Ezh2 inhibition therapy for the treatment of protein 1a (arid1a) mutations cancers comprising at-rich interaction domain

Abstract

Provided herein are methods of treating cancer having at least one ARID1A mutation using (R)-7-chloro-2-((1r, 4R)-4-(3-methoxyazetidin-1-yl) cyclohexyl)-2, 4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1, 2-dihydropyridin-3-yl) methyl) benzo [d] [1, 3] dioxol-5-carboxamide, or a pharmaceutically acceptable salt thereof.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 389,436 filed on July 15, 2022, U.S. Provisional Application No. 63 / 413,323 filed on October 5, 2022, and U.S. Provisional Application No. 63 / 455,024 filed on March 28, 2023, the entire contents of each of which are incorporated herein by reference. Background Art

[0003] Mutations in the ARID1A gene, encoding the essential directional subunit of the SWI / SNF chromatin remodeling complex, have been shown in several groups of cancers, including ovarian, endometrial, and uterine cancer subtypes. ARID1A is a tumor suppressor whose loss results in increased cell proliferation, migration, and invasion, as well as decreased apoptosis and chemosensitivity. ARID1A is mutated in 25% of muscle-invasive bladder cancers, and high frequencies of ARID1A mutations have been reported in many indications, including ovarian clear cell carcinoma (46%-57%), endometrial carcinoma (30%-40%), and gastric cancer (20%).

[0004] Despite efforts to combine multiple cancer immunotherapeutic agents and ongoing clinical exploration of cancer immunotherapies in combination with other therapies, ARID1A mutant cancers, particularly advanced urothelial carcinoma (a subtype of bladder cancer), remain largely incurable, with only a minority of patients responding to second- or subsequent-line treatments with limited survival benefit. Therefore, new approaches to target cancers carrying one or more ARID1A mutations are needed.

[0005] Summarize

[0006] It has now been found that cancers with at least one ARID1A mutation have increased phenotypic sensitivity to the EZH2 inhibitor (R)-7-chloro-2-((1r,4R)-4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide (referred to herein as Compound 1). See, e.g. Figure 1 , Fig.19and Table 1, where growth inhibition was significantly enriched in a panel of 21 bladder cancer cell lines following treatment with compound 1 in cell lines carrying at least one ARID1A loss-of-function (LOF) allele (p=3.7e-6, chi-square test), with 83% (5 of 6) of sensitive cell lines (GI50 3-37 nM after 18 days) carrying truncating mutations (frameshift or nonsense). In contrast, only 6% (1 of 15) of non-responsive cell lines with 18-day GI50>5 μM had an ARID1A LOF allele. In addition, compound 1 treatment significantly inhibited the growth of a patient-derived xenograft (PDX) model of ARID1A LOF endometrial cancer ( Figure 7 ) and ovarian clear cell carcinoma (OCCC) ARID1A mutant TOV21G cell line-derived xenograft (CDX) models ( Figure 8 ). However, surprisingly, colon cancer, lung cancer, and non-clear cell ovarian cancer with ARID1A mutations did not show increased sensitivity to treatment overall. See e.g. Fig.12 , where no clear correlation between mutation status and responsiveness was observed. Clinical efficacy was also observed in cancer subjects carrying ARID1A mutations ( Fig.13 , Fig.14 ). It was found that most OCCC or EC patients had a low tumor mutation load. Therefore, in one aspect, a method of using compound 1 to treat ARID1A mutant cancers with a low tumor mutation load is provided. In one aspect, the tumor mutation load is less than 10mut / Mb.

[0007] It was also found that compound 1 showed higher response rates in some ARID1A mutant PDX cancer models compared to the response rates in the corresponding ARID1A wild-type cancer indications (Table 6). Thus, in one aspect, some ARID1A mutant cancers showed higher response rates than the corresponding ARID1A wild-type cancers.

[0008] Thus, in one aspect, a method of treating a cancer having at least one ARID1A mutation using Compound 1 is provided. Also provided is the use of Compound 1 for the preparation of a medicament for treating said cancer.

[0009] It was also found that compound 1 restored ARID1A expression in ARID1A mutant bladder cancer cells. For example, GSEA of genes upregulated after compound 1 treatment showed enrichment of ARID1A re-expression targets. See e.g. Fig.11Thus, in one aspect, a method of restoring ARID1A expression in a cancer having at least one ARID1A allele using Compound 1 is provided. Also provided is the use of Compound 1 for the preparation of a medicament for restoring ARID1A expression in a cancer having at least one ARID1A allele. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The results of the 18-day viability assay GI50 values ​​of compound 1 in a panel of bladder cancer cell lines are shown. Black bars represent cell lines carrying at least one ARID1A stop gain (indicated by *) or frameshift (fs) allele, as shown in the figure below. Green bars represent cell lines carrying single missense mutations, while gray bars represent those cell lines without mutations in the ARID1A coding region. Data are expressed as the mean ± SD of duplicate wells, representing repeated independent experiments.

[0011] Figure 2 Summary of the mutational status of the major components of the BAF complex and KDM6A in the bladder cancer panel. Mutations in bold are the most common mutations in cancer.

[0012] Figure 3 Represents normalized global H3K27me levels in HT1197 (left) and T24 (right) cell lines after 72 h of treatment over a dose range of compound 1. Data are presented as mean ± SD of triplicate wells and are representative of four independent experiments.

[0013] Figure 4 Cell viability dose response curves for HT1197 (left) and T24 (right) cell lines after 18 days of treatment are shown. Data are presented as mean ± SD of duplicate wells and are representative of repeated independent experiments.

[0014] Figure 5 Tumor growth inhibition of HT1376 bladder cancer xenografts by Compound 1 or vehicle treatment is shown. Data are presented as mean tumor volume + SEM, n = 5 mice per group for all groups except 150 mg / kg where n = 3. p values ​​were calculated using two-way ANOVA until day 30, ns = p>0.05, *p<0.05.

[0015] Figure 6 Tumor growth inhibition in an ARID1A mutant PDX model (BL9209) of bladder cancer treated with compound 1, 75 mg / kg PO, QD is shown. Data are expressed as mean tumor volume + SEM, with n = 3 mice per group. TGI was calculated using the tumor volume on day 28, *p < 0.05, using a two-way ANOVA until day 28, when the vehicle reached the endpoint.

[0016] Figure 7Tumor growth inhibition in an ARID1A mutant PDX model (UT5319) of endometrial cancer treated with compound 1 is shown. Mice were initially treated with 75 mg / kg PO QD, and then the dose was reduced to 50 mg / kg PO QD after day 23. Data are expressed as mean tumor volume + SEM, with n = 3 mice per group. TGI was calculated using the tumor volume on day 21, ***p < 0.0001, using a two-way ANOVA until day 21, when the vehicle reached the endpoint.

[0017] Figure 8 Tumor growth inhibition of TOV21G OCCC xenografts by Compound 1 or vehicle treatment is shown. Data are presented as mean tumor volume + SEM, with n = 6 mice in the vehicle group and n = 4 in the Compound 1 group. p values ​​were calculated using two-way ANOVA until day 42, **p < 0.01.

[0018] Fig. 9 Shown are Western blots of whole cell lysates of HT1376 cells transduced with empty TET-ON vector or TET-ON-ARID1A, treated with or without 50 ng / mL Dox for 5 days, and treated with DMSO or 250 nM Compound 1 for 4 days.

[0019] Fig.10 Shown is a gene set enrichment analysis (GSEA) of EZH2 target genes in HT1376 transduced with empty vector or doxycycline-inducible ARID1A and treated with 50 ng / ml doxycycline for 5 days.

[0020] Fig.11 ARID1A-induced genes (defined above) in HT1376 cells transduced with a doxycycline-inducible ARID1A vector and treated with 50 ng / ml doxycycline for 5 days and DMSO or 250 nM compound 1 for 4 days Figure 3 F) GSEA.

[0021] Fig.12 Shown are responses to Compound 1 treatment in ARID1A mutant PDX or PD organoid type models (as indicated) of colon, lung, gastric, and non-clear cell ovarian cancers.

[0022] Fig.13 The duration of treatment with Compound 1 in subjects with ovarian clear cell carcinoma harboring an ARID1A mutation is shown.

[0023] Fig.14 The duration of treatment with Compound 1 in subjects with endometrial cancer harboring an ARID1A mutation is shown.

[0024] Fig.15Tumor volumes in ARID1A mutant HT1376 xenograft efficacy studies after treatment with various EZH2 and EED inhibitors are shown. Data are expressed as mean + SEM, with n = 6 mice per group for all groups, but n = 12 for compound 1. TGI values ​​for all groups were calculated using the 27th day tumor volume relative to the vehicle. P values ​​were calculated using two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.0001, using two-way ANOVA until day 27 (for TGI) or until day 55 (for p values ​​on the graph).

[0025] Fig.16 Shows Fig.15 H3K27me3 levels in tumor samples collected on day 15 of the study are shown. Data are presented as mean + SD, n = 3 tumors per group. p values ​​were calculated using unpaired Student's t-test, **p < 0.01, ***p < 0.0001.

[0026] Fig.17 Shown is a scatter plot showing the relationship between relative H3K27me3 levels and tumor size. Fig.15 The relative H3K27me3 / H3 ratios of tumor samples collected on day 15 of each group were normalized to those of the vehicle group. The tumor volume of each group was measured on day 27. The p value was calculated by Pearson's correlation coefficient.

[0027] Fig.18 Shows Fig.15 Bar graph of changes in expression of EZH2 target genes (as defined in Table 5) in tumors relative to vehicle at day 15. Data are presented as mean Log2 fold change ± 95% confidence interval.

[0028] Fig.19 Compound 1 is shown to have a 21-day viability assay in a panel of ovarian clear cell carcinoma and endometrioid cell lines. 50 Results are shown in Figure 2. Black bars represent cell lines carrying at least one ARID1A stop gain (indicated by *) or frameshift (fs) allele, as detailed in the figure below. Gray bars represent those cell lines with wild-type (no mutation) coding regions of ARID1A. Data are presented as mean ± SD of triplicate wells in two independent experiments.

[0029] Fig. 20 Tumor growth inhibition in an ARID1A mutant PDX model (UT5326) of endometrial cancer after treatment with compound 1 is shown. Mice were treated with 75 mg / kg PO QD. Data are presented as mean tumor volume + SEM, with n = 5 mice per group. TGI was calculated using the tumor volume on day 49, *p < 0.05, using two-way ANOVA until day 49, when the study reached the endpoint.

[0030] Fig.21 Tumor growth inhibition in an ARID1A mutant PDX model of endometrial cancer (CTG-1280) after treatment with compound 1 is shown. Mice were treated with 75 mg / kg PO QD. Data are presented as mean tumor volume + SEM, with n = 5 mice per group. TGI was calculated using the tumor volume on day 27, *p < 0.05, using two-way ANOVA until day 27, when the vehicle reached the endpoint.

[0031] Fig. 22 Tumor growth inhibition in the ARID1A mutant PDX model (UT5320) of endometrial cancer after treatment with compound 1 is shown. Mice were initially treated with 75 mg / kg PO QD, then the dose was reduced to 50 mg / kg PO QD from day 12 to day 21, after which 75 mg / kg dosing was resumed. Data are expressed as mean tumor volume + SEM, with n = 5 mice per group. TGI was calculated using the tumor volume on day 17, *p < 0.05, using a two-way ANOVA until day 17, when the vehicle reached the endpoint.

[0032] Fig.23 Tumor growth inhibition of ARID1A mutant HT1376 bladder cancer xenografts by Compound 1 or vehicle treatment is shown, treated with 35 or 50 mg / kg PO QD for 48 days. Data are presented as mean tumor volume or mean % change in body weight from day 0 + SEM, with n=8 mice per group for vehicle and 50 mg / kg cohorts and n=6 for 35 mg / kg cohort.

[0033] Fig.24 Global H3K27me3 levels in ARID1A mutant HT1376 xenograft tumors after 12 or 31 days of treatment are shown. Data points represent measurements of individual tumors sampled at a given time point, and error bars reflect mean + SEM. Two tumors were measured on day 12, and five tumors were measured on day 31. All treatment groups were significantly lower than vehicle controls at the same time point, **p<0.01, ***p<0.0001, unpaired Student's t-test. There is no data for 150 mg / kg on day 31 because the tumors were too small to analyze. DETAILED DESCRIPTION

[0034] In a first embodiment, methods of treating cancer in a subject are provided, comprising administering to the subject an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof, wherein the cancer has at least one ARID1A mutation. Such cancers include, but are not limited to, bladder cancer (e.g., urothelial carcinoma), endometrial cancer, ovarian cancer, ovarian clear cell carcinoma, breast cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, bile duct cancer, gastric cancer, hepatocellular carcinoma, liver cancer, lung cancer, and melanoma. In one aspect, the cancer is selected from bladder cancer (e.g., urothelial carcinoma), endometrial cancer, and ovarian clear cell carcinoma.

[0035] As part of the first embodiment, there is also provided the use of an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a cancer having at least one ARID1A mutation. Such cancers include, but are not limited to, bladder cancer (e.g., urothelial carcinoma), endometrial cancer, ovarian cancer, ovarian clear cell carcinoma, breast cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, bile duct cancer, gastric cancer, hepatocellular carcinoma, liver cancer, lung cancer, and melanoma. In one aspect, the cancer is selected from bladder cancer (e.g., urothelial carcinoma), endometrial cancer, and ovarian clear cell carcinoma.

[0036] As part of the first embodiment, there is also provided an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof for treating a cancer having at least one ARID1A mutation. Such cancers include, but are not limited to, bladder cancer (e.g., urothelial carcinoma), endometrial cancer, ovarian cancer, ovarian clear cell carcinoma, breast cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, bile duct cancer, gastric cancer, hepatocellular carcinoma, liver cancer, lung cancer, and melanoma. In one aspect, the cancer is selected from bladder cancer (e.g., urothelial carcinoma), endometrial cancer, and ovarian clear cell carcinoma.

[0037] As part of the first embodiment, a pharmaceutical composition comprising an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof is also provided for treating cancers having at least one ARID1A mutation. Such cancers include, but are not limited to, bladder cancer (e.g., urothelial carcinoma), endometrial cancer, ovarian cancer, ovarian clear cell carcinoma, breast cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, bile duct cancer, gastric cancer, hepatocellular carcinoma, liver cancer, lung cancer, and melanoma. In one aspect, the cancer is selected from bladder cancer (e.g., urothelial carcinoma), endometrial cancer, and ovarian clear cell carcinoma.

[0038] Compound 1 and (R)-7-chloro-2-((1r,4R)-4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide are used interchangeably and each refers to a compound having the following chemical structure.

[0039]

[0040] In one aspect, as part of a second embodiment, the cancer treated by the methods of the present invention is bladder cancer. In another aspect, as part of a second embodiment, the cancer treated by the methods of the present invention is urothelial carcinoma. In another aspect, as part of a second embodiment, the cancer treated by the methods of the present invention is advanced urothelial carcinoma (e.g., urothelial carcinoma that has spread to another part of the body). In another aspect, as part of a second embodiment, the cancer treated by the methods of the present invention is endometrial carcinoma. In yet another aspect, as part of a second embodiment, the cancer treated by the methods of the present invention is ovarian clear cell carcinoma.

[0041] In a third embodiment, at least one ARID1A mutation of the method of the present invention (e.g., as in the first or second embodiment) is a loss of function (LOF) mutation. On the other hand, as part of the third embodiment, at least one ARID1A mutation of the method of the present invention (e.g., as in the first or second embodiment) is a truncated mutation (frameshift or nonsense). On the other hand, as part of the third embodiment, at least one ARID1A mutation of the method of the present invention (e.g., as in the first or second embodiment) is Q557*, and the cancer is urothelial carcinoma. On the other hand, as part of the third embodiment, at least one ARID1A mutation of the method of the present invention (e.g., as in the first or second embodiment) is selected from G1340fs, S301fs, P302fs, P1326fs and R693, Q557*, and the cancer is endometrial cancer. On the other hand, as part of the third embodiment, at least one ARID1A mutation of the method of the present invention (e.g., as in the first or second embodiment) is selected from Q546fs and Q723*, and the cancer is ovarian clear cell carcinoma. In another aspect, as part of the third embodiment, the at least one ARID1A mutation of the methods of the invention (eg, as in the first or second embodiment) is selected from N1216fs and A162Rfs*238, and the cancer is endometrial cancer.

[0042] As used herein, ARID1A LOF mutation refers to a mutation that reduces or eliminates the function of the ARID1A protein. LOF may be due to nonsense-mediated loss of activity or loss of expression due to protein truncation (missing key residues or domains).

[0043] The terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset of one or more symptoms of cancer or diseases described herein, or inhibiting their progression. In some embodiments, treatment (i.e., therapeutic treatment) may be administered after one or more signs or symptoms of cancer have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of cancer. For example, treatment (i.e., preventive treatment) may be administered to susceptible subjects before the onset of symptoms (e.g., based on a history of symptoms and / or based on exposure to pathogens). In further embodiments, treatment includes delaying the onset of at least one symptom of cancer for a period of time. Treatment may also be continued after symptoms subside, for example, to delay or prevent recurrence (i.e., maintenance therapy).

[0044] The terms "subject" and "patient" are used interchangeably and refer to mammals in need of treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Typically, the subject is a human in need of treatment.

[0045] The term "effective amount" or "therapeutically effective amount" refers to the amount of compound 1 or a pharmaceutically acceptable salt thereof that will elicit a biological or medical response in a subject, such as a dose of 0.01-100 mg / kg body weight / day. In one aspect, as part of the fourth embodiment, the effective amount of compound 1 in the method of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 10 mg / kg body weight / day to about 150 mg / kg body weight / day. In another aspect, as part of the fourth embodiment, the effective amount of compound 1 in the method of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 50 mg to about 375 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of compound 1 in the method of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 150 mg to about 350 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of compound 1 in the method of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 175 mg to about 325 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the methods of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 200 mg to about 300 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the methods of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 225 mg to about 375 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the methods of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 325 mg to about 400 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the methods of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 350 mg to about 375 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the methods of the present invention (e.g., as in any one of the first to third embodiments) ranges from about 200 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the methods of the present invention (e.g., as in any one of the first to third embodiments) is about 250 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the method of the present invention (e.g., as in any one of the first to third embodiments) is about 300 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the method of the present invention (e.g., as in any one of the first to third embodiments) is about 350 mg per day. In another aspect, as part of a fourth embodiment, the effective amount of compound 1 in the method of the present invention (e.g., as in any one of the first to third embodiments) is about 375 mg per day.In one aspect, as part of the fourth embodiment, the effective amount of the pharmaceutically acceptable salt of compound 1 in the methods of the present invention (e.g., in any one of the first to third embodiments) is equivalent to an amount of compound 1 in the range of about 10 mg / kg body weight / day to about 150 mg / kg body weight / day. In another aspect, as part of the fourth embodiment, the effective amount of the pharmaceutically acceptable salt of compound 1 in the methods of the present invention (e.g., in any one of the first to third embodiments) is equivalent to an amount of compound 1 in the range of about 50 mg to about 375 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of the pharmaceutically acceptable salt of compound 1 in the methods of the present invention (e.g., in any one of the first to third embodiments) is equivalent to an amount of compound 1 in the range of about 325 mg to about 400 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of the pharmaceutically acceptable salt of compound 1 in the methods of the present invention (e.g., in any one of the first to third embodiments) is equivalent to an amount of compound 1 in the range of about 350 mg to about 375 mg per day. On the other hand, as part of the fourth embodiment, the effective amount of the pharmaceutically acceptable salt of Compound 1 in the method of the present invention (e.g., in any one of the first to third embodiments) is equivalent to an amount of about 350 mg of Compound 1 per day. On the other hand, as part of the fourth embodiment, the effective amount of the pharmaceutically acceptable salt of Compound 1 in the method of the present invention (e.g., in any one of the first to third embodiments) is equivalent to an amount of about 375 mg of Compound 1 per day.

[0046] The method of administration herein can be oral, parenteral, by inhalation spray, topical, rectal, nasal, buccal, vaginal or by implanted drug reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. The sterile injectable form of compound 1 described herein can be an aqueous or oily suspension. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. On the one hand, as part of the fifth embodiment, compound 1 in the method of the present invention (e.g., in any one of the first to fourth embodiments) is orally administered.

[0047] Compound 1 can exist in the form of a pharmaceutically acceptable salt. For use in medicine, a pharmaceutically acceptable salt refers to a non-toxic "pharmaceutically acceptable salt". Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts (if possible).

[0048] Compound 1 or a pharmaceutically acceptable salt thereof can be formulated as part of a pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers (e.g., carriers, adjuvants or vehicles) that can be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

[0049] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant or vehicle that does not adversely affect the pharmacological activity of the compound formulated therewith, and is also safe for human use. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used for the compositions of the present disclosure include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, magnesium stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based materials (e.g., microcrystalline cellulose, hydroxypropyl methylcellulose, lactose monohydrate, sodium lauryl sulfate and cross-linked sodium carboxymethylcellulose), polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

[0050] On the one hand, as part of the sixth embodiment, the compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) exists in a crystalline form. The crystalline form of compound 1 is disclosed in WO 2021 / 016414 and is incorporated herein by reference. On the other hand, as part of the sixth embodiment, the compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is a crystalline form 1, characterized in that at least three X-ray powder diffraction peaks at 2θ angles selected from the following: 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2° and 22.5°. On the other hand, as part of the sixth embodiment, the compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is a crystalline form 1, characterized in that at least four X-ray powder diffraction peaks at 2θ angles selected from the following: 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2° and 22.5°. On the other hand, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of Compound 1 is in crystalline Form 1, characterized by at least five X-ray powder diffraction peaks at 2θ angles selected from the following: 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. On the other hand, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of Compound 1 is in crystalline Form 1, characterized by at least six X-ray powder diffraction peaks at 2θ angles selected from the following: 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. On the other hand, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of compound 1 is in crystalline form 1, characterized by an X-ray powder diffraction peak at 2θ angle selected from the following: 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. On the other hand, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of compound 1 is in crystalline form 1, characterized by an X-ray powder diffraction peak at 2θ angle selected from the following: 10.0°, 10.2°, 12.3°, 12.7°, 13.3°, 14.9°, 15.3°, 20.2°, 20.8°, 21.3°, 22.2°, 22.5°, and 23.8°.In another aspect, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of compound 1 is in crystalline form 1, characterized by an X-ray powder diffraction peak at 2θ angle selected from the following: 10.0°, 10.2°, 11.0°, 11.4°, 11.8°, 12.3°, 12.7°, 13.3°, 14.9°, 15.3°, 16.1°, 17.4°, 20.2°, 20.8°, 21.3°, 22.2°, 22.5°, and 23.8°. In another aspect, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of compound 1 is in crystalline form 1, characterized by an X-ray powder diffraction peak at 2θ angle selected from the following: 14.9°, 20.2°, and 20.8°. In another aspect, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of Compound 1 is in crystalline Form 1, characterized by an x-ray powder diffraction peak at 2θ angles selected from the group consisting of: 10.0°, 14.9°, 20.2°, and 20.8°. In another aspect, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of Compound 1 is in crystalline Form 1, characterized by an x-ray powder diffraction peak at 2θ angles selected from the group consisting of: 10.0°, 14.9°, 20.2°, 20.8°, and 22.2°. In another aspect, as part of a sixth embodiment, the disclosed method (e.g., in any one of the first to fifth embodiments) of Compound 1 is in crystalline Form 1, characterized by an x-ray powder diffraction peak at 2θ angles selected from the group consisting of: 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, and 22.2°.

[0051] In one aspect, as part of the seventh embodiment, the compound 1 or a pharmaceutically acceptable salt thereof in the disclosed method (e.g., in any one of the first to fifth embodiments) is present as a solid dispersion comprising amorphous (R)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable polymer. In some aspects, the pharmaceutically acceptable polymer is selected from polyvinylpyrrolidone (PVP), polyvinylpyrrolidone / vinyl acetate copolymer (PVP-VA), hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose phthalate (HPMC-P) and hydroxypropyl methylcellulose acetate succinate (HPMC-AS), preferably HPMC or HPMC-AS, more preferably M grade HPMC-AS. In some aspects, the weight ratio of the pharmaceutically acceptable polymer to (R)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide ranges from 10:90 wt % to 90:10 wt %, 15:85 wt % to 85:15 wt %, 20:80 wt % to 80:20 wt %, 25:75 wt % to 75:25 wt % %, 30:70 wt % to 70:30 wt %, 35:65 wt % to 65:35 wt %, 40:60 wt % to 60:40 wt %, or 45:55 wt % to 55:45 wt %, preferably 25:75 wt % to 75:25 wt %, 30:70 wt % to 70:30 wt %, 40:60 wt % to 60:40 wt %, or 45:55 wt % to 55:45 wt %, more preferably 20 wt % to 40 wt % or 25 wt % to 35 wt %, or 50%. Other aspects of the solid dispersion are described in WO 2018 / 136596.

[0052] In one aspect, as part of the eighth embodiment, Compound 1 or a pharmaceutically acceptable salt thereof in the disclosed method (e.g., in any one of the first to seventh embodiments) is administered for at least about 4 days, at least about 6 days, at least about 8 days, at least about 12 days, at least about 18 days, at least about 30 days, at least about 60 days, at least about 6 months, or at least about 1 year.

[0053] Examples

[0054] Preparation of compound 1

[0055] (R)-7-Chloro-2-((1r,4R)-4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)benzo[d][1,3]dioxole-5-carboxamide was prepared according to the procedures described in PCT / US2019 / 027932 and PCT / US2020 / 043163, each of which is incorporated herein by reference.

[0056] Cell lines and culture

[0057] The cell lines used were obtained from ATCC (Manassas, VA), DSMZ (Brunswick, Germany), ECACC (Salisbury, UK or through Sigma), or JCRB (Osaka, Japan) and grown in the medium recommended by the supplier (or as indicated in Supplementary Methods Table 1) and kept in a humidified incubator with 5% CO at 37°C. Cell lines were maintained in T75 flasks and subcultured by release from plates with TrypLE solution (ThermoFisher Scientific / Invitrogen #12604021) every 2-4 days, depending on the growth kinetics of the cell line, to maintain growth at subconfluent levels.

[0058] Assessment of H3K27me3 levels in cells and tissues

[0059] H3K27me3 and total H3 expression levels in cells and tumor tissues were analyzed by ELISA from Meso Scale Discovery (MSD). For assays in cultured cell lines, Trypsinized cells were counted using a cell counter (Life Technologies) and plated in 100 μL of cell culture medium onto 96-well tissue culture treated plates containing compound 1 (9 concentrations in a series of 3-fold dilutions) and incubated at 37°C in 5% CO2 for 24-96 hours, depending on the assay.

[0060] In vitro elution assay

[0061] HT1376 bladder cancer cells were used for elution experiments to observe the long-term effects of compound 1 on H3K27me3 levels and gene expression. Cells were plated in T75 flasks for 4 days and treated with a specified dose of compound or DMSO control. After 4 days, the cells were washed twice with PBS and released from the flask with TrypLE solution. A portion of the cells were taken out and quickly frozen for 4-day treatment by Western blotting and qRT-PCR analysis. The remaining cells were counted and plated in 6-well plates for protein extraction and 24-well plates for RNA extraction at a density that allowed sub-confluence to grow for another 1-4 days for continuous compound treatment (treatment) or no compound treatment (elution) in duplicate wells. On the 5th, 6th, 7th and 8th days, cells were harvested from the treatment wells and elution wells of each compound for protein and RNA extraction (the 5th day sample was treated for 5 days or treated for 4 days + elution for 1 day, etc.). For Western blot analysis, cells were released from the plate, washed with PBS and quickly frozen. For qRT-PCR analysis, cells were washed and lysed directly in 24-well plates containing buffer RLT + β-mercaptoethanol (Sigma #M6250), removed into snap-top tubes and frozen for subsequent processing according to the manufacturer's instructions of the QIAGEN mini RNeasy kit.

[0062] Western blotting

[0063] The snap-frozen culture cell pellet was thawed on ice and lysed on ice with RIPA-500 buffer (1X RIPA buffer (Boston BioProducts #BP-116T), 350nM NaCl, 0.1% Benzonase, 1X Complete EDTA-free protease inhibitor (Roche #11873580001); NaCl adjusted to 500nM after initial lysis) for 20-30 minutes. The lysate was centrifuged at 13,000rpm for 15 minutes at 4°C. The supernatant was transferred to a new microcentrifuge tube, and the protein concentration was determined by the BCA method and the absorbance was read at A252nm. If necessary, the protein lysate was diluted to the same concentration and volume in 6x SDS sample buffer + β-mercaptoethanol (Boston BioProducts #BP-111NR or #BP-605) to obtain a 1X final concentration. 12-40ug total protein was loaded on SDS-PAGE gel (NuPAGETM 4%-12% Bis-Tris Midi Protein Gel, Invitrogen #WG1402BOX) and run with 1XNuPAGETM MES SDS running buffer (Invitrogen #NP0002-02). Protein was transferred to PVDF membrane (Immobilon-P, Millipore Sigma #IPVH00010) and washed with 1XTBST (containing 2% skim milk powder) The membranes were blocked with 20X Tris-buffered saline (20X Tris-buffered saline, Boston Bioproducts, #BB-180X) and combined with antibodies for western blot analysis.

[0064] For CUT&RUN experiments, HT1376-TetON control cells and cloned HT1376-TetON-ARID1A cell lines were induced with 50ng / ml doxycycline for 24 hours and then treated with DMSO or 250nM compound 1 for 4 days. Two samples were collected for each condition and treated as duplicate samples for ARID1A and SMARCA4 CUT&RUN. For H3K27me3, H3K27ac, and H3K4me3, each CUT&RUN was repeated once. Cells were fixed by adding formaldehyde to a final concentration of 0.1%. Fix for 1 minute at room temperature. Crosslinking was terminated by adding glycine to a final concentration of 125mM. Fixed cells were quickly frozen and shipped by Epicypher (Durham, North Carolina) for CUT&RUN treatment (see the relevant section of the method for details).

[0065] Xenograft Tumor Studies

[0066] Cell line-derived xenograft tumor experiments were performed at WuXi AppTec, Shanghai, China. Patient-derived xenograft experiments were performed at Crown BioScience Inc., Taicang, Jiangsu Province, China, or Champions Oncology, Rockville, Maryland, USA. All procedures related to animal handling, care, and treatment in xenograft studies were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec or Crown Biotech Co., Ltd., following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Mice were housed in individually ventilated cages at a constant temperature and humidity, with 3-5 animals per cage. Mice were examined daily for any effects of tumor growth and / or treatment on normal behavior, such as activity, food and water consumption, weight gain / loss, eyes, and any other abnormal effects. If death and observed clinical signs occurred, they were recorded, and animals observed to be in a state of continuous deterioration or with a tumor size exceeding 3000 mm3 were euthanized before reaching a moribund state. Animal body weights were monitored regularly as an indirect measure of toxicity. After cell inoculation, all groups were provided with a supplemental diet to help maintain body weight. If an animal lost >15% body weight, treatment was suspended accordingly and then resumed when weight loss returned to <10%.

[0067] Cell line derived xenograft studies:

[0068] HT1376 and HT1197 bladder cancer cell lines and Karpas-422 lymphoma cell lines were amplified in vitro in the medium recommended by the supplier under the conventional subculture procedure, harvested in the exponential growth phase, and counted for tumor inoculation. For HT1376 and Karpas-422 studies, female CB17 SCID mice of 6-8 weeks of age were used to initiate the study; for HT1197 studies, female Balb / c nude mice of 6-8 weeks of age were used. Tumor cells in PBS mixed with Matrigel (BD Biosciences) were inoculated subcutaneously in the right flank of each mouse; 5x106 cells were injected at each time for HT1376 and Karpas-422 cell lines, and 1x107 cells were injected at each time for HT1197 cells. Mice were randomized and drug treatment was initiated 11-15 days after inoculation, when tumors reached an average of 139-160 mm3; animals were distributed so that each treatment group had a similar starting tumor size (3-21 animals per group, depending on the experiment and sampling schedule). Mice that had an extended dosing holiday due to weight loss (>10 days, n=2 mice in the 150 mg / kg compound 1HT1376 group only) were removed from the study and are not included in the data presented.

[0069] Tumor size was measured in two dimensions using a caliper three times a week and the volume was expressed in mm3 using the following formula: V = 0.5 a x b2, where a and b are the long and short diameters of the tumor, respectively. The TGI of each group was calculated using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100; Ti is the average tumor volume of the treatment group on a certain day, T0 is the average tumor volume of the treatment group on the day of treatment start, Vi is the average tumor volume of the vehicle control group on the same day as Ti, and V0 is the average tumor volume of the vehicle group on the day of treatment start.

[0070] Example 1 - Enrichment of phenotypic responses in the context of ARID1A LOF mutations

[0071] A panel of 21 bladder cancer cell lines was evaluated for their response to compound 1. Compound 1 potently inhibited growth in a subset of cell lines after 18 days of compound treatment, GI 50 3-37nM. Figure 1 Table 1. Growth inhibition was significantly enriched in bladder cancer cell lines carrying at least one ARID1A LOF allele (p = 3.7e-6, chi-square test), with 83% (5 of 6) of sensitive cell lines carrying truncating mutations (frameshift or nonsense) in ARID1A. In contrast, only 6% (1 of 15) of the 18-day GI 50 Non-responsive cell lines (hereafter referred to as "resistant") at >5 μM possessed the ARID1A LOF allele. No correlation was observed between Compound 1 sensitivity and baseline levels of EZH1, EZH2, H3K27me3, ARID1A, or ARID1B by Western blot.

[0072] Table 1

[0073]

[0074]

[0075] We also assessed the mutational status of a broader panel of frequently mutated BAF complex components across cancer types and found that only ARID1A genomic alterations dissociated with compound 1 response ( Figure 2 ), supporting the notion that ARID1A LOF mutations are a predictive biomarker for response to EZH2 inhibition. Notably, mutations in the histone demethylase gene KDM6A, which, like ARID1A, is recurrently mutated in bladder cancer and has been suggested to potentially confer sensitivity to EZH2 inhibition, were not associated with compound 1 response in this group ( Figure 2Compound 1 was equally effective in reducing H3K27me3 levels in both resistant and sensitive cell lines, with consistent concentration-dependent reductions in H3K27me3 levels observed at 72 h (Table 2), regardless of phenotype or ARID1A mutation status ( Figure 3 As with EZH2 inhibitors in other cancer cell line settings, the cell viability effects in compound 1-sensitive bladder cancer cell lines were time-dependent. While most bladder cancer cell lines showed little or no viability effects after 6 days of compound 1 treatment, extended treatment for 12 and 18 days significantly increased the sensitivity of ARID1A mutant cell lines ( Figure 4 ).

[0076] Table 2

[0077]

[0078] Phenotypically responsive cell lines such as HT1376 and HT1197 showed induction of cell death at day 12 as evidenced by an increase in the subG1 population, whereas the cell cycle characteristics of resistant cell lines such as T24 remained unchanged even after prolonged treatment with compound 1. Taken together, these results suggest that while compound 1 was equally effective in inhibiting the ability of PRC2 to maintain H3K27me3 levels, only the sensitive cell lines showed induction of cell death and subsequent loss of cell viability over time.

[0079] To further explore the potential dose-dependency of compound 1 on tumor growth inhibition in vivo, a HT1376 xenograft study was conducted using compound 1 doses of 10 to 150 mg / kg, QD, orally once a day (PO, QD). Compound 1 treatment achieved a dose-dependent TGI, from 30% at 10 mg / kg to 98% at 150 mg / kg by day 30, with doses <75 mg / kg well tolerated. All dose levels ≥75 mg / kg resulted in significant reductions in tumor volume compared to vehicle ( Figure 5 ). Global H3K27me3 levels in HT1376 tumors were reduced in a dose-dependent manner following Compound 1 treatment, with at least an 80% reduction at 10 mg / kg and >90% at 25 mg / kg and higher doses at day 10, maintaining comparable levels at day 31. A subsequent HT1376 xenograft study with extended treatment duration showed reproducible TGI levels at 30 days, followed by a reduction in tumor volume at both the 35 mg / kg QD and 50 mg / kg QD dose levels at time points beyond 35 days ( Fig.23 ).

[0080] We next evaluated the in vitro and in vivo efficacy of compound 1 in ARID1A LOF models of other solid cancers. Compound 1 was shown to have an in vitro and in vivo efficacy in ARID1A LOF patient-derived xenograft (PDX) models of bladder cancer and endometrial cancer ( Figure 6 , Figure 7 , Fig. 20 , Fig.21 and Fig. 22 ), and also achieved significant antitumor activity as a single agent in the ARID1A mutant TOV21G cell line-derived xenograft (CDX) model of ovarian clear cell carcinoma (OCCC), consistent with its in vitro sensitivity to compound 1 ( Figure 8 ). In summary, these data demonstrate that compound 1 is highly effective at a well-tolerated dose in ARID1A LOF solid tumor models in indications that exhibit a high frequency of ARID1A mutations. In addition, a comparative study was performed between ARID1A wild-type and ARID1A mutant PDX cancer models (Table 6).

[0081] Table 6

[0082]

[0083] Example 2 - Phenotypic restoration of ARID1A expression in ARID1A mutant bladder cancer cells by compound 1 treatment

[0084] To investigate molecular changes that may increase EZH2 dependency in the context of ARID1A mutation, we used a doxycycline inducible system to express wild-type ARID1A in the ARID1A mutant cell model HT1376. A significant decrease in H3K27me3 levels was observed in HT1376 cells treated with compound 1, regardless of ectopic ARID1A expression. Although EZH1 levels increased with compound 1 treatment, no changes in EZH2 were seen under any of the conditions tested ( Fig. 9). Restoration of ARID1A function in HT1376 cells resulted in a loss of cell viability to a similar extent as treatment with compound 1 alone, and the combination of ARID1A re-expression and compound 1 treatment did not result in a combined growth defect, suggesting that both EZH2 inhibition and ARID1A re-expression significantly affect the viability of cells that adapt to an altered epigenetic state due to ARID1A loss. Next, we sought to explore changes in chromatin binding profiles and gene expression in HT1376 cells following ARID1A re-expression and / or compound 1 treatment. We identified three clusters of active enhancers by positivity for histone H3 lysine 27 acetylation (H3K27ac), which had different behaviors in response to ARID1A or compound 1. Enhancer cluster 1 is uniquely defined by low H3K27ac and high H3K27me3 levels and shows increased enhancer activity after compound 1 treatment, as evidenced by increased H3K27ac levels and significantly reduced baseline H3K27me3 levels. Enhancer clusters 2 and 3 also showed increased enhancer activity in response to compound 1 treatment, but to a lesser extent than enhancer cluster 1. Clusters 2 and 3 (to a lesser extent) were defined primarily by increases in both ARID1A and SMARCA4 binding in response to ARID1A re-expression. When cells re-expressed ARID1A and were treated with compound 1, a combined increase in ARID1A and SMARCA4 occupancy was observed in enhancer cluster 1, suggesting that these enhancers are associated with gene targets co-regulated by PRC2 and BAF. Unsupervised clustering revealed 3 subclusters: a small subset of genes repressed by ARID1A but not altered by compound 1 (enhancer proximal subcluster 1A), genes induced by compound 1 but not ARID1A (enhancer proximal subcluster 1B), and genes induced by compound 1, ARID1A, or both (enhancer proximal subcluster 1C). Compound 1-induced genes within enhancer proximal subcluster 1C were enriched for PRC2 targets, targets of the chimeric oncogenic transcription factor PAX3-FOXO1, and p53 targets. Enhancer proximal subcluster 1B was enriched for PRC2 targets, but no gene sets were significantly enriched in enhancer proximal subcluster 1A.

[0085] Analysis of CUT&RUN peaks in gene bodies and flanking regions identified two clusters of genes, both of which shifted (to varying degrees) from a repressed state (high H3K27me3 / low H3K27ac) to a more permissive state (low H3K27me3 / high H3K27ac) following treatment with compound 1. Genes in promoter cluster 1 also showed an increase in ARID1A and SMARCA4 binding following compound 1 treatment, suggesting that inhibition of EZH2 promotes recruitment of the BAF complex to these promoters. Expression analysis of genes within promoter cluster 1 showed that the majority of these genes were induced by compound 1, consistent with a dominant PRC2 function in repressing these genes, and were minimally responsive to ARID1A re-expression. Notably, while genes in promoter subcluster 1A showed an additive induction of gene expression similar to genes in enhancer proximal subcluster 1C following compound 1 treatment and ARID1A re-expression, the two gene sets had only a few genes in common.

[0086] Principal component analysis (PCA) of global gene expression patterns in samples indicated that compound 1 treatment and ARID1A re-expression were the main drivers of changes in gene expression. The vast majority (>94%) of genes altered by compound 1 treatment were significantly upregulated (Log2 fold change (L2FC) ≥ 1), consistent with a role for PRC2 in gene repression. ARID1A re-expression resulted in variable changes in gene expression, with approximately 62% of significantly altered genes being downregulated by ARID1A and approximately 38% being upregulated, consistent with a role for ARID1A in both transcriptional repression and activation. GSEA of genes upregulated by ARID1A re-expression revealed enrichment for EZH2 targets (defined as targets induced by compound 1 treatment and having EZH2 and H3K27me3 peaks). See Fig.10 Similarly, GSEA of genes upregulated after compound 1 treatment showed enrichment of ARID1A re-expression targets. Fig.11 . Comprehensive GSEA analysis using the HallmarkCollection showed that more than half of the enriched gene sets were commonly enriched by compound 1 treatment and re-expression of ARID1A, including pathways regulating cell differentiation, immune signaling, and inflammation. Taken together, these data suggest that ARID1A LOF mutations lead to an imbalance in epigenetic gene regulation within a subset of BAF- and PRC2-regulated pathways that promote cancer cell growth. These cancer cells rely on PRC2-mediated repression of these gene targets, and compound 1-mediated EZH2 inhibition may allow re-expression of these genes to affect cell viability in the context of ARID1A LOF.

[0087] Example 3 - Clinical Efficacy

[0088] As part of a human Phase 2 clinical study (NCT04104776), subjects with ovarian clear cell carcinoma (cohort M2) and endometrial cancer (cohort M3) were administered 350 mg / day of compound 1 for the duration of treatment, respectively. Fig.13 and Fig.14 As shown. Compound 1 was administered orally as a single agent (monotherapy). Each subject's cancer was known to have an ARID1A mutation, as determined by next-generation sequencing (NGS) prior to treatment. The responses of efficacy-evaluable patients by cancer cohort at the intermediate cutoff date are shown in Table 3. The assessment of changes in tumor burden was based on RECIST 1.1 criteria. Complete remission was characterized by the disappearance of all lesions, and partial remission was characterized by a reduction of at least 30% in the sum of the longest diameters (LD) of target lesions, with the baseline LD sum as the reference; stable disease was characterized by neither sufficient shrinkage to meet the conditions for partial remission nor sufficient increase to meet the conditions for progressive disease, with the minimum diameter sum as the reference; progressive disease was characterized by an increase of at least 20% in the LD sum of target lesions, with the minimum LD sum recorded since the start of treatment or the appearance of one or more new lesions as the reference. In order to be designated as a status of partial or complete remission, changes in tumor measurements must be confirmed by repeated assessments no less than 4 weeks after the response criteria were first met.

[0089] Table 3

[0090]

[0091] In addition, most patients with OCCC or EC were found to have low tumor mutational burden. TMB-low status was defined as <10mut / Mb and assessed by NGS (Tempus xT & Predicine ATLAS targeted panel sequencing).

[0092] Example 4 - In vivo comparison of Compound I with other PRC2 inhibitors in an ARID1A mutant bladder cancer xenograft model

[0093] The responses to compound 1 treatment relative to other EZH2, dual EZH1 and EZH2, or EED inhibitors were analyzed in vitro and in vivo. In an in vitro long-term cell viability assay, compound 1 had a GI 50 The potency was comparable to PF-06821497 (approximately 5 nM), while valemetostat and the EED inhibitor MAK683 were 3-10 times less potent (GI 50 : 18 and 73 nM, respectively). The first generation of EZH2 inhibitors, such as tazemetostat (GI 50 =385 nM) and CPI-1205 (GI 50=574nM), with lower potency in the viability assay, consistent with their lower target affinity and shorter residence time. When administered at the same dose (75mg / kg QD), Compound 1, valmetostostat, and MAK683 showed >50% tumor growth inhibition (TGI) compared to vehicle in the ARID1A mutant HT1376 xenograft mouse model ( Fig.15 ). At this dose, compound 1 exhibited significantly more robust and durable antitumor activity than valmetuxostat (p=0.0001), with tumor growth in the valmetuxostat group rebounding after 35 days of treatment, while compound 1-treated tumors continued to regress to below initial tumor volume on day 37. Tazemetostat, CPI-1205, and PF-06821497 resulted in TGI <50% (32%, 40%, and 41% TGI on day 27, respectively). Overall, compound 1 caused complete tumor regression in the ARID1A mutant bladder cancer xenograft model. In mice treated with valmetuxostat, tumor volume decreased within the first 35 days of treatment, but complete regression was not achieved, while mice treated with tazemetostat showed an increase in volume during treatment ( Fig.15 All compounds were well tolerated at 75 mg / kg QD, and the plasma exposure of compound 1 was no greater than that of any other compound (Table 4).

[0094] Table 4. Pharmacokinetic properties of EZH2 inhibitors in the ARID1A mutant HT1376 xenograft mouse model

[0095]

[0096] Compounds that resulted in >50% TGI strongly reduced H3K27me3 (>90%) in tumors collected on day 15, whereas compounds with weaker TGI retained higher levels of H3K27me3 ( Fig.16 ), indicating a link between tumor response and the extent of H3K27me3 reduction ( Fig.17 ). However, a >90% reduction in global H3K27me3 levels could not distinguish TGI differences between the most potent compounds, compound 1, valmetostostat, and MAK683. Gene expression profiling of day 15 tumors revealed that compound 1 induced significantly more EZH2 / PRC2 target genes than any other compound (Table 5), and the overall changes in gene expression levels were consistent with reduced H3K27me3 and tumor response ( Fig.18 and Fig.24 ). In contrast to global changes in H3K27me3 levels, a greater number of altered genes was associated with a greater magnitude of TGI, suggesting that gene expression changes may be a preferred biomarker for correlating target inhibition and efficacy.

[0097] Table 5 Significantly enriched gene sets in enhancer subclusters from the C2-curated gene set list from MSigDB (FDR<0.01).

[0098]

[0099]

[0100] The contents of all references cited in this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly hereby incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein are given the meanings commonly recognized by those of ordinary skill in the art.

Claims

1. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a compound having the formula: or a pharmaceutically acceptable salt thereof, wherein the cancer has at least one ARID1A mutation.

2. The method of claim 1, wherein the cancer is selected from bladder cancer, breast cancer, endometrial cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, bile duct cancer, gastric cancer, hepatocellular carcinoma, liver cancer, lung cancer, melanoma, and ovarian cancer.

3. The method of claim 1 or 2, wherein the cancer is selected from bladder cancer, endometrial cancer, and ovarian clear cell carcinoma.

4. The method of any one of claims 1 to 3, wherein the cancer is bladder cancer.

5. The method of claim 4, wherein the bladder cancer is urothelial carcinoma.

6. The method of claim 4 or 5, wherein the bladder cancer is advanced urothelial carcinoma.

7. The method of any one of claims 1 to 3, wherein the cancer is endometrial cancer.

8. The method of any one of claims 1 to 3, wherein the cancer is ovarian cancer.

9. The method of claim 8, wherein the cancer is ovarian clear cell carcinoma.

10. The method of any one of claims 1 to 9, wherein the at least one ARID1A mutation is a loss-of-function (LOF) mutation.

11. The method of any one of claims 1 to 10, wherein the compound or pharmaceutically acceptable salt is administered for at least 6 days.

12. The method of any one of claims 1 to 11, wherein administration results in restoration of ARID1A expression.

13. The method of any one of claims 1 to 12, wherein administration results in increased SMARCA4 binding.

14. The method of any one of claims 1 to 13, wherein administration results in increased ARID1A binding.

15. The method of any one of claims 1 to 14, wherein administration results in a decrease in global H3K27me3 levels.

16. The method of any one of claims 1 to 15, wherein administration results in a sustained reduction in tumor volume.

17. The method of any one of claims 1 to 16, wherein administration drives re-expression of a repressor gene.

18. The method of any one of claims 1 to 17, wherein the cancer is characterized by a low tumor mutational burden.

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