some skeletal (SkMII) and smooth (SmMII) muscle myosin inhibition, safety testing has identified CMII as a key target for toxicity. Additional in vitro DMPK data indicate that, although bound to proteins, compound 1 is highly cell permeable and is not a P-glycoprotein (Pgp) substrate, which is consistent with the brain / plasma ratio in mice (blebbistatin (0.9), compound 1 (2.3) at 10 mg / kg). Compound 1 at concentrations >30 μM is also nontoxic to healthy cells in culture.

[0137] Example 3: Combined inhibition of NMIIA and IIB with Compound 1 is cytotoxic to GBM cells in vitro.

[0138] Dose-response studies were performed by treating murine and human primary GBM cell lines in vitro for 72 hours and measuring cell viability. Figure 1A Compound 1 sensitivity of two murine GBM lines is shown: these are a PDGF-driven proneural line deficient in Trp53 and a PDGF-driven mesenchymal line (provided by Dr. Hambardzumyan at Mt. Sinai) deficient in Nf-1 and inhibiting Trp53, respectively. Figure 1BEight low-passage human GBM cell lines were shown to be sensitive to compound 1, of which three cell lines had tumor initiating cell (TIC) capabilities (L0, L1, GBM1A) (Deleyrolle, LP, et al., Evidence for label-retaining tumor-initiating cells in human glioblastoma. Brain, 2011. 134(Pt 5): p. 1331-43; Tilghman, J., et al., Regulation of Glioblastoma Tumor-Propagating Cells by the Integrin Partner Tetraspanin CD151. Neoplasia, 2016. 18(3): p. 185-98). All tested cell lines were sensitive to EC 50 The values were in the low micromolar range and were sensitive to compound 1.

[0139] Example 4. Compound 1 has synergistic effects with kinase inhibitors in GBM

[0140] General Approach

[0141] Mice. All mouse procedures were performed in accordance with the guidelines of the Mayo Clinic Institutional Animal Care and Use Committee. Homozygous floxed Trp53 mice (stock number 008462) were obtained from Jackson Laboratories. Equal numbers of male and female mice aged 8 to 20 weeks were studied.

[0142] Glioma cell lines were isolated from mouse GBM tumors and cultured. A protocol for isolating tumor cells from Trp53(- / -) mouse tumor cells has been described (Lei, L. et al. (2011)).

[0143] Dose response curve / cell viability assay. 5000 cells / well were plated in 96-well plates and treated with a series of doses of compound 1, sunitinib, sacatinib, or a combination of 5 μM compound 1 and a series of concentrations of sunitinib or sacatinib after 48 hours. The cells were treated with drugs for 72 to 96 hours, and cell viability was measured using CellTiter-Glo (Promega, catalog number G9242). For experiments involving MuSyc analysis to measure drug synergy (Meyer CT, et al., Quantifying Drug Combination Synergy along Potency and Efficacy Axes. Cell Syst., 2019.8, 97-108), a combination of compound 1 and sunitinib was added to 96-well plates, and viable cell counts were measured with CellTiter-Glo as described above. Cell count data were normalized to the signal of the control with only vehicle.

[0144] Retroviral production and intracerebral injection. PDGF-IRES-cre retrovirus was generated and injected intracranially according to previously described methods (Lei, L. et al. (2011); Kenchappa RS, et al., Myosin 10 regulates invasion, mitosis, and metabolic signaling in gliobloblastoma. iScience, 2020. Nov 13; 23(12): 101802). For pharmacological studies, 5 days after retroviral injection, mice were treated with the following (10 mice per treatment group):

[0145] Carrier;

[0146] Compound 1 (10 mg / kg IP, 7 days per week);

[0147] Saracatinib (25 mg / kg by oral gavage, 5 days per week);

[0148] Sunitinib (40 mg / kg by oral gavage, 5 days per week);

[0149] Compound 1 + sunitinib (10 mg / kg by IP, 7 days per week + 40 mg / kg by oral gavage, 5 days per week); or

[0150] • Compound 1 + saracatinib (10 mg / kg by IP, 7 days per week + 25 mg / kg by oral gavage, 5 days per week).

[0151] Treatment was continued until tumor onset.Survival data were analyzed by Kaplan-Meier analysis, and the log-rank test (Prizm) was used to assess the statistical significance of survival differences.

[0152] Drug synergy. Synergy was calculated using the MuSyC algorithm as previously described (Meyer, CT et al. (2019; Wooten DJ, et al., MuSyC is a consensus framework that unifies multi-drugsynergy metrics for combinatorial drug discovery. Nat Commun., 2021.12, 4607). MuSyC quantifies two types of drug synergy, namely synergistic potency and synergistic efficacy, both of which are related to geometric transformations of the dose-response surface, which is similar to the 1D Hill equation for potency (EC 50 horizontal offset in) and effectiveness (E max Synergy was calculated by fitting a dose-response surface relating drug effect (cell growth rate or static cell count at 96 hours) to the concentrations of drug 1 and drug 2. To aid convergence of the growth rate-based fit, the bounds for E0 (no drug effect) and E1, E2, and E3 (maximum effects of drugs 1, 2, and the combination) were set to [0.005, 0.002] and [0.01, -0.01], respectively.

[0153] A. In vitro synergistic effects with RTK inhibitors

[0154] This example examines the effect of 5 μM Compound 1 on the dose response of Trp53-deficient (Trp53(- / -)) mouse GBM cells to two FDA-approved CNS-penetrant therapeutics, sacatinib (a combined SRC / EGFR inhibitor) and sunitinib (a PDGFR, VEGFR, and c-KIT inhibitor). Compound 1 increases the EC of sunitinib by 50 Shifted to the left by about 20 times ( Figure 4A ), and make the EC of saracatinib 50 Shifted to the left by a factor of >1000 ( Figure 4B ). Taken together, these in vitro results demonstrate a synergistic effect of co-administration of Compound 1 and an RTK inhibitor in a preclinical model of GBM.

[0155] The in vitro synergy between sunitinib and compound 1 was analyzed using MuSyC, which fits a dose-response surface to the drug combination data to calculate the degree of synergy in potency (β) and synergy in potency (log(α12) and log(α21)). Unlike isobolograms or combination index analysis, MuSyC captures synergy in both potency and efficacy. Figure 2A The MuSyC dose response surface for the combination of sunitinib and compound 1 is shown, showing that the combination increased potency by 42% (β=0.42) compared to either drug alone, and that the combination increased the potency of sunitinib, whose EC 50 Reduced by >8.5 times ( Figure 2B ).

[0156] B. In vivo synergy with RTK inhibitors and GBM survival

[0157] In this example, a genetically engineered model (GEMM) of GBM with Trp53 deletion was used to test the therapeutic benefit of Compound 1, as this model is uniformly lethal 35 days after retroviral injection. Administration of Compound 1 was started on day 5 after retroviral injection and stopped at tumor onset. The Kaplan Meier survival curves of these mice showed that in this group, single-agent Compound 1 improved median survival by 32% ( 0.05%) compared to vehicle treatment. Figure 3 ; p < 0.0001, log-rank test).

[0158] To confirm the in vitro synergistic effect between compound 1 and RTK inhibitors, compound 1 was co-administered with sunitinib in Trp53(- / -) GEMMs in vivo. Similar to compound 1, single-agent sunitinib enhanced survival by approximately 32% relative to vehicle. However, the combination of the two drugs significantly enhanced survival, exceeding either drug alone, i.e., >50% of the sum of each drug alone ( Figure 3 ).

[0159] In this example, which represents two combination experiments for replication purposes, mice were treated until they developed tumor onset or until day 85 after retroviral injection, whichever occurred first. Median survival was doubled with the synergistic combination compared to either drug alone. In addition, approximately 35% of the mice were still alive at day 100 (15 days after stopping active treatment). Autopsies of these mice showed no signs of tumors, as measured by immunohistochemistry using the HA epitope to observe PDGF-secreting cells. Thus, the results indicate unexpectedly long-term survival, especially in this highly aggressive GBM model.

[0160] C. In vivo synergy with PI3K inhibitors and GBM survival

[0161] Following the above procedure, p53-deficient GBM was induced in mice with homozygous conditional knockout of p53 by in situ injection of PDGF-IRES-cre retrovirus. Seven days later, the animals were injected daily with vehicle, compound 1, paxalisib, or a combination of compound 1 and paxalisib, and survival was tracked. The results showed that the combination of compound 1 and paxalisib achieved an unexpected improvement in mouse survival compared to mice treated with compound 1 or paxalisib alone ( Figure 5 ).

[0162] Example 5. Compound 1 has a synergistic effect with radiation

[0163] The purpose of this example is to show that compound 1 synergistically sensitizes GBM to radiotherapy. Figure 6A ) and p53-deficient genetically engineered mouse models (GEMM) ( Figure 6B ) of the two, the combination of Compound 1 (5 mg / kg, SC) and radiation (2 Gy / dose x 5 daily doses) prolonged mouse survival to a greater extent than the sum of each treatment.

[0164] Example 6. Compound 1 has synergistic effects with kinase inhibitors in pancreatic cancer and TNBC

[0165] General Methods. Cancer cells were seeded in 96-well culture plates at a density of 1000 to 2000 cells per well. After a 24-hour incubation period, the cells were treated with the specified compounds, maintaining a constant concentration ratio or a non-constant concentration ratio between the two compounds. Cell viability was assessed using a luminescent cell viability assay. The data were analyzed and visualized using GraphPad Prism 10. To quantify synergy, the Combination Index (CI) was calculated using CompuSyn computer software. A CI value below 1 indicates synergy, with lower CI values reflecting stronger synergy. More detailed information and analysis tools can be found at https: / / www.combosyn.com / .

[0166] A. Pancreatic cancer

[0167] This example demonstrates the synergistic effect of Compound 1 in combination with three inhibitors (everolimus, sunitinib, and olaparib) in pancreatic adenocarcinoma cell lines. Cells were treated with Compound 1 alone, an inhibitor alone, and a combination of Compound 1 and each inhibitor, maintaining the specified constant concentration ratios as shown in the accompanying figures over a 5-day period. Synergy was evaluated using CompuSyn computer software described herein.

[0168] Everolimus / PANC-1. For compound 1 and everolimus in PANC-1, a combination index (CI) below 1 was achieved in the viability fraction range of 0.3 to 0.85 ( Figure 7A and Figure 7B ).

[0169] Sunitinib / PANC-1. For compound 1 and sunitinib in PANC-1, a CI below 1 was achieved when the viability score was less than 0.3 ( Figure 8A and Figure 8B ).

[0170] Olaparib / PANC-1. For compound 1 and olaparib in PANC-1, a CI below 1 was achieved when the viability score exceeded 0.3 ( Figure 9A and Figure 9B ).

[0171] Sunitinib / MIAPaCa-2. For compound 1 and sunitinib in MIAPaCa-2, a CI below 1 was achieved when the viability score was less than 0.2 ( Figure 10A and Figure 10B ).

[0172] Olaparib / MIAPaCa-2. For compound 1 and olaparib in MIAPaCa-2, a CI below 1 was achieved when the viability score was less than 0.7 ( Figure 11A and Figure 11B ).

[0173] B. Triple-negative breast cancer (TNBC)

[0174] The present embodiment shows the synergistic effect of the combination of compound 1 and everolimus for TNBC cell line (MDA-MB-468). Cells are treated with a combination of independent compound 1, independent everolimus and compound 1 (5 μM) and everolimus. The combination utilizes a non-constant concentration ratio and is applied for 5 days. The evaluation of synergy is performed using CompuSyn computer software described herein. Compound 1 and everolimus in MDA-MB-468 show a combination index (CI) lower than 1 within the entire test range, with a viability score of 0.52 to 0.38 ( Figure 11A and Figure 11B ).

[0175] Example 7. Synergistic Effects of Compound 1 and FDA-Approved Drugs

[0176] This example provides additional data demonstrating the synergistic combination of Compound 1 with additional anticancer therapeutics against multiple tumor cell lines.

[0177] The cells shown in Table 2 below were treated with compound 1 alone, an anticancer drug alone, and a combination of compound 1 and each drug. Compound 1 was maintained at a concentration of 5 μM over a period of 2, 3, or 5 days, at a non-constant ratio of the two compounds or at a specific constant concentration ratio. To assess synergy, the combination index (CI) was calculated using the following formula:

[0178] CI=(D)1 / (Dx)1+(D)2 / (Dx)2+(D)1(D)2 / (Dx)1(Dx)2

[0179] Here, (D)1 and (D)2 represent the doses of compound 1 and the drug that produce 50% efficacy in combination (Fa = 0.5), while (Dx)1 and (Dx)2 represent the doses of compound 1 and the test drug that produce 50% efficacy each. A CI value below 1 indicates synergy, and lower CI values reflect stronger synergy.

[0180] Table 2

[0181]

Claims

1. A method for treating cancer in a subject having cancer, wherein the cancer is characterized by rapid tumor cell proliferation and vigorous invasion of surrounding tissues, comprising administering to the subject a combination of Compound 1 or a pharmaceutically acceptable salt thereof and at least one anti-cancer treatment.

2. The method of claim 1 , wherein the cancer is selected from the group consisting of glioblastoma (GBM), pancreatic adenocarcinoma, triple-negative breast cancer (TNBC), bile duct carcinoma, head and neck squamous cell carcinoma, testicular germ cell tumor, hepatocellular carcinoma, gastric adenocarcinoma, renal chromophobe cell carcinoma, esophageal cancer, brain low-grade glioma, ovarian serous cystadenocarcinoma, thyroid cancer, bladder urothelial carcinoma, skin melanoma, renal clear cell carcinoma, prostate adenocarcinoma, cervical squamous cell carcinoma, adrenal cortical carcinoma, lung adenocarcinoma, rectal adenocarcinoma, lung squamous cell carcinoma, invasive breast cancer, colon adenocarcinoma, uterine corpus endometrial cancer, pheochromocytoma and paraganglioma, renal papillary cell carcinoma, uterine carcinosarcoma, acute myeloid leukemia, lymphoid neoplasms diffuse large B-cell lymphoma, mesothelioma, oropharyngeal squamous cell carcinoma, sarcoma, thymoma, and uveal melanoma.

3. The method of claim 1 or 2, wherein the cancer is GBM.

4. The method of claim 1 or 2, wherein the cancer is TNBC.

5. The method of claim 1 or 2, wherein the cancer is pancreatic adenocarcinoma.

6. The method according to any one of claims 1 to 5, wherein Compound 1 is administered as its monohydrochloride salt.

7. The method according to any one of claims 1 to 6, wherein the anti-cancer treatment is selected from: (a) at least one inhibitor of at least one kinase in the RTK-PI3K-mTOR signaling pathway, or a pharmaceutically acceptable salt thereof, (b) at least one inhibitor of at least one kinase in the mitogen-activated protein kinase (MAPK) signaling pathway, or a pharmaceutically acceptable salt thereof, (c) inhibitors of poly (ADP-ribose) polymerase 1 (PARP1), and (d) Therapeutic doses of radiation.

8. The method of any one of claims 1 to 7, wherein the anti-cancer treatment is (a) at least one inhibitor of at least one kinase in the RTK-PI3K-mTOR signaling pathway, or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the kinase in the RTK-PI3K-mTOR signaling pathway is a receptor tyrosine kinase (RTK), and the inhibitor is a RTK inhibitor.

10. The method of claim 9, wherein the RTK inhibitor or a pharmaceutically acceptable salt thereof targets two or more receptor tyrosine kinases.

11. The method of claim 9 or 10, wherein the RTK inhibitor or a pharmaceutically acceptable salt thereof targets three or more receptor tyrosine kinases.

12. The method of any one of claims 9 to 11, wherein the RTK inhibitor or a pharmaceutically acceptable salt thereof targets four or more receptor tyrosine kinases.

13. The method according to any one of claims 9 to 12, wherein the receptor tyrosine kinase is selected from: RTK class I (EGF receptor family; ErbB family); RTK class II (insulin receptor family); RTK class III (PDGF receptor family); RTK class IV (VEGF receptor family); RTK class V (FGF receptor family); RTK class VI (CCK receptor family); RTK class VII (NGF receptor family); RTK class VIII (HGF receptor family); RTK class IX (Eph receptor family); RTK class X (AXL receptor family); RTK class XI (TIE receptor family); RTK class XII (RYK receptor family); RTK class XIII (DDR receptor family); RTK class XIV (RET receptor family); RTK class XV (ROS receptor family); RTK class XVI (LTK receptor family); RTK class XVII (ROR receptor family); RTK class XVIII (MuSK receptor family); and RTK class XIX (LMR receptors).

14. The method according to any one of claims 9 to 13, wherein the RTK inhibitor is selected from fasudil (Erilu), sirolimus (Rapamune), imatinib, gefitinib, erlotinib, sorafenib, sunitinib, sacatinib, dasatinib, lapatinib, nilotinib, temsirolimus, everolimus, pazopanib, ruxolitinib, vandetanib, vemurafenib, crizotinib, icotinib, axitinib, tofacitinib, bosutinib, cabozantinib, ponatinib, regorafenib , afatinib, dabrafenib, trametinib, ibrutinib, nintedanib, idelalisib, alectinib, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, bosutinib, dacomitinib, neratinib, osimertinib, gilteritinib, midostaurin, erdafitinib, larotrectinib, axitinib, cabozantinib, lenvatinib, regorafenib, dabrafenib, conacalcitonib, bimetinib, cobimetinib, abemaciclib, palbociclib, ribociclib, pharmaceutically acceptable salts thereof, and combinations thereof.

15. The method of any one of claims 9 to 14, wherein the RTK inhibitor is sunitinib.

16. The method of claim 8, wherein the kinase in the RTK-PI3K-mTOR signaling pathway is phosphoinositide 3-kinase (PI3K), and the inhibitor is a PI3K inhibitor.

17. The method of claim 16, wherein the PI3K inhibitor is selected from the group consisting of idelalisib, cupanisib, duvilisib, apellisib, erbulisib, leniolisib, bupanisib, datolicoxib, parsaclisib, paxalisib, tacelisiib, zandelisib, inarlisib, apitolisib, bimilisib, eganelisib, feminostat, gidalisib, limplisib, nemilisib, pitilisib, pilaralisib, samoto lisib, selelisib, ceralisib, sonolisib, tenalisib, voxtalisib, AMG319, AZD8186, GSK2636771, SF1126, LY294002, acalisib, amelisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, ZSTK474, AEZS-136, B591, GNE-477, hibiscone C, IC87114, PI-103, wortmannin, pharmaceutically acceptable salts thereof, and combinations thereof.

18. The method of claim 16 or 17, wherein the PI3K inhibitor is paxalisib.

19. The method of claim 8, wherein the kinase in the RTK-PI3K-mTOR signaling pathway is a mechanistic target of rapamycin (mTOR) kinase and the inhibitor is an mTOR inhibitor.

20. The method of claim 19, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, temsirolimus, everolimus, defostiolimus, sirolimus, defostiolimus, urolimus, zotarolimus, torin-1, torin-2, veltorin, pharmaceutically acceptable salts thereof, and combinations thereof.

21. The method of claim 19 or 20, wherein the mTOR inhibitor is everolimus.

22. The method of any one of claims 1 to 7, wherein the anti-cancer treatment is (b) at least one inhibitor of at least one kinase in the MAPK signaling pathway, or a pharmaceutically acceptable salt thereof.

23. The method of claim 22, wherein the kinase in the MAPK signaling pathway is a Src kinase and the inhibitor is a Src inhibitor.

24. The method of claim 23, wherein the Src inhibitor is selected from the group consisting of KX2-391, bosutinib, saracatinib, dasatinib, PP1, PP2, pharmaceutically acceptable salts thereof, and combinations thereof.

25. The method of claim 23 or 24, wherein the Src inhibitor is saracatinib.

26. The method of claim 22, wherein the kinase in the MAPK signaling pathway is mitogen-activated protein kinase (MEK) and the inhibitor is a MEK inhibitor.

27. The method of claim 26, wherein the MEK inhibitor is selected from bimetinib, cobimetinib, selumetinib, trametinib, pharmaceutically acceptable salts thereof, and combinations thereof.

28. The method of claim 22, wherein the kinase in the MAPK signaling pathway is extracellular signal-regulated kinase (ERK), and the inhibitor is an ERK inhibitor.

29. The method of claim 28, wherein the ERK inhibitor is selected from the group consisting of ravoxertinib, MK-8353, uritinib, temuterkib, KO-947, CC-90003, ONC201, tizaterkib, SCH772984, pluripotin, VX-Ile, DEL-22379, FR 180204, ERK5-IN-1, pharmaceutically acceptable salts thereof, and combinations thereof.

30. The method of any one of claims 1 to 7, wherein the anti-cancer treatment is (c) a PARP1 inhibitor.

31. The method of claim 30, wherein the PARP1 inhibitor is selected from the group consisting of rucaparib, iniparib, olaparib, veliparib, niraparib, talazoparib, CEP-9722, and E7016.

32. The method of claim 30 or 31 , wherein the PARP1 inhibitor is olaparib.

33. The method of any one of claims 1 to 7, wherein (d) a therapeutic dose of radiation is administered to the subject.

34. The method of any one of claims 1 to 30, wherein the anti-cancer therapy and Compound 1, or a pharmaceutically acceptable salt thereof, are administered concurrently.

35. The method of any one of claims 1 to 34, wherein the anti-cancer therapy and Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject once daily, four days a week.

36. The method of any one of claims 1 to 34, wherein the anti-cancer therapy and Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject once daily, 5 days a week.

37. The method of any one of claims 1 to 34, wherein the anti-cancer therapy and Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject once daily for 6 days per week.

38. The method of any one of claims 1 to 34, wherein the anti-cancer therapy and Compound 1, or a pharmaceutically acceptable salt thereof, is administered to the subject once daily, 7 days a week.

39. The method of any one of claims 1 to 38, wherein the dose of the anti-cancer therapy or Compound 1, or a pharmaceutically acceptable salt thereof, in the combination is less than the dose of the anti-cancer therapy or Compound 1, or a pharmaceutically acceptable salt thereof, respectively, that would be required when administered alone to the subject to achieve the same therapeutic endpoint.

40. The method of any one of claims 1 to 39, wherein the therapeutic endpoint of treatment with the combination is survival of the subject longer than survival of a subject administered the anti-cancer therapy or Compound 1, or a pharmaceutically acceptable salt thereof, alone.

41. The method of claim 40, wherein the therapeutic endpoint of treatment with the combination is that the subject survives for at least about twice the duration of survival of a subject administered the anti-cancer therapy or Compound 1, or a pharmaceutically acceptable salt thereof, alone.

42. The method of any one of claims 1 to 41, wherein each dose of Compound 1, or a pharmaceutically acceptable salt thereof, in the combination is about 0.1 to about 15 mg / kg.

43. The method of any one of claims 1 to 31 and 34 to 42, wherein each dose of (a), (b), or (c) in the combination is about 0.1 to about 200 mg / kg.

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