EZH2 Inhibitor Therapy for Treating ARID1A-Mutated Cancers

Abstract

Provided herein is a method of treating cancer having at least one ARID1A mutation with (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 or a pharmaceutically acceptable salt thereof.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 389,436, filed July 15, 2022; U.S. Provisional Patent Application No. 63 / 413,323, filed October 5, 2022; and U.S. Provisional Patent Application No. 63 / 455,024, filed March 28, 2023, the entire contents of each of which are incorporated herein by reference.

Background Art

[0002] Mutations in the ARID1A gene, which encodes the fundamental orientation subunit of the SWI / SNF chromatin remodeling complex, have emerged in several groups of cancers, including subtypes of ovarian, endometrial, and uterine cancers. ARID1A is a tumor suppressor, and its deficiency causes increased cell proliferation, migration, and invasion, as well as decreased cell apoptosis and chemosensitivity. ARID1A is mutated in 25% of muscle-invasive bladder cancers, and high frequencies of ARID1A mutations have been reported in several indications, including ovarian clear cell carcinoma (46 - 57%), endometrial cancer (30 - 40%), and gastric cancer (20%).

Summary of the Invention

Problems to be Solved by the Invention

[0003] Despite efforts to combine ongoing clinical explorations of cancer immunotherapy in combination with multiple cancer immunotherapy agents and other therapies, ARID1A mutant cancers, particularly advanced urothelial carcinoma, a subtype of bladder cancer, generally remain incurable, and only a small number of patients respond to second-line or later treatment options with limited life-prolonging effects. Thus, new approaches to target cancers harboring one or more ARID1A mutations are needed.

Means for Solving the Problems

[0004] Here, it has been found that cancers having at least one ARID1A mutation exhibit 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. For example, in cell lines having at least one ARID1A loss-of-function (LOF) allele, in which 83% (5 out of 6) of the sensitive cell lines (GI50 of 3-37 nM after 18 days) harbor truncating mutations (frameshift or nonsense), after treatment with Compound 1, the growth inhibitory effect was significantly enriched in a panel of 21 bladder cancer cell lines (p = 3.7e-6, chi-square test); see FIGS. 1, 19, and Table 1. In contrast, only 6% (1 out of 15) of the non-responsive cell lines with a GI50 > 5 μM on day 18 had an ARID1A LOF allele. Additionally, treatment with Compound 1 significantly inhibited tumor growth in patient-derived xenograft (PDX) models of ARID1A LOF endometrial cancer (FIG. 7) and in xenograft (CDX) models derived from the ARID1A mutant TOV21G cell line of ovarian clear cell carcinoma (OCCC) (FIG. 8). However, unexpectedly, colorectal cancers, lung cancers, and non-clear cell ovarian cancers having ARID1A mutations did not generally exhibit increased sensitivity to treatment. For example, no clear correlation was observed between the mutation status and responsiveness; see FIG. 12. Clinical efficacy was also observed in subjects having cancers harboring ARID1A mutations (FIGS. 13, 14). The amount of tumor gene mutations was found to be low for most patients having OCCC or EC. Thus, in one aspect, provided is a method of treating ARID1A mutant cancers having a low amount of tumor gene mutations using Compound 1. In one aspect, the amount of tumor gene mutations is less than 10 mut / Mb.

[0005] Compound 1 was also found to exhibit a higher efficacy in some ARID1A mutant PDX cancer models compared to the efficacy in the corresponding ARID1A wild-type cancer indications (Table 6). Thus, in one aspect, some ARID1A mutant cancers exhibit a higher efficacy than the corresponding ARID1A wild-type cancers.

[0006] Thus, in one aspect, provided is a method of treating cancer having at least one ARID1A mutation using Compound 1. Also provided is the use of Compound 1 for the manufacture of a medicament for treating said cancer.

[0007] Compound 1 was also found to restore ARID1A expression in ARID1A mutant bladder cancer cells. For example, GSEA of genes upregulated after Compound 1 treatment showed enrichment in ARID1A re-expression targets. See, for example, FIG. 11. Thus, in one aspect, provided is a method of restoring ARID1A expression in cancer having at least one ARID1A allele using Compound 1. Also provided is the use of Compound 1 for the manufacture of a medicament for restoring ARID1A expression in cancer having at least one ARID1A allele. BRIEF DESCRIPTION OF THE DRAWINGS

[0008]

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Mode for Carrying Out the Invention

[0009] In a first embodiment, there is provided a method of treating cancer in a subject, 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 cancer), endometrial cancer, ovarian cancer, ovarian clear cell carcinoma, breast cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, stomach cancer, hepatocellular carcinoma, liver cancer, lung cancer, and melanoma. In one aspect, the cancer is selected from bladder cancer (e.g., urothelial cancer), endometrial cancer, and ovarian clear cell carcinoma.

[0010] 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 manufacture of a medicament for treating cancer having at least one ARID1A mutation. Such cancers include, but are not limited to, bladder cancer (e.g., urothelial cancer), endometrial cancer, ovarian cancer, ovarian clear cell carcinoma, breast cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, stomach cancer, hepatocellular carcinoma, liver cancer, lung cancer, and melanoma. In one aspect, the cancer is selected from bladder cancer (e.g., urothelial cancer), endometrial cancer, and ovarian clear cell carcinoma.

[0011] 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 treating 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, cholangiocarcinoma, stomach 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.

[0012] As part of the first embodiment, there is also provided a pharmaceutical composition comprising an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof for treating 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, cholangiocarcinoma, stomach 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.

[0013] 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 refer to compounds having the following chemical structures, respectively. [Chemical formula]

[0014] In one aspect, as part of the second embodiment, the cancer treated by the method is bladder cancer. In another aspect, as part of the second embodiment, the cancer treated by the method is urothelial cancer. In another aspect, as part of the second embodiment, the cancer treated by the method is advanced urothelial cancer (e.g., urothelial cancer that has spread to another part of the body). In another aspect, as part of the second embodiment, the cancer treated by the method is endometrial cancer. In yet another aspect, as part of the second embodiment, the cancer treated by the method is ovarian clear cell carcinoma.

[0015] In the third embodiment, at least one ARID1A mutation of the method (e.g., in the first or second embodiment) is a loss-of-function (LOF) mutation. In another aspect, as part of the third embodiment, at least one ARID1A mutation of the method (e.g., in the first or second embodiment) is a truncating mutation (frameshift or nonsense). In another aspect, as part of the third embodiment, at least one ARID1A mutation of the method (e.g., in the first or second embodiment) is Q557 * and the cancer is urothelial cancer. In another aspect, as part of the third embodiment, at least one ARID1A mutation of the method (e.g., in the first or second embodiment) is selected from G1340fs, S301fs, P302fs, P1326fs and R693, Q557 * and the cancer is endometrial cancer. In another aspect, as part of the third embodiment, at least one ARID1A mutation of the method (e.g., 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, at least one ARID1A mutation of the method (e.g., in the first or second embodiment) is selected from N1216fs and A162Rfs * 238 and the cancer is endometrial cancer.

[0016] As used herein, an ARID1A LOF mutation refers to a mutation that reduces or abolishes ARID1A protein function. The LOF can be due to nonsense-mediated decay defects in activity or loss of expression due to protein truncation (loss of critical residues or domains).

[0017] The terms "treating," "treat," and "treatment" refer to reversing, reducing, delaying the onset of, or inhibiting the progression of cancer or one or more symptoms of a disease described herein. In some embodiments, treatment can be administered after one or more signs or symptoms of cancer have developed or been recognized (i.e., therapeutic treatment). In other embodiments, treatment can be administered in the absence of signs or symptoms of cancer. For example, treatment can be administered to a susceptible subject prior to the onset of symptoms (i.e., prophylactic treatment) (e.g., based on a medical history of symptoms and / or exposure to a pathogen). In further embodiments, treatment includes delaying the onset of at least one symptom of cancer for a period of time. Treatment can also continue after symptoms have resolved, for example, to delay or prevent recurrence (i.e., maintenance treatment).

[0018] The terms "subject" and "patient" can be used interchangeably and refer to a mammal in need of treatment, such as a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, pig, horse, sheep, goat, etc.), and a laboratory animal (e.g., rat, mouse, guinea pig, etc.). Typically, the subject is a human in need of treatment.

[0019] The term "effective amount" or "therapeutically effective amount" refers to an amount of Compound 1 or a pharmaceutically acceptable salt thereof that will induce a biological or medical response in a subject, for example, a dosage in the range of 0.01 to 100 mg / kg body weight / day. In one aspect, as part of the fourth embodiment, the effective amount of Compound 1 in the method (e.g., in any one of the first to third embodiments) is 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 Compound 1 in the method (e.g., in any one of the first to third embodiments) is 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 Compound 1 in the method (e.g., in any one of the first to third embodiments) is in the range of 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 (e.g., in any one of the first to third embodiments) is in the range of about 175 mg to about 325 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of Compound 1 in the method (e.g., in any one of the first to third embodiments) is in the range of about 200 mg to about 300 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of Compound 1 in the method (e.g., in any one of the first to third embodiments) is in the range of about 225 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 (e.g., in any one of the first to third embodiments) is 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 Compound 1 in the method (e.g., in any one of the first to third embodiments) is in the range of about 350 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 (e.g., in any one of the first to third embodiments) is about 200 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of Compound 1 in the method (e.g., in any one of the first to third embodiments) is about 250 mg per day.In another aspect, as part of the fourth embodiment, the effective amount of Compound 1 in the present method (e.g., in any one of the first to third embodiments) is about 300 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of Compound 1 in the present method (e.g., in any one of the first to third embodiments) is about 350 mg per day. In another aspect, as part of the fourth embodiment, the effective amount of Compound 1 in the present method (e.g., 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 present method (e.g., in any one of the first to third embodiments) is equal to the 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 present method (e.g., in any one of the first to third embodiments) is equal to the 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 present method (e.g., in any one of the first to third embodiments) is equal to the 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 present method (e.g., in any one of the first to third embodiments) is equal to the amount of Compound 1 in the range of about 350 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 present method (e.g., in any one of the first to third embodiments) is equal to the amount of Compound 1 of about 350 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 present method (e.g., in any one of the first to third embodiments) is equal to the amount of Compound 1 of about 375 mg per day.

[0020] The administration methods described in this specification can be carried out orally, parenterally, by inhalation spray, locally, rectally, nasally, buccally, vaginally or via an implant reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intramedullary, intrahepatic, intralesional and intracranial injection or infusion techniques. The sterilizable injectable form of Compound 1 described herein can be an aqueous or oily suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. In one aspect, as part of the fifth embodiment, Compound 1 in the method (for example, in any one of the first to fourth embodiments) is administered orally.

[0021] Compound 1 can exist in the form of a pharmaceutically acceptable salt. For use in a medicament, a pharmaceutically acceptable salt refers to a non-toxic "pharmaceutically acceptable salt". Pharmaceutically acceptable salt forms include, where possible, pharmaceutically acceptable acidic / anionic salts or basic / cationic salts.

[0022] 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 together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used in the compositions described herein (for example, carriers, adjuvants or vehicles) include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, etc.), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0023] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant, or vehicle that, when formulated, does not adversely affect the pharmacological activity of the compound and is safe for human use. Pharmaceutically acceptable carriers, adjuvants, or vehicles that can be used in the compositions of the present disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., microcrystalline cellulose, hydroxypropylmethylcellulose, lactose monohydrate, sodium lauryl sulfate and croscarmellose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, and lanolin. In one aspect, as part of the sixth embodiment, compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) exists in a certain crystalline form. The crystalline form of compound 1 is disclosed in WO 2021 / 016414 pamphlet and incorporated herein by reference. In another aspect, as part of the sixth embodiment, compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is crystalline form 1 characterized by at least three X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. In another aspect, as part of the sixth embodiment, compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is crystalline form 1 characterized by at least four X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°.In another aspect, as part of the sixth embodiment, Compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is Crystal Form 1 characterized by at least five X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. In another aspect, as part of the sixth embodiment, Compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is Crystal Form 1 characterized by at least six X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. In another aspect, as part of the sixth embodiment, Compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is Crystal Form 1 characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, 22.2°, and 22.5°. In another aspect, as part of the sixth embodiment, Compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is Crystal Form 1 characterized by X-ray powder diffraction peaks at 2Θ angles selected from 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 the sixth embodiment, Compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is Crystal Form 1 characterized by X-ray powder diffraction peaks at 2Θ angles selected from 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 the sixth embodiment, Compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is Crystal Form 1 characterized by X-ray powder diffraction peaks at 2Θ angles selected from 14.9°, 20.2°, and 20.8°.In another aspect, as part of the sixth embodiment, compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is crystalline form 1 characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 14.9°, 20.2°, and 20.8°. In another aspect, as part of the sixth embodiment, compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is crystalline form 1 characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 14.9°, 20.2°, 20.8°, and 22.2°. In another aspect, as part of the sixth embodiment, compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) is crystalline form 1 characterized by X-ray powder diffraction peaks at 2Θ angles selected from 10.0°, 13.3°, 14.9°, 20.2°, 20.8°, and 22.2°. In one aspect, as part of the seventh embodiment, compound 1 in the disclosed method (e.g., in any one of the first to fifth embodiments) or a pharmaceutically acceptable salt thereof exists 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), hydroxypropylmethylcellulose (HPMC), hypromellose phthalate (HPMC-P), and hypromellose acetate succinate (HPMC-AS), preferably HPMC or HPMC-AS, more preferably HPMC-AS grade M.In some embodiments, the weight ratio of the pharmaceutically acceptable polymer pair 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 is 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 in the range of 20 wt% to 40 wt% or 25 wt% to 35 wt% or 50%. Other embodiments of the solid dispersant are described in WO 2018 / 136596.

[0024] In one embodiment, as part of the eighth embodiment, compound 1 (e.g., in any one of the first to seventh embodiments) or a pharmaceutically acceptable salt thereof in the disclosed method is administered over a period of 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.

Example

[0025] Preparation of Compound 1 (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.

[0026] Cell lines and culture The cell lines used were obtained from ATCC (Manassas, VA), DSMZ (Braunschweig, Germany), ECACC (through Salisbury, UK or Sigma), or JCRB (Osaka, Japan), grown in the medium recommended by the vendor (or as shown in the supplementary method in Table 1), and maintained at 37 °C in a humidified incubator with 5% CO2. The cell lines were maintained in T75 flasks and passaged every 2 - 4 days by releasing them from plates with TrypLE solution (Thermo Fisher Scientific / Invitrogen #12604021) according to the growth kinetics of the cell lines, and growth was maintained at sub-confluent levels.

[0027] Evaluation of H3K27me3 levels in cells and tissues The levels of H3K27me3 and total H3 expression in cells and tumor tissues were analyzed by Meso Scale Discovery (MSD) ELISA. For assays using cultured cell lines, trypsinized cells were counted using a Countess® cell counter (Life Technologies) according to the assay, and seeded at 9 concentrations in a series of 3-fold dilutions in 100 μL of cell culture medium on 96-well tissue culture-treated plates containing compound 1, and incubated at 37 °C in 5% CO2 for 24 - 96 hours.

[0028] In vitro washout assay HT1376 bladder cancer cells were used in the washout experiment to observe the extended effects of compound 1 on H3K27me3 levels and gene expression. Cells were seeded in T75 flasks for 4 days and treated with the designated dose of the 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 was removed and snap-frozen for analysis during the 4-day treatment by Western blot and qRT-PCR. The remaining cells were counted and seeded into two types of wells, and compound treatment was continued (during treatment) or not (washout) in 6-well plates for protein extraction and 24-well plates for RNA extraction at a density that allowed for further sub-confluent growth for 1 - 4 days. For each compound, cells were collected on days 5, 6, 7, and 8 for both protein and RNA extraction from the during-treatment wells and washout wells (the day 5 sample was, for example, during 5-day treatment or 4-day treatment + 1-day washout, etc.). For Western blot analysis, the cells were released from the plates, washed with PBS, and snap-frozen. For qRT-PCR analysis, following the manufacturer's instructions for the QIAGEN mini RNeasy kit, the cells were washed and directly lysed in 24-well plates with buffer RLT + β-mercaptoethanol (Sigma #M6250), removed to snap-cap tubes, and frozen for later processing.

[0029] Western blotting The rapidly frozen pellet of cultured cells was thawed on ice and dissolved in RIPA-500 buffer (1×RIPA buffer (Boston BioProducts #BP-116T), 350 nM NaCl, 0.1% Benzonase, 1×complete EDTA-free protease inhibitor (Roche #11873580001); after initial dissolution, NaCl adjusted to 500 nM) on ice for 20 - 30 minutes. The lysate was centrifuged at 4°C and 13,000 rpm for 15 minutes. The supernatant was transferred to a new microtube, and the protein concentration was measured by the BCA method with absorbance readings at A252 nm. If necessary, the protein lysate was diluted to the same concentration and the volume was made up to 6×SDS sample buffer + β-mercaptoethanol (Boston BioProducts #BP-111NR or #BP-605) to obtain a final concentration of 1×. 12 - 40 μg of total protein was loaded onto an SDS-PAGE gel (NuPAGE™ 4 - 12% Bis-Tris Midi Protein Gels, Invitrogen #WG1402BOX) and run with 1×NuPAGE™ MES SDS running buffer (Invitrogen #NP0002-02). The protein was transferred to a PVDF membrane (Immobilon-P, Millipore Sigma #IPVH00010), blocked with 1×TBST (20× Tris-buffered saline with Tween® 20, Boston BioProducts #BB-180X) containing 2% non-fat dry milk, bound to the antibody, and prepared for Western blot analysis.

[0030] For CUT&RUN experiments, HT1376-TetON control cells and the cloned HT1376-TetON-ARID1A cell line were induced with 50 ng / ml doxycycline for 24 hours and then treated with DMSO or 250 nM compound 1 for 4 days. Two samples were collected per condition and processed as replicates for ARID1A and SMARCA4 CUT&RUN. For H3K27me3, H3K27ac, and H3K4me3, a single replicate was performed for each CUT&RUN. Cells were fixed by adding formaldehyde to a final concentration of 0.1%. Fixation was carried out for 1 minute at room temperature. Crosslinking was stopped by adding glycine to a final concentration of 125 mM. Fixed cells were snap-frozen and shipped by Epicypher (Durham, NC) in preparation for CUT&RUN treatment (see the relevant section of the methods for details).

[0031] Xenograft tumor assay Xenograft tumor experiments using cell lines were conducted at WuXi AppTec, Shanghai, China. Patient-derived xenograft experiments were conducted at Crown BioScience Inc., Taicang, Jiangsu Province, China or Champions Oncology, Rockville, MD, US. All procedures regarding the handling, care, and treatment of animals in xenograft studies were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec or Crown BioScience Inc. in accordance with the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Mice were maintained in individual ventilated cages at a constant temperature and humidity, with 3 - 5 mice per cage. Mice were examined daily for tumor growth and / or any effects of treatment on normal behavior such as mobility, food and water consumption, weight gain / loss, eyes, and any other abnormal effects. Deaths and observed clinical signs were recorded if they occurred, and animals that were found to be in a deteriorating condition or whose tumor size exceeded 3000 mm3 were euthanized before reaching a moribund state. As an indirect measure of toxicity, the body weight of animals was monitored regularly. To assist with weight maintenance for all groups, dietary supplementation was provided after cell inoculation. Therefore, if an animal lost >15% of its body weight, the treatment was discontinued and then restarted if the weight loss recovered to <10%.

[0032] Cell line-derived xenograft study: The HT1376 and HT1197 bladder cancer cell lines and the Karpas-422 lymphoma cell line were expanded in vitro in the medium recommended by the supplier under normal subculture procedures, collected during the logarithmic growth phase, and counted for tumor inoculation. For the HT1376 and Karpas-422 tests, 6- to 8-week-old female CB17 SCID mice were used at the start of the test, and for the HT1197 test, 6- to 8-week-old female Balb / c nude mice were used. Tumor cells in 0.2 ml of PBS mixed with Matrigel (BD Biosciences) were subcutaneously inoculated into the right flank of each mouse, using 5×106 cells per injection for the HT1376 and Karpas-422 cell lines and 1×107 cells per injection for the HT1197 cells. The mice were randomized, and drug treatment was started 11 to 15 days after inoculation when the tumors reached an average of 139 to 160 mm3, and the animals were distributed so that each treatment arm had a similar starting tumor size (3 to 21 animals per arm, depending on the experiment and sampling schedule). Mice receiving extended dosing on holidays due to weight loss (>10 days, n = 2 mice in the HT1376 group with 150 mg / kg of Compound 1) were terminated from the study and not included in the presented data.

[0033] Tumor size was measured in two dimensions using calipers three times a week, and the volume was expressed in mm3 using the formula: V = 0.5a × b2, where a and b are the long and short diameters of the tumor, respectively. For each group, the TGI was calculated using the formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti is the mean tumor volume of the treatment group on a specific day, T0 is the mean tumor volume of the treatment group on the treatment start day, Vi is the mean tumor volume of the vehicle control group on the same day as Ti, and V0 is the mean tumor volume of the vehicle group on the treatment start day.

[0034] Example 1 - Phenotypic responses are enriched in association with ARID1A LOF mutations A panel of 21 bladder cancer cell lines was evaluated for their responses to Compound 1. Compound 1 had a GI of 3 - 37 nM 18 days after compound treatment 50The growth of a subset of stocks having it was effectively inhibited. See FIGS. 1 and 1. The growth inhibitory effect was significantly enriched in bladder cancer cell lines having at least one ARID1A LOF allele (p = 3.7e-6, chi-square test), and 83% (5 out of 6) of the sensitive cell lines contained a truncating mutation (frameshift or nonsense) in ARID1A. In contrast, only 6% (1 out of 15) of the non-responsive cell lines with GI 50 >5 μM have an ARID1A LOF allele. No association was found between the sensitivity of Compound 1 and the baseline levels of EZH1, EZH2, H3K27me3, ARID1A or ARID1B by Western blot.

[0035]

Table 1

[0036] The inventors also evaluated the mutational status of a broader set of BAF complex components that were frequently mutated across cancer types and found that only ARID1A genomic alterations segregated from Compound 1 response (FIG. 2), supporting the concept that ARID1A LOF mutations are predictive biomarkers for response to EZH2 inhibition. In particular, similar to ARID1A, mutations in the histone demethylase gene KDM6A, which are recurrently mutated in bladder cancer and are thought to potentially confer sensitivity to EZH2 inhibition, are not associated with Compound 1 response in this panel (FIG. 2). Compound 1 is equally effective at reducing H3K27me3 levels in both resistant and sensitive cell lines, and a consistent concentration-dependent decrease in H3K27me3 levels was observed after 72 hours (Table 2), which is independent of phenotype or ARID1A mutational status (FIG. 3). In relation to other cancer cell lines, similar to EZH2 inhibitors, the cell viability effect in Compound 1-sensitive bladder cancer cell lines is time-dependent. Most bladder cancer cell lines show little or near-zero viability effects 6 days after Compound 1 treatment, while extended treatment for 12 and 18 days substantially increased the sensitivity of ARID1A mutant cell lines (FIG. 4).

[0037]

Table 2

[0038] Cell lines showing phenotypic responses such as HT1376 and HT1197 showed induction of cell death on day 12, as evident from the increase in the sub-G1 population, while the cell cycle characteristics of resistant cell lines such as T24 remained unchanged even after extended treatment with Compound 1. Collectively, these results indicate that Compound 1 is equally effective in inhibiting the ability to maintain H3K27me3 levels of PRC2, while only sensitive cell lines show induction of cell death and subsequent time-dependent decrease in cell viability.

[0039] To further explore the potential dose-dependence of Compound 1 on tumor growth inhibition in vivo, an HT1376 xenograft study was conducted using oral doses (PO, QD) of Compound 1 at 10 - 150 mg / kg QD once daily. Dose-dependent TGI was obtained with Compound 1 treatment, ranging from 30% at 10 mg / kg to 98% at 150 mg / kg up to 30 days, and doses < 75 mg / kg had sufficient tolerance. All dose levels ≧ 75 mg / kg resulted in a significant decrease in tumor volume compared to the vehicle (Figure 5). The overall H3K27me3 levels in HT1376 tumors decreased in a dose-dependent manner in response to Compound 1 treatment, with at least an 80% decrease at 10 mg / kg on day 10 and > 90% at 25 mg / kg and higher doses, and were maintained at comparable levels on day 31. The HT1376 xenograft study with a subsequent extended treatment duration showed reproducible levels of TGI on day 30, and then at 35 days, a decrease in tumor volume was shown at both dose levels of 35 mg / kg QD and 50 mg / kg QD (Figure 23).

[0040] Next, the inventors evaluated the in vitro and in vivo efficacy of Compound 1 in other solid cancer ARID1A LOF models. Compound 1 achieved significant antitumor activity as a single agent in ARID1A LOF bladder cancer and endometrial cancer patient-derived xenograft (PDX) models (Figures 6, 7, 20, 21, and 22), as well as in an ARID1A mutant TOV21G cell line-derived xenograft (CDX) model of ovarian clear cell carcinoma (OCCC) (Figure 8), which is consistent with its sensitivity to Compound 1 in vitro. Collectively, these data indicate that Compound 1 is highly effective at a sufficient dose in ARID1A LOF solid tumor models through indications showing a high frequency of ARID1A mutations. Furthermore, a comparative study of ARID1A wild-type PDX cancer models and ARID1A mutant PDX cancer models was conducted (Table 6).

[0041]

Table 3

[0042] Example 2 - Restoration of phenotypic mimicry of ARID1A expression by treatment with Compound 1 in bladder cancer cells with ARID1A mutants To test for molecular changes that support an increased likelihood of EZH2-dependence in relation to ARID1A mutations, the inventors expressed wild-type ARID1A in the ARID1A mutant cell model HT1376 using a doxycycline-inducible system. A significant decrease in H3K27me3 levels was observed within HT1376 cells treated with Compound 1, independent of ectopic ARID1A expression. While EZH1 levels increased with Compound 1 treatment, no change in EZH2 was observed under any of the test conditions (Figure 9). Restoration of ARID1A function in HT1376 cells resulted in a decrease in cell viability similar to that by treatment with Compound 1 alone, and the combination of ARID1A re-expression and Compound 1 treatment did not result in combinatorial growth abnormalities. Both EZH2 inhibition and ARID1A re-expression were suggested to dramatically affect the viability of cells adapted to the epigenetic state modified due to the decrease in ARID1A. The inventors then sought to explore changes in chromatin-binding properties and gene expression within HT1376 cells following ARID1A re-expression and / or Compound 1 treatment. The inventors identified three clusters of active enhancers that behaved differently in response to ARID1A or Compound 1, as determined by positivity for histone H3 lysine 27 acetylation (H3K27ac). Enhancer cluster 1 was uniquely defined by low H3K27ac levels and high H3K27me3 levels, and exhibited increased enhancer activity after Compound 1 treatment, as evident from the increased levels of H3K27ac and the dramatic decrease in the H3K27me3 baseline level. Enhancer clusters 2 and 3 also showed increased enhancer activity in response to Compound 1 treatment, although to a lesser extent than enhancer cluster 1. Clusters 2 and 3 (to a lesser extent) were defined primarily by an increase in the binding of both ARID1A and SMARCA4 in response to ARID1A re-expression.Combinatorial increases in occupancy of ARID1A and SMARCA4 were observed at enhancer cluster 1 when both re-expression of ARID1A in cells and treatment with compound 1 were done, suggesting that these enhancers are associated with gene targets co-regulated by PRC2 and BAF. In unsupervised clustering, three subclusters were shown: a small subset of genes repressed by ARID1A but unchanged by compound 1 (proximal subcluster 1A of enhancer), genes induced by compound 1 but not ARID1A (proximal subcluster 1B of enhancer) and genes induced by either compound 1, ARID1A or both (proximal subcluster 1C of enhancer). Genes induced by compound 1 within proximal subcluster 1C of enhancer are enriched for PRC2 targets, targets of the chimeric oncogenic transcription factor PAX3-FOXO1 and p53 targets. Proximal subcluster 1B of enhancer is enriched for PRC2 targets, but there is no gene set significantly enriched within proximal subcluster 1A of enhancer.

[0043] Analysis of CUT&RUN peaks against the gene body and adjacent regions identified a cluster of two genes that both (to different extents) changed from a repressive state (high H3K27me3 / low H3K27ac) to a more permissive state (low H3K27me3 / high H3K27ac) upon treatment with Compound 1. Genes within promoter cluster 1 also showed increased ARID1A and SMARCA4 binding after Compound 1 treatment, suggesting that inhibition of EZH2 promotes the recruitment of these BAF complex components to these promoters. Expression analysis of genes within promoter cluster 1 showed that most of these genes were induced by Compound 1, consistent with a dominant PRC2 function that represses these genes, and a minimal response to re-expression of ARID1A. Notably, genes within promoter subcluster 1A showed additive induction of gene expression similar to genes within enhancer proximal subcluster 1C after Compound 1 treatment and re-expression of ARID1A, while only these two gene sets shared a small number of genes in common.

[0044] According to the principal component analysis (PCA) of the overall gene expression pattern in the sample, it was shown that compound 1 treatment and the re-expression of ARID1A were the major drivers of gene expression changes. The majority (>94%) of the genes modified by compound 1 treatment were significantly upregulated (Log2 fold change (L2FC) ≥ 1), which is consistent with the role of PRC2 in gene repression. The re-expression of ARID1A caused variable changes in gene expression, with approximately 62% of the significantly modified genes being downregulated by ARID1A and approximately 38% being upregulated, which is consistent with the role of ARID1A in both transcriptional repression and activation. Gene set enrichment analysis (GSEA) of the genes upregulated by the re-expression of ARID1A showed enrichment of EZH2 targets (defined as those induced by compound 1 treatment and having EZH2 and H3K27me3 peaks). See Figure 10. Similarly, GSEA of the genes upregulated after compound 1 treatment showed enrichment of the re-expression targets of ARID1A. See Figure 11. According to the comprehensive GSEA analysis using the Hallmark Collection, more than half of the enriched gene sets were commonly enriched by both compound 1 treatment and the re-expression of ARID1A, including pathways regulating cell differentiation, immune signaling, and inflammation. In summary, these data suggest that ARID1A LOF mutations cause epigenetic gene regulatory imbalances within the subsets of BAF and PRC2 regulatory pathways that promote cancer cell proliferation. These cancer cells rely on PRC2-mediated repression of these gene targets, and compound 1-mediated EZH2 inhibition may enable the re-expression of these genes to affect cell viability in relation to ARID1A LOF.

[0045] Example 3 - Clinical Efficacy As part of a human clinical phase 2 trial (NCT04104776), 350 mg / day of Compound 1 was administered to each of the subjects with ovarian clear cell carcinoma (cohort M2) and subjects with endometrial carcinoma (cohort M3) over the treatment durations shown in Figures 13 and 14. Compound 1 was administered orally as a single agent (monotherapy). The cancer in each subject was found to have an ARID1A mutation as determined by next-generation sequencing (NGS) prior to treatment. Table 3 shows the responses by cancer cohort for efficacy-evaluable patients at the intermediate cutoff date. The evaluation of changes in tumor volume was based on the RECIST 1.1 criteria. Complete response was characterized by the disappearance of all lesions, partial response was characterized by at least a 30% decrease in the sum of the longest diameters (LD) of the target lesions, considering the baseline total LD as a reference, stable was characterized by neither a reduction sufficient to be recognized as a partial response nor an increase sufficient to be recognized as disease progression, considering the minimum total diameter as a reference, and disease progression was characterized by at least a 20% increase in the sum of the LD of the target lesions, considering the minimum total LD recorded after treatment initiation or the appearance of one or more new lesions as a reference. For assignment, the status of a partial response change or complete response change in tumor measurement must be confirmed by repeated evaluation for more than 4 weeks after the criteria for response are first met.

[0046]

Table 4

[0047] Furthermore, the amount of tumor gene mutations was found to be low in most patients with OCCC or EC. The state of low TMB was defined as <10 mut / Mb and evaluated by NGS (Tempus xT & Predicine ATLAS targeted panel sequencing).

[0048] Example 4 - Comparison of Compound 1 with Other PRC2 Inhibitors in a Bladder Cancer Xenograft Model of ARID1A Mutants In Vivo The responses to other EZH2, dual EZH1 and EZH2, or EED inhibitors in addition to compound 1 treatment were analyzed in vitro and in vivo. In the long-term cell viability assay in vitro, compound 1 had a GI 50 equivalent to that of PF-06821497 (about 5 nM), while valemetostat and the EED inhibitor MAK683 were not more than 3 - 10 times more potent (GI 50 : 18 nM and 73 nM respectively). First-generation EZH2 inhibitors such as tazemetostat (GI 50 = 385 nM) and CPI-1205 (GI 50 = 574 nM) were not more potent in the viability assay, consistent with their lower target affinity and shorter residence time. Compound 1, valemetostat, and MAK683 showed > 50% tumor growth inhibition (TGI) compared to vehicle when administered at the same dose (75 mg / kg QD) in an HT1376 xenograft mouse model with ARID1A mutation (Figure 15). At this dose, compound 1 showed significantly stronger and longer-lasting antitumor activity than valemetostat (tumor growth in the valemetostat arm rebounded against treatment after 35 days) (p = 0.0001), while tumors treated with compound 1 continued to show suppression below the initial tumor volume until day 37. Tazemetostat, CPI-1205, and PF-06821497 resulted in < 50% TGI (32%, 40%, and 41% TGI respectively on day 27). Overall, compound 1 caused complete tumor regression in an ARID1A-mutated bladder cancer xenograft model. In mice treated with valemetostat, the tumor volume decreased from the beginning of treatment but did not reach complete regression over 35 days, while mice treated with tazemetostat showed an increase in volume during treatment (Figure 15). All compounds were well tolerated at 75 mg / kg QD, and the plasma exposure of compound 1 was not greater than that of any of the other compounds (Table 4).

[0049]

Table 5

[0050] Compounds that resulted in >50% TGI had a strong (>90%) reduction of H3K27me3 in tumors collected on day 15, while compounds with weaker TGI retained higher levels of H3K27me3 (Figure 16), showing a correlation between tumor response and the degree of H3K27me3 reduction (Figure 17). However, at a reduction scale of >90% in overall H3K27me3 level changes, differences in TGI among the most effective compounds, compound 1, valemetostat, and MAK683, could not be distinguished. According to gene expression profiling from tumors on day 15, compound 1 induced significantly more EZH2 / PRC2 target genes than any other compound (Table 5), showing that the overall changes in gene expression levels were consistent with reduced H3K27me3 and tumor response (Figures 18 and 24). Contrary to the overall changes in H3K27me3 levels, a larger number of modified genes correlated more extensively with TGI, suggesting that gene expression changes could be a preferred biomarker for associating target inhibition with efficacy.

[0051]

Table 6

[0052] The content of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) is hereby expressly incorporated by reference in its entirety. Unless otherwise defined, all scientific and technical terms used herein shall conform to the meanings commonly known to those skilled in the art.

Claims

1. A pharmaceutical composition for use in the treatment of cancer in a subject, wherein the pharmaceutical composition has the formula: 【Chemistry 1】 A pharmaceutical composition comprising a compound having or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is administered to a subject, and the cancer has at least one ARID1A mutation.

2. The pharmaceutical composition according to 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 pharmaceutical composition according to claim 1, wherein the cancer is selected from bladder cancer, endometrial cancer, and ovarian clear cell carcinoma.

4. The pharmaceutical composition according to claim 1, wherein the cancer is bladder cancer.

5. The pharmaceutical composition according to claim 4, wherein the bladder cancer is urothelial carcinoma.

6. The pharmaceutical composition according to claim 4, wherein the bladder cancer is advanced urothelial carcinoma.

7. The pharmaceutical composition according to claim 1, wherein the cancer is endometrial cancer.

8. The pharmaceutical composition according to claim 1, wherein the cancer is ovarian cancer.

9. The pharmaceutical composition according to claim 8, wherein the cancer is ovarian clear cell carcinoma.

10. The pharmaceutical composition according to claim 1, wherein the at least one ARID1A mutation is a loss-of-function (LOF) mutation.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the compound or a pharmaceutically acceptable salt is administered over a period of at least six days.

12. The pharmaceutical composition according to any one of claims 1 to 10, wherein the administration restores ARID1A expression.

13. The pharmaceutical composition according to any one of claims 1 to 10, wherein the administration increases SMARCA4 binding.

14. The pharmaceutical composition according to any one of claims 1 to 10, wherein the administration increases ARID1A binding.

15. The pharmaceutical composition according to any one of claims 1 to 10, wherein the administration reduces the overall H3K27me3 level.

16. The pharmaceutical composition according to any one of claims 1 to 10, wherein the administration results in a long-term reduction in tumor volume.

17. The pharmaceutical composition according to any one of claims 1 to 10, wherein the administration drives the re-expression of the suppressed gene.

18. The pharmaceutical composition according to any one of claims 1 to 10, wherein the cancer is characterized by a low tumor gene mutational load.

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