KAT6 inhibitor methods and combinations for cancer treatment

Abstract

This invention relates to methods and combination therapies for treating cancer, which involve administering a KAT6 inhibitor to a patient in need.

Description

[Technical Field]

[0001] This invention relates to methods and combination therapies for treating cancer. Specifically, this invention relates to methods for treating cancer by administering a combination of a KAT6 inhibitor and a CDK4 inhibitor and / or an anti-estrogen. Furthermore, this invention relates to methods for overcoming clinical drug resistance by administering a KAT6 inhibitor as a single agent or in combination. The pharmaceutical uses of the methods and combinations of this invention are also described. [Existing Technology]

[0002] Histone acetylation is a reversible protein modification essential for chromatin organization and function (Lee, KK et al., Histone acetyltransferase complexes: one size doesn't fit all. Nat. Rev. Mol. Cell Biol. 2007, 8, 284-295). Histone acetylation is catalyzed by histone lysine acetyltransferases (HAT or KAT) using acetyl-CoA (AcCoA) as a cofactor (Roth, SY et al., Histone acetyltransferases. Annu. Rev. Biochem. 2001, 70, 81-120; Furdas, SD et al., Small molecule inhibitors of histone acetyltransferases as epigenetic tools and drug candidates. Arch. Pharm. 2012, 345, 7-21). Lysine acetyltransferase 6A (KAT6A, also known as MOZ or MYST3) and lysine acetyltransferase 6B (KAT6B, also known as MORF) are intraspecies homologs. Their protein products form complexes with ING5, EAF6 and BRPF1, BRPF2 or BRPF3 to acetylate H3K23Ac (Doyon, Y. et al., ING tumorsuppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation. Mol. Cell. 2006, 21(1):51-64; Mullah, M. et al., Molecular architexture of quartet MOZ / MORF histone acetyltransferase complexes. Mol. Cell. Biol. 2008, 28(22):6828-6843).KAT6A and KAT6B acetyltransferases are involved in basic cellular processes, including gene transcription, cell senescence, tissue development, and the maintenance of normal hematopoietic stem cells (Huang, F. et al., Regulation of KAT6 acetyltransferase and their roles in cell cycle progression, stem cell maintenance, and human disease. Mol. Cell. Biol. 2016, 36, 1900-1907).

[0003] KAT enzymes perform important regulatory functions in cancer and are therefore frequently targeted by mutations, translocations, and amplifications (Hu, Z. et al., Genomic characterization of genes encoding histone acetylation modulator proteins identifies therapeutic targets for cancer treatment. Nat Commun. 2019 Feb 13; 10(1):733). KAT6A was identified in 1996 as part of the chromosomal translocation t(8;16)(p11;p13) of CREBBP (CREB-binding protein) in a subtype of acute myeloid leukemia (Borrow, J. et al., The translocation t(8;16)(p11;p13) of acute myeloid leukemia fuses a putative acetyltransferase to the CREB-binding protein. Nat. Genet. 1996, 14, 33-41). Subsequently, additional KAT6A and KAT6B translocations were identified in more AML patients, resulting in fusions with other HATs such as EP300 (adenovirus EIA-associated protein p300), NCOA2 (nuclear receptor coactivator 2), and NCOA3 (Huang et al.).

[0004] In human cancers, especially breast cancer, KAT6A is identified as part of the 8p11-12 repeat amplification region found in 10-15% of breast cancers. J. et al., Chromosome region 8p11-p21: refined mapping and molecular alterations in breast cancer. Genes Chromosomes Cancer. July 1998; 22(3):186-99). Breast cancer cell lines with KAT6A amplification overexpress KAT6A, suggesting that KAT6A is a presumed breast cancer susceptibility gene. Turner-Ivey et al. used a genome-scale shRNA screening strategy to identify KAT6A as a significant contributor in 8p11 amplified breast cancer cell lines with KAT6A overexpression (Turner-Ivey et al., KAT6A, a chromatin modifier from the 8p11-p12 amplicon is a candidate oncogene in luminal breast cancer. Neoplasia. August 2014; 16(8):644-55). Yu et al. subsequently confirmed in 8p11 amplified breast cancer cells that KAT6A is located at the estrogen receptor promoter, and that shRNA-mediated KAT6A knockdown reduced ERα ESR1 mRNA and protein levels (Yu, L. et al., Identification of MYST3 as a novel epigenetic activator of ERα frequently amplified in breast cancer. Oncogene 2017, 36, 2910-2918). Furthermore, they demonstrated that growth defects caused by KAT6A depletion in 8p11 amplified breast cancer cells could be partially rescued by ESR1 reexpression. These findings indicate the important role of KAT6A in the regulation of ERα genes required for ER+ breast cancer cell growth.

[0005] Chromosome 8p11-12 amplification and KAT6A overexpression are present in other tumor types, including ovarian cancer, cervical cancer, lung adenocarcinoma, colorectal adenocarcinoma, and medulloblastoma (Zack TI et al., Pan-cancer patterns of somatic copy number alteration. Nat Genet 2013 45:1134-1140; Northcott PA et al., Multiple recurrent genetic events converge on control of histone lysinemethylation in medulloblastoma. Nat Genet 2009 41:465-472). Additional KAT6A-dependent cancers, including prostate cancer, have been identified (Yu C et al., High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor celllines. Nat Biotechnol. 2016; Meyers RM et al., Computational correction of copynumber effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells. Nat Genet. 2017; and Tsherniak A et al., Defining a cancer dependency map. Cell. 2017). In summary, these data demonstrate broader therapeutic opportunities targeting KAT6A in other cancer types.

[0006] In addition to its catalytic function mediated by the histone acetyltransferase (HAT) domain, the KAT6A protein also includes other domains such as the PHD domain, an acidic domain, and a serine / methionine-rich domain. KAT6A regulation of gene expression independent of its catalytic activity has been reported (Kitabayashi, I. et al., Activation of AML1-mediated transcription by MOZ and inhibition by the MOZ-CBP fusion protein. EMBO J. 2001, 20(24):7184-7196). Knockdown of KAT6A protein levels using RNA interference has confirmed the dependence of ER+ breast cancer cells on KAT6A (Turner-Ivey B. et al. and Yu, L. et al.). However, the requirement of ERα expression and ER+ breast cancer cell proliferation for KAT6A catalytic activity remains unclear. Researchers at Monash University have reported two advanced KAT6A / 6B inhibitors that effectively kill MYC-driven lymphoma cells, demonstrating that KAT6A / 6B enzyme activity is essential for the proliferation of this type of cancer (Kitabayashi, I. et al.). However, these two reported KAT6A / 6B inhibitors exhibit high human liver microsomal clearance, strong DDI potential, and acid instability due to the presence of a key pharmacophore (acylsulfonylhydrazide moiety) in their chemical structure. To date, no KAT6A / 6B enzyme inhibitors have been reported to be effective in treating solid tumors (including HR+ breast cancer and AR+ prostate cancer). Currently, no known KAT6A / 6B catalytic inhibitors have been tested in human clinical trials.

[0007] Compound N′-(4-fluoro-5-methyl-[1,1′-biphenyl]-3-carbonyl)benzenesulfonylhydrazine (also referred to as “Compound A” in this document) is a KAT6A inhibitor with the following structure:

[0008]

[0009] Compound A and its pharmaceutically acceptable salts are disclosed in International Publication No. WO 2016 / 198507, the contents of which are incorporated herein by reference in their entirety.

[0010] Compound N′-(5-chloro-4-fluoro-[1,1′-biphenyl]-3-carbonyl)benzenesulfonylhydrazine (also referred to herein as "compound B") is a KAT6A inhibitor with the following structure:

[0011]

[0012] Compound B and its pharmaceutically acceptable salts are disclosed in International Publication No. WO 2016 / 198507, the contents of which are incorporated herein by reference in their entirety.

[0013] Compound 2,6-dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (also referred to herein as "compound C") is a potent and selective catalytic inhibitor of KAT6 histone acetyltransferases KAT6A and KAT6B, and has the following structure:

[0014]

[0015] Compound C and its pharmaceutically acceptable salts are disclosed in U.S. Patent Application Serial No. 16 / 902,515, the contents of which are incorporated herein by reference in their entirety.

[0016] Compound 2-fluoro-N′-(3-fluoro-5-(pyridin-2-yl)benzoyl)benzenesulfonylhydrazine (also referred to herein as "Compound D") is a KAT6A inhibitor with the following structure:

[0017]

[0018] Compound D and its pharmaceutically acceptable salts are disclosed in International Publication No. WO 2016 / 198507, the contents of which are incorporated herein by reference in their entirety.

[0019] Compound 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (also referred to herein as "Compound E") is a potent and selective catalytic inhibitor of KAT6 histone acetyltransferases KAT6A and KAT6B, and has the following structure:

[0020]

[0021] Compound E and its pharmaceutically acceptable salts are disclosed in U.S. Patent Application Serial No. 16 / 902,515, the contents of which are incorporated herein by reference in their entirety.

[0022] Cyclin-dependent kinases (CDKs) and their associated serine / threonine protein kinases are essential cellular enzymes that perform fundamental functions in regulating eukaryotic cell division and proliferation. The CDK catalytic unit is activated by a regulatory subunit called a cyclin. At least 16 mammalian cyclins have been identified (Johnson DG, Walker CL. Cyclins and Cell Cycle Checkpoints. Annu. Rev. Pharmacol. Toxicol. (1999) 39: 295-312). Cyclin B / CDK1, cyclin A / CDK2, cyclin E / CDK2, cyclin D / CDK4, cyclin D / CDK6, and possible heterodynes are important regulators of cell cycle progression. Additional functions of cyclin / CDK hybrids include regulation of transcription, DNA repair, differentiation, and apoptosis (Morgan DO, Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu. Rev. Cell. Dev. Biol. (1997) 13: 261-291).

[0023] CDK inhibitors have been shown to be useful in the treatment of cancer. It has been shown that increased activity or transient abnormal activation of cyclin-dependent kinases can lead to the development of human tumors, and the development of human tumors is usually associated with alterations in CDK proteins themselves or their regulators (Cordon-Cardo C. Mutations of cell cycle regulators: biological and clinical implications for human neoplasia. Am. J. Pathol. (1995) 147: 545-560; Karp JE, Broder S. Molecular foundations of cancer: new targets for intervention. Nat. Med. (1995) 1: 309-320; Hall M, Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv. Cancer Res. (1996) 68: 67-108).

[0024] CDK4 and CDK6 are important regulators of cell cycle progression at the G1-S checkpoint, and are influenced by D-cyclin and endogenous CDK inhibitors of INK4 (such as p16). INK4a (CDKN2A) control. Dysregulation of the cyclin D-CDK4 / 6–INK4–retinoblastoma (Rb) pathway has been reported to be associated with the development of endocrine therapy resistance. Furthermore, CDK4 has been identified as a single oncogen for many breast cancers, and emerging data suggest that cyclin D3-CDK6 inhibition may be associated with hematologic toxicity, indicating the role of selective CDK4 inhibitors.

[0025] Clinical trials of the CDK4 / 6 inhibitors palbociclib, ribociclib, and abemaciclib, as monotherapy or in combination with other therapeutic agents, are ongoing for breast cancer and other cancers. The combination of CDK4 / 6 inhibitors with endocrine therapy has demonstrated significant efficacy in treating hormone receptor (HR)-positive, human epidermal growth factor 2 (HER2)-negative advanced or metastatic breast cancer, and combinations of CDK4 / 6 inhibitors (including palbociclib, ribociclib, and abemaciclib) with endocrine therapy have been approved in first-line or second-line settings. Palbociclib, ribociclib, and abemaciclib have been approved in first-line settings in combination with aromatase inhibitors (such as letrozole), and in certain patients in second-line or later-line treatment in combination with fulvestrant for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer. (O'Leary et al., Treating cancer with selective CDK4 / 6 inhbitors. Nature Reviews (2016) 13:417-430).

[0026] Palbociclib, or 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (also known as PD-0332991), is a potent and selective inhibitor of CDK4 and CDK6, and has the following structure:

[0027]

[0028] Palbociclib is described in WHO Drug Information, Volume 27, Issue 2, page 172 (2013). Palbociclib and its pharmaceutically acceptable salts are disclosed in International Publication No. WO 2003 / 062236 and U.S. Patents Nos. 6,936,612, 7,456,168, and RE47,739; International Publication No. WO 2005 / 005426 and U.S. Patents Nos. 7,345,171 and 7,863,278; International Publication No. WO 2008 / 032157 and U.S. Patent No. 7,781,583; and International Publication No. WO 2014 / 128588. The contents of each of the foregoing references are incorporated herein by reference in their entirety.

[0029] Compound 1,5-dehydr-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1Hbenzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentanol (also referred to herein as "compound F") is a potent and selective inhibitor of CDK4 and has the following structure:

[0030]

[0031] Compound F and its pharmaceutically acceptable salts are disclosed in International Publication No. WO2019 / 207463, published on October 31, 2019, the contents of which are incorporated herein by reference in their entirety.

[0032] Although CDK4 / 6 inhibitors have shown significant clinical efficacy in ER-positive metastatic breast cancer, like other kinases, their effectiveness may be limited over time by the development of primary or acquired resistance. The selective CDK4 / 6 inhibitor palbociclib has been shown to be clinically effective in breast cancer (DeMichele A, Clark AS, Tan KS et al., CDK4 / 6 inhibitor palbociclib (PD-0332991) in Rb+ advanced breast cancer: phase II activity, safety, and predictive biomarker assessment. Clin Cancer Res 2015; 21(5):995-1001; Finn RS, Martin M, Rugo HS et al., Palbociclib and Letrozole in Advanced Breast Cancer. New Engl J Med 2016; 375(20):1925-36; Cristofanilli M, Turner NC, Bondarenko I et al., Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on previous endocrine). Therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol 2016; 17(4):425-39). However, after initial clinical benefit, acquired resistance to palbociclib may occur (Knudsen Erik S., Witkiewicz Agnieszka K., The Strange Case of CDK4 / 6 Inhibitors: Mechanisms, Resistance, and Combination Strategies. Trends Cancer 2017; 3(1):39-55).

[0033] Therefore, there remains a need for improved therapies for treating cancer. The combinations and methods of the present invention are believed to have one or more advantages, such as greater efficacy; the potential to reduce side effects; the potential to reduce drug-drug interactions; or the potential to overcome drug resistance mechanisms (such as breast cancer resistant to endocrine therapy); and so on. [Invention Overview]

[0035] Each of the embodiments described below may be combined with any other embodiment described herein, provided that such other embodiment does not contradict the embodiments to which it is combined. Furthermore, each embodiment described herein includes, within its scope, a pharmaceutically acceptable salt of the compound described herein. Therefore, the phrase "or a pharmaceutically acceptable salt thereof" is implied in the description of all compounds described herein.

[0036] This invention relates to a method for treating cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a) a certain amount of a CDK4 inhibitor; b) a certain amount of an anti-estrogen; or c) a certain amount of a CDK4 inhibitor and a certain amount of an anti-estrogen; wherein these amounts together are effective in treating cancer.

[0037] The present invention relates to a method for treating cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 inhibitor, wherein these amounts together are effective in treating cancer.

[0038] The present invention relates to a method for treating cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of an anti-estrogen, wherein these amounts together are effective in treating cancer.

[0039] This invention relates to a method for treating cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor, a certain amount of a CDK4 inhibitor, and a certain amount of an anti-estrogen, wherein these amounts together are effective in treating cancer.

[0040] In an embodiment of the aforementioned method of the present invention, the KAT6 inhibitor is a KAT6A inhibitor.

[0041] In an embodiment of the method of the present invention, the KAT6 inhibitor is selected from:

[0042] N′-(4-fluoro-5-methyl-[1,1′-biphenyl]-3-carbonyl)benzenesulfonylhydrazine;

[0043] N′-(5-chloro-4-fluoro-[1,1′-biphenyl]-3-carbonyl)benzenesulfonylhydrazine;

[0044] 2,6-Dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide;

[0045] 2-Fluoro-N′-(3-Fluoro-5-(pyridin-2-yl)benzoyl)benzenesulfonylhydrazine; and

[0046] 2-Methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide,

[0047] Or its pharmaceutically acceptable salt.

[0048] In embodiments of the method of the present invention, the KAT6 inhibitor is 2,6-dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof.

[0049] In a preferred embodiment of the method of the present invention, the KAT6 inhibitor is 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof.

[0050] In an embodiment of the method of the present invention, the CDK4 inhibitor is a CDK4 selective inhibitor or a CDK4 / 6 inhibitor.

[0051] In an embodiment of the method of the present invention, the CDK4 inhibitor is a selective CDK4 inhibitor.

[0052] In an embodiment of the method of the present invention, the CDK4 selective inhibitor is 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentol or a pharmaceutically acceptable salt thereof.

[0053] In an embodiment of the method of the present invention, the CDK4 inhibitor is a CDK4 / 6 inhibitor.

[0054] In embodiments of the method of the present invention, the CDK4 / 6 inhibitor is abeciclib, ribociclib, and palbociclib, or a pharmaceutically acceptable salt thereof.

[0055] In a preferred embodiment of the method of the present invention, the CDK4 / 6 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

[0056] In embodiments of the method of the present invention, the anti-estrogen is an aromatase inhibitor, a selective estrogen receptor degrader (SERD), or a selective estrogen receptor modulator (SERM).

[0057] In embodiments of the method of the present invention, the anti-estrogen is fulvestrant or letrozole; the anti-estrogen is fulvestrant; or the anti-estrogen is letrozole.

[0058] In an embodiment of the method of the present invention, a certain amount of KAT6 inhibitor, a certain amount of CDK4 inhibitor, and a certain amount of anti-estrogen are administered, wherein the anti-estrogen is fulvestrant or letrozole, or the anti-estrogen is letrozole.

[0059] In any implementation of the method of this invention, the patient is a human.

[0060] The present invention relates to a method for treating cancer, comprising administering to a patient in need a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of palbociclib, wherein these amounts together are effective in treating cancer.

[0061] This invention relates to a method for treating cancer, comprising administering to a patient in need an amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and an amount of 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof, wherein these amounts together are therapeutically effective in treating cancer.

[0062] The present invention relates to a method for treating cancer, comprising administering to a patient in need a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of fulvestrant, wherein these amounts together are effective in treating cancer.

[0063] This invention relates to a method for treating cancer, comprising administering to a patient in need a quantity of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, a quantity of palbociclib or a pharmaceutically acceptable salt thereof, and a quantity of fulvestrant, wherein these quantities together are effective in treating cancer.

[0064] This invention relates to a method for treating cancer, comprising administering to a patient in need a quantity of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, a quantity of palbociclib or a pharmaceutically acceptable salt thereof, and a quantity of letrozole, wherein these quantities together are effective in treating cancer.

[0065] In any embodiment of the method of the present invention, the cancer treated by the method of the present invention is breast cancer, lung cancer, colon cancer, brain cancer, head and neck cancer, prostate cancer, stomach cancer, pancreatic cancer, ovarian cancer, melanoma, endocrine cancer, uterine cancer, testicular cancer, or bladder cancer.

[0066] In any embodiment of the method of the present invention, the cancer treated by the method of the present invention is breast cancer, lung cancer, prostate cancer, pancreatic cancer, or ovarian cancer.

[0067] In any embodiment of the method of the present invention, the cancer treated by the method of the present invention is breast cancer, lung cancer, or prostate cancer.

[0068] In any embodiment of the method of the present invention, the cancer to be treated by the method of the present invention is breast cancer.

[0069] In any embodiment of the method of the present invention, the breast cancer to be treated by the method of the present invention is hormone receptor-positive (HR+) breast cancer; the hormone receptor-positive (HR+) breast cancer is selected from progesterone receptor-positive (PR+) breast cancer and estrogen receptor-positive (ER+) breast cancer; and the hormone receptor-positive (HR+) breast cancer is progesterone receptor-positive (PR+) breast cancer.

[0070] In any embodiment of the method of the present invention, the breast cancer to be treated by the method of the present invention is estrogen receptor-positive (ER+) breast cancer; estrogen receptor-positive (ER+) breast cancer is human epidermal growth factor receptor 2 negative (HER2-); estrogen receptor-positive (ER+) breast cancer is human epidermal growth factor receptor 2 positive (HER2+).

[0071] The method of the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and palbociclib or a pharmaceutically acceptable salt thereof for the treatment of cancer.

[0072] The method of the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzyl-1-sulfonamide or a pharmaceutically acceptable salt thereof and 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof, for the treatment of cancer.

[0073] The method of the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and fulvestrant for the treatment of cancer.

[0074] The method of the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof; palbociclib or a pharmaceutically acceptable salt thereof; and fulvestrant for the treatment of cancer.

[0075] The method of the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof; palbociclib or a pharmaceutically acceptable salt thereof; and letrozole for the treatment of cancer.

[0076] This invention relates to a method for overcoming clinical resistance to endocrine therapy, comprising administering to a patient in need a dose of a KAT6 inhibitor that is effective in overcoming clinical resistance to endocrine therapy.

[0077] The present invention relates to a method for overcoming clinical resistance to endocrine therapy, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and optionally a certain amount of a CDK4 inhibitor, wherein these amounts together are therapeutically effective in overcoming clinical resistance to endocrine therapy.

[0078] In an implementation plan for a method to overcome clinical resistance to endocrine therapy, the endocrine therapy is used to treat cancer; the cancer is breast cancer, lung cancer, colon cancer, brain cancer, head and neck cancer, prostate cancer, gastric cancer, pancreatic cancer, ovarian cancer, melanoma, endocrine cancer, uterine cancer, testicular cancer, or bladder cancer; the cancer is breast cancer, lung cancer, prostate cancer, pancreatic cancer, or ovarian cancer; the cancer is breast cancer, lung cancer, or prostate cancer; the cancer is breast cancer; the breast cancer is hormone receptor-positive (HR+) breast cancer; hormone receptor-positive (HR+) breast cancer is selected from: progesterone receptor-positive (PR+) breast cancer and estrogen receptor-positive (ER+) breast cancer; hormone receptor-positive (HR+) breast cancer is progesterone receptor-positive (PR+) breast cancer; hormone receptor-positive (HR+) breast cancer is estrogen receptor-positive (ER+) breast cancer; estrogen receptor-positive (ER+) breast cancer is human epidermal growth factor receptor 2 negative (HER2-); and estrogen receptor-positive (ER+) breast cancer is human epidermal growth factor receptor 2 negative (HER2-).

[0079] In the implementation of methods for overcoming clinical resistance to endocrine therapy, the KAT6 inhibitor is a KAT6A inhibitor.

[0080] In the implementation of methods for overcoming clinical resistance to endocrine therapy, KAT6 inhibitors are selected from:

[0081] N′-(4-fluoro-5-methyl-[1,1′-biphenyl]-3-carbonyl)benzenesulfonylhydrazine;

[0082] N′-(5-chloro-4-fluoro-[1,1′-biphenyl]-3-carbonyl)benzenesulfonylhydrazine;

[0083] 2,6-Dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide;

[0084] 2-Fluoro-N′-(3-Fluoro-5-(pyridin-2-yl)benzoyl)benzenesulfonylhydrazine; and

[0085] 2-Methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide,

[0086] Or its pharmaceutically acceptable salt.

[0087] In an implementation of a method for overcoming clinical resistance to endocrine therapy, the KAT6 inhibitor is 2,6-dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof.

[0088] In an implementation of a method for overcoming clinical resistance to endocrine therapy, the KAT6 inhibitor is 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof.

[0089] In implementation methods for overcoming clinical resistance to endocrine therapy, the CDK4 inhibitor is a CDK4 selective inhibitor or a CDK4 / 6 inhibitor.

[0090] In implementation methods for overcoming clinical resistance to endocrine therapy, CDK4 inhibitors are selective CDK4 inhibitors.

[0091] In an implementation of a method for overcoming clinical resistance to endocrine therapy, a CDK4 selective inhibitor is 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threopentanediol or a pharmaceutically acceptable salt thereof.

[0092] In implementation methods for overcoming clinical resistance to endocrine therapy, CDK4 inhibitors are CDK4 / 6 inhibitors.

[0093] In implementation schemes for methods to overcome clinical resistance to endocrine therapy, the CDK4 / 6 inhibitor is abeciclib, ribociclib, and palbociclib, or a pharmaceutically acceptable salt thereof.

[0094] In implementation schemes for methods to overcome clinical resistance to endocrine therapy, the CDK4 / 6 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof. [Attached Image Description]

[0095] Figure 1A The number of cells proliferating in T747D ER+ breast cancer cells after removal of palbociclib treatment is shown.

[0096] Figure 1B The number of cells proliferating in MCF7 ER+ breast cancer cells after removal of palbociclib treatment is shown.

[0097] Figure 2 This shows a summary of the pooled RNAi screening used to identify genes required for reversible proliferation arrest during palbociclib treatment.

[0098] Figure 3A This demonstrates the workflow for validating KAT6A as an epigenetic enzyme required for the proliferation and recovery from palbociclib arrest in ER+ breast cancer cells.

[0099] Figure 3B The results of proliferation and colony formation analysis and palbociclib-restored colony formation analysis are shown in T47D and ZR75-1 ER+ breast cancer cells.

[0100] Figure 4A This shows the results of colony formation analysis in MCF7 ER+ breast cancer cells.

[0101] Figure 4B This shows the results of palbociclib recovery analysis in MCF7 ER+ breast cancer cells.

[0102] Figure 5A This shows the results of colony formation analysis in CAMA1 ER+ breast cancer cells.

[0103] Figure 5B The number of cells per culture of KAT6A_5, KAT6A_6, shCB3, and KAT6A_10_1u in CAMA1 ER+ breast cancer cells 14 days after KAT6A knockdown.

[0104] Figure 6 The morphology of CAMA1 cells after KAT6A knockdown is shown.

[0105] Figure 7A KAT6A knockdown showed a moderate effect on proliferation in EFM192A ER+ breast cancer cells, as well as a substantial synthetic lethal effect when combined with 100 nM palbociclib.

[0106] Figure 7B This demonstrates the effective knockdown of KAT6A at the mRNA level in EFM192A ER+ breast cancer cells via shKAT6A_5 and shKAT6A_6.

[0107] Figure 7C This demonstrates that EFM192A ER+ breast cancer cells effectively knock down KAT6A at the protein level using both shKAT6A_5 and shKAT6A_6.

[0108] Figure 8A Compound A showed that it inhibited the proliferation of T47D ER+ breast cancer cells.

[0109] Figure 8B The combination of compound A and palbociclib showed that it blocked the recovery of T47D ER+ breast cancer cells from growth arrest caused by palbociclib.

[0110] Figure 8C Compound B showed that it inhibited the proliferation of T47D ER+ breast cancer cells.

[0111] Figure 8DThe combination of compound B and palbociclib showed that it blocked the recovery of T47D ER+ breast cancer cells from growth arrest caused by palbociclib.

[0112] Figure 9 Compound A was shown to inhibit proliferation and block the recovery of ZR-75-1ER+ breast cancer cells from growth arrest caused by palbociclib.

[0113] Figure 10 Compound A, as a single agent, and its combination with palbociclib, showed that it inhibited the recovery of MCF7 ER+ breast cancer cells from growth arrest caused by palbociclib.

[0114] Figure 11A The results show the knockdown of KAT6A in T47D, ZR75-1, and CAMA1 ER+ breast cancer cells.

[0115] Figure 11B The study showed a decrease in ESR1 mRNA in T47D, ZR75-1, and CAMA1 ER+ breast cancer cells.

[0116] Figure 11C The study showed a decrease in ERα protein in T47D, ZR75-1, and CAMA1 ER+ breast cancer cells.

[0117] Figure 12A The study showed that KAT6A knockdown resulted in a significant decrease in ERα protein in T47D ER+ breast cancer cells, and re-expression of ESR1 from the ectopic promoter restored ERα protein levels.

[0118] Figure 12B The results showed that KAT6A knockdown led to a significant reduction in ESR1 transcripts in T47D ER+ breast cancer cells, and reexpression of ESR1 from the ectopic promoter restored ESR1 transcript levels.

[0119] Figure 12C The results showed that KAT6A knockdown led to a significant reduction in cell growth in T47D ER+ breast cancer cells, and that reexpression of ESR1 from the ectopic promoter restored cell growth in the presence of KAT6A depletion.

[0120] Figure 12D The re-expression of ESR1 showed that it partially restored cell cycle arrest in T47D cells caused by the combination of impabociclib and KAT6A depletion.

[0121] Figure 13A The combination of compound C and palbociclib showed that it inhibited cell growth and blocked the recovery of T47D ER+ breast cancer cells.

[0122] Figure 13BThe combination of compound C and palbociclib showed that it inhibited cell growth and blocked the recovery of ZR75-1 ER+ breast cancer cells.

[0123] Figure 13C The combination of compound D and palbociclib showed that it inhibited cell growth and blocked the recovery of T47D ER+ breast cancer cells.

[0124] Figure 13D The combination of compound D and palbociclib showed that it inhibited cell growth and blocked the recovery of ZR75-1 ER+ breast cancer cells.

[0125] Figure 14A Treatment with compound C resulted in dose-dependent depletion of both ERα and cyclin D1 in T47D ER+ breast cancer cells.

[0126] Figure 14B Treatment with compound D resulted in dose-dependent depletion of both ERα and cyclin D1 in T47D ER+ breast cancer cells.

[0127] Figure 14C Compound D showed that it caused dose-dependent depletion of ESR1 and CCND1 mRNA in T47D ER+ breast cancer cells, as measured by qPCR.

[0128] Figure 14D Compound D showed that it caused dose-dependent depletion of ESR1 and CCND1 mRNA in T47D ER+ breast cancer cells, as measured by qPCR.

[0129] Figure 15A The sensitivity of T47D ER+ breast cancer cells to Y537S, D538G, and Y537S / D538 mutations to fulvestrant, palbociclib, compound C as a single agent, and its combination with palbociclib.

[0130] Figure 15B The sensitivity of T47D ER+ breast cancer cells to Y537S, D538G, and Y537S / D538 mutations to fulvestrant, palbociclib, compound D as a single agent, and its combination with palbociclib.

[0131] Figure 16A The study showed that palbociclib, as a single agent and in combination with compound A, reduced ESRI mRNA in T47D ER+ breast cancer cells.

[0132] Figure 16B The study showed that palbociclib, as a single agent, and its combination with compound C reduced ESRI mRNA in T47D ER+ breast cancer cells.

[0133] Figure 16C Palbociclib as a single agent and in combination with compound C were shown to reduce ERα levels in T47D and MCF7 ER+ breast cancer cells.

[0134] Figure 16D The reduction in ESR1 mRNA levels was demonstrated in T47D and MCF7 ER+ breast cancer cells by palbociclib as a single agent and in combination with compound C, as measured by qPCR.

[0135] Figure 17A The study demonstrated the inhibition and delay of tumor growth in the ST340PDX model by compound E, palbociclib, and fulvestrant as single agents and in dual and triple combinations.

[0136] Figure 17B This study demonstrates the tolerability of compounds E, palbociclib, and fulvestrant as single agents and in dual and triple combinations in the ST340PDX model.

[0137] Figure 18A The study demonstrated the inhibitory effect on tumor growth and the delay of growth in the ST941 PDX model by compound E, palbociclib, and fulvestrant as single agents and in dual and triple combinations.

[0138] Figure 18B This study demonstrates the tolerability of compounds E, palbociclib, and fulvestrant as single agents and in dual and triple combinations in the ST941PDX model. [Detailed Description of the Invention]

[0140] The invention can be more readily understood by referring to the following detailed description of preferred embodiments and examples included therein. It should be understood that the terminology used herein is for the purpose of illustrating particular embodiments only and is not intended to limit the invention. Furthermore, it should be understood that unless expressly defined herein, the terminology used herein is given in its conventional meaning as known in the related art.

[0141] As used herein, unless otherwise indicated, the singular forms “a,” “an,” and “the” include plural references. For example, an excipient includes one or more excipients.

[0142] As used herein, the term “about” when used to modify a parameter defined numerically (e.g., the dose of a KAT6 inhibitor or CDK inhibitor) means that the parameter may vary by up to 10% below or above the stated value for that parameter. For example, a dose of about 5 mg means 5% ± 10%, that is, it may vary between 4.5 mg and 5.5 mg.

[0143] As used herein, the terms “pharmaceutical,” “component,” “composition,” “compound,” “drug,” “targeting agent,” “targeted therapeutic agent,” and “therapeutic agent” are used interchangeably to refer to the compounds of the present invention, particularly KAT6 inhibitors and CDK4 inhibitors.

[0144] The following abbreviations may be used in this document: BID (twice a day, twice daily, two times daily); Dox (doxycycline); DMEM (Dulbecco's Modified Eagle's Medium); DMSO (dimethyl sulfoxide); dNTP (deoxyribonucleotide triphosphate); FBS (fetal bovine serum); RPMI (Roswell Park Memorial Institute); PBS (phosphate-buffered saline); PCR (polymerase chain reaction); PEG300 (polyethylene glycol 300); mpk (mg / kg or mg drug per kg of animal body weight); PO (oral); Q7D (once every 7 days, once a week); QD (once daily); and SC (subcutaneous).

[0145] As used herein, “KAT6 inhibitor” includes KAT6A inhibitors, KAT6B inhibitors, and both KAT6A and KAT6B inhibitors. KAT6 inhibitors are disclosed in International Patent Application Nos. WO2019 / 043139A1, WO2019 / 243491A1, WO2020 / 002587, and PCT / IB2020 / 055667. The contents of each of the foregoing references are incorporated herein by reference in their entirety.

[0146] The preparation of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) is described below.

[0147]

[0148] 1-(methanesulfonyl)-1H-pyrazole (Int-13) was synthesized according to route 1.

[0149] Route 1:

[0150]

[0151] MsCl (73.9 g, 645 mmol) was slowly added to a solution of 1H-pyrazole (8a) (33.0 g, 485 mmol) and TEA (73.6 mg, 727 mmol) in DCM at 0 °C. The mixture was stirred at 0 °C for 10 min, followed by stirring at room temperature for 1 h. TLC analysis (1:1 ethyl acetate (EtOAc) / petroleum ether) showed the consumption of the starting materials. The reactants were diluted with saturated NH4Cl aqueous solution (200 mL) and the mixture was separated. The aqueous layer was extracted with DCM (200 mL). The combined organic layers were washed with brine (300 mL) and saturated Na2CO3 aqueous solution (300 mL), dried over anhydrous Na2SO4, filtered, and concentrated to provide 1-(methanesulfonyl)-1H-pyrazole (Int-13) (64 g, 90% yield) as a pale yellow oil. 1 H NMR (400MHz, CDCl3) δ8.04 (d, J = 2.6 Hz, 1H), 7.86–7.79 (m, 1H), 6.46 (dd, J = 1.6, 2.7 Hz, 1H), 3.33 (s, 3H).

[0152] 4-Methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazole-3-amine was synthesized according to route 2.

[0153] Route 2:

[0154]

[0155] Step 1: Synthesis of 2-fluoro-6-methoxy-4-[(1H-pyrazol-1-yl)methyl]benzyl nitrile (A-1).

[0156] Cs₂CO₃ (18.9 g, 58 mmol) was added to a solution of 2-fluoro-4-(hydroxymethyl)-6-methoxybenzyl nitrile (Int-01) (7.0 g, 38.6 mmol) and 1-(methanesulfonyl)-1H-pyrazole (Int-13) (6.2 g, 42.5 mmol) in MeCN (150 mL). The mixture was stirred at 70 °C for 2 h. LC-MS analysis showed the consumption of the starting material. The reaction mixture was filtered and the filtrate was concentrated to dryness. The crude residue was purified by rapid chromatography (40 g SiO₂, 1:1 EtOAc / petroleum ether) to provide 2-fluoro-6-methoxy-4-[(1H-pyrazole-1-yl)methyl]benzyl nitrile (A-1) (7.0 g, 78% yield) as a yellow solid. m / z (ESI+) 231.8 (M+H) + .

[0157] Step 2: Synthesis of 4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazole-3-amine (A-2).

[0158] K₂CO₃ (25.1 g, 182 mmol) was added to a solution of 2-fluoro-6-methoxy-4-[(1H-pyrazol-1-yl)methyl]benzylnitrile (A-1) (7.0 g, 30.3 mmol) and N-hydroxyacetamide (6.8 g, 90.8 mmol) in DMF (200 mL) and H₂O (30 mL). The mixture was stirred at 60 °C for 16 h. TLC analysis (EtOAc) showed the consumption of the starting materials. The reaction mixture was concentrated to remove most of the DMF, and then diluted with H₂O (100 mL). The resulting precipitate was collected by filtration. The filter cake was washed with H₂O (3 × 20 mL) and dried under vacuum to provide 4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-amine (A-2) (6.0 g). The filtrate was extracted with EtOAc (2 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid chromatography (SiO2, EtOAc) to give another batch of 4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazole-3-amine (A-2) (0.5 g). The two batches were combined and dried under vacuum to give 4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazole-3-amine (A-2) (6.5 g, 88% yield) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ7.88(d,J=2.0Hz,1H),7.51(d,J=1.3Hz,1H),6.70(s,1H),6.63 (s,1H),6.31(t,J=2.0Hz,1H),6.08–5.78(m,2H),5.52–5.31(m,2H),3.93–3.73(m,3H). m / z(ESI+)244.8(M+H) + .

[0159] 2-Methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) was prepared according to route 3 (pathway A).

[0160] Route 3:

[0161]

[0162] 2-Methoxybenzene-1-sulfonyl chloride (3.17 g, 15.4 mmol) was added to a suspension of 4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-ylamine (A-2) (2.5 g, 10 mmol) in pyridine (8.0 mL). The reaction mixture was stirred at 120 °C for 1.5 h. The mixture was cooled to room temperature and diluted with MeOH. The resulting suspension was filtered and the filter cake was washed with MeOH (30 mL). The solid was dissolved in DCM (50 mL) and MeOH (30 mL) was added. The DCM was removed under vacuum and the precipitate was collected by filtration. The filter cake was lyophilized to provide 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) (2.5 g, 59% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ10.18(s,1H),7.87(d,J=2.0Hz,1H),7.80(dd,J=1.6,7.9Hz,1H),7.66–7.59(m,1H),7.49(d,J=1.5Hz,1H),7.19(d,J=8 .3Hz,1H),7.09(t,J=7.7Hz,1H),6.83(s,1H),6.74(s,1H),6.30(t,J=2 .0Hz,1H),5.44(s,2H),3.82(s,3H),3.78(s,3H); m / z(ESI+)415.0(M+H) + .

[0163] The alternative preparation of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) was carried out according to route 4.

[0164]

[0165] Route 4:

[0166]

[0167] 4-Methoxy-6-(1H-pyrazol-1-ylmethyl)-1,2-benzoxazol-3-amine (A-2) (10.00 g, 40.94 mmol), 2-methoxybenzenesulfonyl chloride (10.15 g, 49.13 mmol), and acetonitrile (100 mL) were charged into a 100 mL reactor equipped with a top stirrer. The resulting suspension was stirred at 25 °C for 55 min. Dimethyl sulfoxide (0.36 mL, 4.09 mmol) was added in a single addition via pipette. 3,5-Dimethylpyridine (14.8 mL, 122.82 mmol) was added dropwise over 15 min via syringe. The resulting pale yellow suspension was stirred at 25 °C for 18 h to achieve a conversion of >98%, as determined by LCMS. The reaction mixture was acidified with 1 M HCl aqueous solution (100 mL) and then concentrated to approximately 80 mL (rotary evaporator, 40 °C, 85 μg). The slurry was treated with a separate 1M HCl aqueous solution (40 mL) to rinse the container walls, followed by stirring at 20°C for 2.5 hours. The resulting precipitate was collected by suction filtration. The filter cake was washed with water (2 × 50 mL) and then dried under vacuum at 35°C for 48 hours to give crude 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) as a solid (15.2 g, 90% yield, 98% purity as determined by LCMS). m / z 415.1 (M+H) + .

[0168] To purify the crude product, a suspension of crude 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) (14.00 g, 33.78 mmol) in dichloromethane (210 mL) was heated in a 40 °C bath until a clear solution was obtained (10 min). The mixture was filtered, and the filtrate was returned to a clean reaction vessel for quantitative transfer using additional dichloromethane (70 mL). Ethyl acetate (140 mL) was added to the solution over 2 min, and the mixture was stirred for 2.5 h. No crystallization was observed, so the solution was concentrated under reduced pressure (200 mbar) to remove the dichloromethane (reducing the volume by approximately 70 mL). Ethyl acetate (140 mL) was then added to the residue, and the mixture was stirred at room temperature for 21 h. The resulting suspension was concentrated under reduced pressure (40°C, 200 mbar) to approximately 280 mL, followed by stirring at room temperature for 3 hours. The solid was collected by filtration, and the reaction vessel and filter cake were washed with additional ethyl acetate (70 mL). The filter cake was dried in a vacuum oven at 35°C for 23 hours to give 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) as a solid (12.0 g, 85% yield, 97.9% purity as determined by UPLC, free from single impurities greater than 0.5%). m / z 415.1 (M+H) + .

[0169] For further purification, a suspension of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) (2.0 g, 4.73 mmol) in acetone (80 mL) was heated to reflux (bath temperature 55 °C) with stirring and maintained for 2 hours. While the mixture was still being heated, ethyl acetate (30 mL) was slowly added to maintain the internal temperature above 45 °C. The resulting slurry was concentrated to approximately 30 mL under moderate vacuum (bath temperature 65 °C), and then slowly cooled to 20 °C at a rate of 1 °C / min (approximately 31 min). The resulting precipitate was collected by suction filtration. The filter cake was dried under vacuum at 50°C for 22 hours to obtain 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound E) (1.825 g, 93% yield, 99.5% purity determined by UPLC) as a crystalline solid.

[0170] 1¹H NMR (400MHz, chloroform-d) δ 8.14 (dd, J = 1.7, 7.8 Hz, 1H), 8.04 (s, 1H), 7.59–7.51 (m, 2H), 7.44 (d, J = 2.2 Hz, 1H), 7.14–7.06 (m, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 0.6 Hz, 1H), 6.45 (s, 1H), 6.32 (t, J = 2.1 Hz, 1H), 5.38 (s, 2H), 3.97 (s, 3H), 3.91 (s, 3H).

[0171] The anhydrous free base of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (form 1) (compound E) was prepared according to route 5 (pathway B).

[0172]

[0173] Route 5:

[0174]

[0175] 2-Methoxybenzene-1-sulfonyl chloride (7.6 g, 37 mmol) was placed in a two-necked round-bottom flask equipped with an internal thermometer. 4-Methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-amine (A-2) (8.18 g, 33.5 mmol) was added, and the contents were dissolved in pyridine (55 mL, 0.6 M) under gentle heating. Heating was initiated at an oil bath temperature of 110 °C and an internal temperature of 101 °C. The reaction was complete after 5 h of heating, as determined by LCMS analysis. The reactants were cooled to room temperature and partitioned between DCM (200 mL), 6N HCl (100 mL), and ice water (100 mL). The product was extracted into DCM (×3), and the combined DCM extracts were washed with 1N HCl (×3) to remove trace amounts of pyridine. The DCM extracts were dried over MgSO4 and concentrated into a dark oil. The oily substance was purified by rapid chromatography (eluting with a gradient of 40-100% EtOAc in heptane) to obtain 4.6 g of product, which was confirmed by NMR. The 4.6 g product was recrystallized by first dissolving it in CH3CN (60 mL) under reflux until most of the solid was dissolved. The hot solution was filtered using a preheated / hot glass funnel fitted with grooved filter paper. This step removed any inorganic or silica gel impurities. The filter paper was washed with small amounts of CH3CN, totaling 10 mL of washings. The filtrate was collected in a 250 mL beaker equipped with a stir bar. MTBE (45 mL) was added to the hot filtrate and stirring was initiated. After stirring for 30 seconds, a white precipitate began to form. Stirring was continued at 400 rpm while a gentle stream of N2 was forced through the top of the solution to help accelerate the evaporation process. Forced N2 evaporation continued for 3 h until the total volume was 50 mL. The white solid was filtered and washed with MTBE (×2) and heptane (×2). The white powder was placed in a 3-inch diameter crystallizing dish, covered with filter paper, and heated in a vacuum oven at 70°C for 48 hours. A slow flow of N2 was used to assist the drying process. After drying, 3.9 g of crystalline product was obtained, which was confirmed by NMR. Melting point = 203-204°C. (Regarding C...) 19 H 18 Analytical values ​​of N4O5S: C, 55.06; H, 4.38; N, 13.52. Measured values: C, 55.09; H, 4.41; N, 13.57.

[0176] The preparation of 2,6-dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound C) is described below.

[0177]

[0178] Preparation of 2,6-dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide according to route 6.

[0179] Route 6:

[0180]

[0181] Step 1: Substitutional synthesis of 4-((1H-pyrazol-1-yl)methyl)-2-fluoro-6-methoxybenzyl nitrile (A-1) from 14b

[0182] A solution of 1H-pyrazole (2.0 g, 29.6 mmol) and sodium hydride (NaH) (60% w / w dispersion in mineral oil, 1.5 g, 37.1 mmol) in DMF (520 mL) was stirred at 0 °C for 1 h. Then, a solution of 4-(bromomethyl)-2-fluoro-6-methoxybenzyl nitrile (14b) (6.0 g, 24.7 mmol) in DMF (80 mL) was added, and the mixture was stirred overnight at room temperature (RT). The reaction was quenched with water, and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate (Na₂SO₄), filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / EtOAc = 6 / 1) to give 4-((1H-pyrazole-1-yl)methyl)-2-fluoro-6-methoxybenzyl nitrile (A-1) (2.4 g, 42%) as a yellow solid. m / z 232.0 [M+H] + .

[0183] Step 2: Substitutional synthesis of 6-((1H-pyrazol-1-yl)methyl)-4-methoxybenzo[d]isoxazole-3-amine (A-2) using potassium tert-butoxide

[0184] Potassium tert-butoxide (5.6 g, 49.5 mmol) was added to a solution of acetohydroxamic acid (3.7 g, 49.5 mmol) in anhydrous DMF (150 mL) under RT, and the mixture was stirred at RT for 1 h. Then, 4-((1H-pyrazol-1-yl)methyl)-2-fluoro-6-methoxybenzyl nitrile (A-1) (3.8 g, 16.5 mmol) was added, and the mixture was stirred at 60 °C for another 4 h. Water was added, and the mixture was extracted with EtOAc. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / EtOAc = 5 / 1) to give 6-((1H-pyrazol-1-yl)methyl)-4-methoxybenzo[d]isoxazole-3-amine (A-2) (2.1 g, 53%) as a yellow solid. m / z 245.0 [M+H] + .1 H NMR (400MHz, DMSO-d6) δ7.87 (dd, J=1.6, 0.4Hz, 1H), 7.50 (dd, J=1.6, 0.4Hz, 1H), 6.6 9(s,1H),6.62(s,1H),6.30(t,J=2.1Hz,1H),5.93(s,2H),5.41(s,2H),3.86(s,3H).

[0185] Step 3: Synthesis of N-(6-((1H-pyrazol-1-yl)methyl)-4-methoxybenzo[d]isoxazol-3-yl)-2,6-dimethoxybenzenesulfonamide (Example 98)

[0186] A mixture of 6-((1H-pyrazol-1-yl)methyl)-4-methoxybenzo[d]isoxazole-3-amine (A-2) (50 mg, 0.205 mmol) and 2,6-dimethoxybenzenesulfonyl chloride (Int-26) (73 mg, 0.308 mmol) in pyridine (1 mL) was heated at 120 °C under microwave irradiation for 2 h (batch 1).

[0187] A mixture of 6-((1H-pyrazol-1-yl)methyl)-4-methoxybenzo[d]isoxazole-3-amine (A-2) (500 mg, 2.1 mmol) and 2,6-dimethoxybenzenesulfonyl chloride (Int-26) (746 mg, 3.2 mmol) in pyridine (5 mL) was heated at 120 °C under microwave irradiation for 2 h (batch 2).

[0188] Repeat the reaction on the exact same scale (batch 3).

[0189] A mixture of 6-((1H-pyrazol-1-yl)methyl)-4-methoxybenzo[d]isoxazole-3-amine (A-2) (350 mg, 1.4 mmol) and 2,6-dimethoxybenzenesulfonyl chloride (Int-26) (509 mg, 2.2 mmol) in pyridine (4 mL) was heated at 120 °C under microwave irradiation for 2 h (batch 4).

[0190] The four reaction mixtures were combined, diluted with water, adjusted to pH 5-6 with 2M HCl aqueous solution, and extracted with EtOAc (300 mL × 3). The combined organic extracts were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / EtOAc = 2 / 1) to give N-(6-((1H-pyrazol-1-yl)methyl)-4-methoxybenzo[d]isoxazol-3-yl)-2,6-dimethoxybenzenesulfonamide (compound C) (1.07 g, 43%) as a white solid. m / z 445.0 [M+H] + .1 H NMR (400MHz, DMSO-d6) δ9.58 (s, 1H), 7.87 (d, J = 2.0Hz, 1H), 7.50-7.46 (m, 2H), 6.83(s,1H),6.76(m,3H),6.30(s,1H),5.44(s,2H),3.87(s,3H),3.76(s,6H).

[0191] The alternative preparation of 2,6-dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide was carried out according to route 7.

[0192] Route 7:

[0193]

[0194] Step 1: Substitutional synthesis of 4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazole-3-amine (A-2) using 1,1,3,3-tetramethylguanidine.

[0195] A suspension of 2-fluoro-6-methoxy-4-(1H-pyrazol-1-ylmethyl)benzyl nitrile (A-1) (15.43 g, 66.7 mmol), N-hydroxyacetamide (15.0 g, 200 mmol), and 1,1,3,3-TMG (46.1 g, 400 mmol) in acetonitrile (270 mL) and deionized water (30 mL) was heated to 60 °C and held for 7 hours. Acetonitrile was removed under vacuum, and the remaining viscous oil was partitioned between ethyl acetate (300 mL) and deionized water (250 mL). The aqueous layer was extracted with ethyl acetate (2 × 150 mL). All organic layers were combined and washed with a saturated aqueous NaCl solution. Some solids began to form in the organic layer; therefore, methanol (approximately 10 mL) was added, and the suspension was heated until homogenized. After cooling to room temperature, the organic layer was dried over sodium sulfate, filtered, and concentrated. The resulting pale yellow solid was suspended in ethyl acetate (125 mL) and briefly heated to reflux. The suspension was cooled to room temperature, and the resulting solid was collected by filtration, with the filter cake washed with heptane. The filtrate and heptane wash were concentrated to dryness, and the remaining solid was suspended in ethyl acetate (15 mL). The suspension was briefly heated to reflux, and the second batch of precipitate was collected as described above. The combined precipitate harvests were dried under vacuum to give 4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-amine (A-2) (11.86 g, 48.6 mmol) as a pale yellow powder. 1H NMR (400MHz, DMSO-d6) δ7.87(d,J=1.8Hz,1H),7.49(d,J=1.2Hz,1H),6.69(s,1 H), 6.62 (s, 1H), 6.30 (t, J = 2.1Hz, 1H), 5.93 (s, 2H), 5.41 (s, 2H), 3.86 (s, 3H). LCMS:[M+H] + 245.

[0196] Step 2: Synthesis of 2,6-dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide (compound C)

[0197] A mixture of 4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-amine (A-2) (9.5 g, 39 mmol) and 2,6-dimethoxybenzenesulfonyl chloride (Int-26) (12.1 g, 51.1 mmol) in pyridine (20 mL) was heated to 97 °C (internal temperature) and maintained for 1 hour. After cooling to 50 °C, the solution was poured into a flask containing crushed ice (200 g) and 6N HCl (100 mL). The reaction flask was rinsed with dichloromethane for quantitative transfer. The resulting aqueous mixture was extracted with dichloromethane (4 × 100 mL). The combined organic extracts were washed with deionized water and saturated NaCl aqueous solution, dried over magnesium sulfate, filtered, and concentrated to a yellow foam. Methyl acetate (50 mL) was added to the foam, and the suspension was stirred at room temperature for 1 hour. The solid was collected by suction filtration and washed with heptane. After drying under vacuum, crude 2,6-dimethoxy-N-[4-methoxy-6-(1H-pyrazol-1-ylmethyl)-1,2-benzoxazol-3-yl]benzenesulfonamide (compound C) (16.1 g, 95%) was obtained as an orange-brown solid. Grinding the crude solid twice with methyl acetate did not remove the orange color; therefore, the crude product was ground in warm dichloromethane, cooled to room temperature, and filtered to obtain a milky white solid. The dichloromethane mother liquor was further purified by chromatography (330 g silica column, eluted with 60-100% ethyl acetate in heptane) to obtain a white solid. The solids from the DCM grinding and DCM filtrate were combined, stirred in refluxed methyl acetate, and cooled to room temperature for 2 hours. The obtained solid was collected by suction filtration and dried overnight in a vacuum oven at 100°C to obtain purified 2,6-dimethoxy-N-[4-methoxy-6-(1H-pyrazol-1-ylmethyl)-1,2-benzoxazol-3-yl]benzenesulfonamide (compound C) (15.3 g, 89%) as a grayish-white powder.

[0198] The three batches of compound C prepared as described above (total 54.3 g) were combined, suspended in methyl acetate (250 mL), and heated to reflux for 1 hour. After being removed from the self-heating bath, the mixture was stirred for 4 hours while cooling to room temperature. The resulting precipitate was collected by filtration and washed with heptane. The solid was dried under vacuum at room temperature for 2 hours, followed by further drying in a vacuum oven at 130 °C for 16 hours to give 2,6-dimethoxy-N-[4-methoxy-6-(1H-pyrazol-1-ylmethyl)-1,2-benzoxazol-3-yl]benzenesulfonamide (compound C) (53.55 g, 99%) as a grayish-white solid. 1 HNMR(400MHz,DMSO-d6)δ9.60(s,1H),7.88(d,J=1.7Hz,1H),7.45-7.52(m,2H),6.83(s, 1H), 6.77 (d, J = 8.4Hz, 3H), 6.30 (t, J = 2.1Hz, 1H), 5.44 (s, 2H), 3.87 (s, 3H), 3.76 (s, 6H). LCMS:[M+H] + 445. Regarding C 20 H 20 Analytical values ​​of N4O6S: C, 54.05; H, 4.54; N, 12.61; S, 7.21. Measured values: C, 53.91; H, 4.58; N, 12.51; S, 7.09.

[0199] Cyclin-dependent kinases (CDKs) and their associated serine / threonine kinases are important cellular enzymes that perform essential functions in regulating cell division and proliferation. CDK inhibitors include pan-CDK inhibitors that target a broad spectrum of CDKs or selective CDK inhibitors that target one or more specific CDKs.

[0200] As used herein, “CDK4 inhibitor” includes both selective CDK4 inhibitors and CDK4 / 6 inhibitors. Selective CDK4 inhibitors are disclosed in International Publication No. WO 2019 / 207463. Examples of CDK4 / 6 inhibitors include, but are not limited to, abeciclib, ribociclib, and palbociclib. Other examples of CDK4 / 6 inhibitors include lerociclib (also known as G1T38) and trilaciclib (also known as GTI128).

[0201] In embodiments, the CDK4 selective inhibitor of the present invention comprises 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentol or a pharmaceutically acceptable salt thereof.

[0202] In embodiments, preferred CDK4 / 6 inhibitors of the present invention include palbociclib. Unless otherwise indicated herein, palbociclib (also referred to herein as “palbo” or “Palbo”) means 6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one or a pharmaceutically acceptable salt thereof.

[0203] As used herein, the term "anti-estrogen" refers to a class of drugs that prevent the mediating biological effects of estrogens such as estradiol. Anti-estrogens work by blocking estrogen receptors (ER) and / or inhibiting or suppressing estrogen production. In some embodiments, anti-estrogens are aromatase inhibitors, selective estrogen receptor degraders (SERDs), or selective estrogen receptor modulators (SERMs). Examples of aromatase inhibitors include, but are not limited to, anastrozole. Examples of SERDs include, but are not limited to, fulvestrant. Other SERDs include elacestrant (RAD-1901, Radius Health), SAR439859 (Sanofi), RG6171 (Roche), AZD9833 (AstraZeneca), AZD9496 (AstraZeneca), rintodestrant (G1 therapy), ZN-c5 (Zentalis), LSZ102 (Novartis), D-0502 (Inventisbio), LY3484356 (Lilly), and SHR9549 (Jiansu Hengrui Medicine). Examples of SERMs include, but are not limited to, tamoxifen, clomifene, and raloxifene. Other SERMs include toremifene, lasofoxifene, bazedoxifene, and afimoxifene.

[0204] In the implementation scheme, aromatase inhibitors include letrozole, exemestane, and anastrozole. SERMs include tamoxifen, clomiphene, and raloxifene.

[0205] In some embodiments, the anti-estrogens of the present invention include fulvestrant and letrozole. In some embodiments, the preferred anti-estrogens of the present invention include fulvestrant. In some embodiments, the preferred anti-estrogens of the present invention include letrozole.

[0206] Some embodiments involve pharmaceutically acceptable salts of the compounds described herein. Pharmaceutically acceptable salts of the compounds described herein include their acid addition salts and base addition salts.

[0207] Some embodiments also involve pharmaceutically acceptable acid addition salts of the compounds described herein. Suitable acid addition salts are formed from acids that form non-toxic salts. Non-limiting examples of suitable acid addition salts (i.e., salts containing pharmacologically acceptable anions) include, but are not limited to, acetates, acid citrates, adipates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, bisulfates / sulfates, tartrates, borates, camphor sulfonates, citrates, cyclohexylamine sulfonates, ethanedisulfonates, esylate, ethanesulfonates, formates, fumarates, gluconate, glucuronates, hexafluorophosphates, hydantoinates, hydrochlorides / chlorides. Hydrobromide / bromine, hydroiodide / iodide, hydroxyethanesulfonate, lactate, malate, maleate, malonate, mesylate, methanesulfonate, methyl sulfate, naphthate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, dihydroxynaphthalate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, glycosides, stearate, succinate, tannate, tartrate, p-toluenesulfonate, toluenesulfonate, trifluoroacetate, and xinafoate.

[0208] Other embodiments involve base addition salts of the compounds described herein. Suitable base addition salts are formed from bases that form non-toxic salts. Non-limiting examples of suitable basic salts include aluminum salts, arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, glycerol salts, and zinc salts.

[0209] The compounds described herein, being basic in nature, can form a wide variety of salts with various inorganic and organic acids. Pharmaceutically acceptable acid addition salts that can be used to prepare these basic compounds are those that form non-toxic acid addition salts, such as salts containing pharmacologically acceptable anions, including hydrochlorides, hydrobromides, hydroiodides, nitrates, sulfates, hydrogen sulfates, phosphates, acid phosphates, isonicotinates, acetates, lactates, salicylates, citrates, acid citrates, tartrates, pantothenates, hydrogen tartrates, ascorbic acid salts, succinates, maleates, gentisinates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and bis(hydroxynaphthyl)ate [i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthyl)ate]. In addition to the acids mentioned above, compounds described herein that contain a basic moiety (such as an amino group) can also form pharmaceutically acceptable salts with various amino acids.

[0210] The chemical base that can be used as a reagent for preparing pharmaceutically acceptable basic salts of those compounds described herein that are acidic in nature is that which form non-toxic basic salts with such compounds. These non-toxic basic salts include, but are not limited to, those salts derived from such pharmaceutically acceptable cations (such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium)), ammonium or water-soluble amine addition salts (such as N-methylglucosamine-(glucamine)) and other basic salts of low-carbon alkanol ammonium and pharmaceutically acceptable organic amines.

[0211] It can also form half-salts of acids and bases, such as half-sulfates and half-calcium salts.

[0212] For a review of suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds described herein are known to those skilled in the art.

[0213] The compounds described herein, containing one or more asymmetric carbon atoms, can exist in two or more stereoisomeric forms. In the case of compounds containing an alkenyl or alkenyl group, geometric cis / trans (or Z / E) isomers are possible. Tautomeric isomerism can occur where the structural isomers can interconvert via low energy barriers. In compounds described herein containing, for example, imino, ketone, or oxime groups, proton tautomerism can occur, or in compounds containing aromatic moieties, so-called valence tautomerism can occur. A single compound can exhibit more than one type of isomerism.

[0214] The compounds described herein include all stereoisomers (e.g., cis and trans isomers) and all optical isomers (e.g., R and S enantiomers), as well as racemic mixtures, diastereomeric mixtures, and other mixtures of these isomers. Although all stereoisomers are covered within the scope of our claims, those skilled in the art will recognize that certain stereoisomers may be preferred.

[0215] In some embodiments, the compounds described herein may exist in several tautomeric forms, including enol and imine forms, ketone and enamine forms, and their geometric isomers and mixtures. All such tautomeric forms are included within the scope of these embodiments. The tautomeric forms exist as mixtures of the tautomeric groups in solution. In solid form, typically one tautomeric form is dominant. Even if one tautomeric form is described, these embodiments still include all tautomeric forms of the compounds of the invention.

[0216] The scope of this embodiment includes all stereoisomers, geometric isomers, and tautomers of the compounds described herein, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. It also includes acid addition salts or basic salts wherein the counterion is optically active, such as d-lactate or l-lysine, or is racemic, such as dl-tartrate or dl-arginine.

[0217] This embodiment also includes the rotation-restricted isomers of the compounds described herein. A rotation-restricted isomer is a compound that can be isolated into a rotation-restricted isomer.

[0218] Cis / trans isomers can be separated using conventional techniques known to those skilled in the art, such as chromatography and fractional crystallization.

[0219] Conventional techniques for preparing / separating enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC).

[0220] Alternatively, the racemic mixture (or racemic precursor) can react with a suitable optically active compound (e.g., an alcohol), or, in the case where the compound described herein contains an acidic or basic moiety, with a base or acid (such as 1-phenylethylamine or tartaric acid). The resulting diastereomeric mixture can be separated by chromatography and / or fractional crystallization, and one or two of these diastereomeric isomers can be converted into one or more corresponding pure enantiomers by means well known to those skilled in the art.

[0221] Unless otherwise indicated, as used herein, the term "treating" means reversing, alleviating, suppressing, or preventing the progression of one or more symptoms of the condition to which the term applies. Unless otherwise indicated, as used herein, the term "treatment" refers to a therapeutic action as defined above.

[0222] The "patient" to be treated according to the present invention includes any warm-blooded animal, such as, but not limited to, humans, monkeys or other lower primates, horses, dogs, rabbits, guinea pigs, or mice. For example, the patient is a human. Those skilled in the medical field can easily identify individual patients suffering from cancer (e.g., breast cancer, particularly estrogen receptor-positive breast cancer) who require treatment.

[0223] Unless otherwise stated, the term "combination" as used herein means a fixed-dose combination or combination of pharmaceutical agents administered intermittently, simultaneously, or sequentially according to the same or different routes of administration and according to the same or different schedules. As used herein, an "effective" amount or "therapeuticly effective" amount refers to an amount of an agent, compound, or composition sufficient to reduce the severity of disease symptoms, increase the frequency and duration of asymptomatic periods of disease, or prevent damage or disability caused by disease suffering, whether used as a single dose or according to a multi-dose regimen, alone or in combination with other agents. Those skilled in the art will be able to determine these amounts based on factors such as the patient's physical condition, the severity of the patient's symptoms, and the specific combination, composition, or route of administration chosen. The patient or individual may be a human or a non-human mammal requiring treatment. In one embodiment, the patient is a human.

[0224] As used herein, the term "locally advanced" refers to cancer that may or may not be treated with the aim of curing. As used herein, the term "metastatic" refers to cancer that cannot be treated with the aim of curing. Those skilled in the art will be able to identify and diagnose locally advanced and metastatic cancer in patients.

[0225] For convenience, certain well-known abbreviations may be used in this article, including: castration-resistant prostate cancer (CRPC), estrogen receptor positive (ER+), human epidermal growth factor receptor 2 negative (HER2-), hormone receptor (HR), human epidermal growth factor receptor 2 positive (HER2+), non-small cell lung cancer (NSCLC), and progesterone receptor (PR).

[0226] In some implementation schemes, the cancer is selected from: lung cancer, mesothelioma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, liver cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, hematologic malignancies, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, spinal tumors, glioblastoma, brainstem glioma, pituitary adenoma, head and neck cancer, or a combination of two or more of the above cancers.

[0227] Other implementation methods involve methods for treating a patient's cancer. Some implementation methods involve treating a patient's cancer by administering to the patient a certain amount of the compound described herein that is effective in treating cancer.

[0228] In one implementation scheme, the cancer is breast cancer, lung cancer, colon cancer, brain cancer, head and neck cancer, prostate cancer, stomach cancer, pancreatic cancer, ovarian cancer, melanoma, endocrine cancer, uterine cancer, testicular cancer, or bladder cancer.

[0229] In one implementation plan, the cancer is breast cancer, lung cancer, prostate cancer, pancreatic cancer, or ovarian cancer.

[0230] In one implementation plan, the cancer is breast cancer, lung cancer, or prostate cancer.

[0231] In one implementation, the cancer is breast cancer.

[0232] In one implementation plan, breast cancer is HR+ breast cancer.

[0233] In one implementation, HR+ breast cancer is PR+ and / or ER+ breast cancer.

[0234] In one implementation, breast cancer is PR+ breast cancer.

[0235] In one implementation, the breast cancer is ER+ breast cancer.

[0236] In one implementation, breast cancer is ER+HER2- breast cancer.

[0237] In one implementation, breast cancer is ER+HER2+ breast cancer.

[0238] In one implementation, the breast cancer is locally advanced or metastatic ER+ breast cancer.

[0239] In one implementation, the breast cancer is locally advanced or metastatic ER+HER2- breast cancer.

[0240] In one implementation, the breast cancer is locally advanced or metastatic ER+HER2+ breast cancer.

[0241] In one implementation plan, the lung cancer is non-small cell lung cancer.

[0242] In one implementation scheme, the lung cancer is locally advanced or metastatic non-small cell lung cancer.

[0243] In one implementation, the prostate cancer is castration-resistant prostate cancer.

[0244] In one implementation, the prostate cancer is locally advanced or metastatic castration-resistant prostate cancer.

[0245] Other embodiments relate to methods of treating a patient's solid tumor. Some embodiments involve treating a patient's solid tumor by administering to the patient a certain amount of the compound described herein that is effective in treating solid tumors.

[0246] In one implementation, a solid tumor is a tumor of the breast, lung, colon, brain, head and neck, prostate, stomach, pancreas, ovary, melanoma, endocrine system, uterus, testicle, or bladder.

[0247] In one implementation, a solid tumor is a tumor of the breast, lung, prostate, pancreas, or ovary.

[0248] In one implementation, a solid tumor is a tumor of the breast, lung, or prostate.

[0249] In one embodiment, the solid tumor is breast cancer; in another embodiment, the breast cancer is HR+ breast cancer; and in yet another embodiment, the HR+ breast cancer is PR+ and / or ER+ breast cancer.

[0250] In one implementation, the solid tumor is breast cancer, and in another implementation, the breast cancer is ER+HER2- breast cancer.

[0251] In one implementation, the solid tumor is breast cancer, and in another implementation, the breast cancer is ER+HER2+ breast cancer.

[0252] In one implementation, the solid tumor is breast cancer, and in another implementation, the breast cancer is locally advanced or metastatic ER+HER2- breast cancer.

[0253] In one implementation, the solid tumor is breast cancer, and in another implementation, the breast cancer is locally advanced or metastatic ER+HER2+ breast cancer.

[0254] In one implementation, the solid tumor is lung cancer, and in another implementation, the lung cancer is non-small cell lung cancer.

[0255] In one implementation, the solid tumor is lung cancer, and in another implementation, the lung cancer is locally advanced or metastatic non-small cell lung cancer.

[0256] In one implementation, the solid tumor is prostate cancer, and in another implementation, the prostate cancer is castration-resistant prostate cancer.

[0257] In one implementation, the solid tumor is prostate cancer, and in another implementation, the prostate cancer is locally advanced or metastatic castration-resistant prostate cancer.

[0258] Other implementation methods relate to methods of treating hematologic malignancies in patients. Some implementation methods involve treating hematologic malignancies in patients, which includes administering to the patient a certain amount of the compounds described herein that are effective in treating hematologic malignancies.

[0259] In one implementation, the hematologic malignancy is leukemia, lymphoma, or multiple myeloma.

[0260] In one implementation, the hematologic malignancy is leukemia or lymphoma.

[0261] The term "additive" is used to mean that the result of combining two compounds, components, or targeted agents is no greater than the sum of the individual compounds, components, or targeted agents. The term "additive" also means that there is no improvement in the disease, condition, or symptom treated relative to the use of each compound, component, or targeted agent individually.

[0262] The term "synergy" or "synergy" is used to mean that the result of combining two compounds, components, or targeted agents is greater than the sum of the effects of each agent alone. The term "synergy" or "synergy" also refers to an improvement in the treated disease, condition, or symptom compared to the use of each compound, component, or targeted agent individually. This improvement in the treated disease, condition, or symptom is a "synergistic effect." A "synergistic amount" is the amount by which the combination of two compounds, components, or targeted agents produces a synergistic effect, as defined herein.

[0263] When determining synergistic interactions between one or two components, the optimal range for achieving the effect and the absolute dose range for each component to achieve the effect can be clearly measured by administering these components to patients requiring treatment at different weight / weight ratio ranges and doses. However, observing synergies in in vitro or in vivo models can predict effects in humans and other species, and as described herein, in vitro or in vivo models exist for measuring synergistic effects. Furthermore, the results of these studies can be used to predict the required effective dose and plasma concentration ratio range, as well as absolute dose and plasma concentration, in humans and other species using pharmacokinetic / pharmacodynamic methods.

[0264] According to the present invention, a certain amount of a first compound or component is combined with a certain amount of a second compound or component, and optionally with a certain amount of a third compound or component, and these amounts together are effective or therapeutically effective in treating cancer (e.g., breast cancer). These amounts that are effective or therapeutically effective together will alleviate one or more symptoms of the treated disease to a certain extent. Regarding the treatment of cancer, an effective or therapeutically effective amount refers to an amount that has the following effects: (1) reducing tumor size, (2) inhibiting (i.e., to a certain extent slowing down, preferably terminating) the occurrence of tumor metastasis, (3) to a certain extent inhibiting (i.e., to a certain extent slowing down, preferably terminating) tumor growth or tumor invasiveness and / or (4) to a certain extent alleviating (or preferably eliminating) one or more cancer-related signs or symptoms. The therapeutic or pharmacological effectiveness of the dosage and administration regimen can also be characterized by the ability to induce, enhance, maintain, or prolong disease control and / or overall survival in patients with these specific tumors, which can be measured as a prolongation of the time before disease progression.

[0265] In its implementation, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a combination of a KAT6 inhibitor and a CDK4 inhibitor or an anti-estrogen, said amount being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 inhibitor or anti-estrogen, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 inhibitor or an anti-estrogen for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 inhibitor or anti-estrogen, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2 breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 inhibitor or an anti-estrogen for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the amount of the CDK4 inhibitor in the method and use of the present invention further comprises administering a certain amount of an anti-estrogen, such as fulvestrant or letrozole. In an embodiment, the amount of the CDK4 inhibitor in the method and use of the present invention further comprises administering a certain amount of letrozole. In an embodiment, the amount of the CDK4 inhibitor in the method and use of the present invention further comprises administering a certain amount of fulvestrant.

[0266] The KAT6 inhibitors mentioned in the methods, uses and combinations in the previous paragraph include KAT6A inhibitors.

[0267] In its implementation, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a combination of a KAT6 inhibitor and a CDK4 inhibitor, said amount being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 inhibitor, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 inhibitor for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 inhibitor, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 inhibitor for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the amount of the CDK4 inhibitor in the method and use of the present invention further comprises administering a certain amount of an anti-estrogen, such as fulvestrant or letrozole. In an embodiment, the amount of the CDK4 inhibitor in the method and use of the present invention further comprises administering a certain amount of letrozole. In an embodiment, the amount of the CDK4 inhibitor in the method and use of the present invention further comprises administering a certain amount of fulvestrant.

[0268] The KAT6 inhibitors mentioned in the methods, uses and combinations in the previous paragraph include KAT6A inhibitors.

[0269] In its implementation, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a combination of a KAT6 inhibitor and an anti-estrogen, said amount being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of an anti-estrogen, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and an anti-estrogen for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of an anti-estrogen, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and an anti-estrogen for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic.

[0270] The KAT6 inhibitors mentioned in the methods, uses and combinations in the previous paragraph include KAT6A inhibitors.

[0271] In its implementation, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a combination of a KAT6 inhibitor and a CDK4 selective inhibitor, said amount being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 selective inhibitor, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 selective inhibitor for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 selective inhibitor, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 selective inhibitor for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the amount of the CDK4 selective inhibitor in the method and use of the present invention further comprises administering a certain amount of an anti-estrogen, such as fulvestrant or letrozole. In an embodiment, the amount of the CDK4 selective inhibitor in the method and use of the present invention further comprises administering a certain amount of letrozole. In an embodiment, the amount of the CDK4 selective inhibitor in the method and use of the present invention further comprises administering a certain amount of fulvestrant.

[0272] The KAT6 inhibitors mentioned in the methods, uses and combinations in the previous paragraph include KAT6A inhibitors.

[0273] In its implementation, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a combination of a KAT6 inhibitor and a CDK4 / 6 inhibitor, said amounts being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 / 6 inhibitor, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of KAT6 inhibitors and CDK4 / 6 inhibitors for the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor and a certain amount of a CDK4 / 6 inhibitor, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 / 6 inhibitor for the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the amount of the CDK4 / 6 inhibitor in the method and use of the present invention further comprises administering a certain amount of an anti-estrogen, such as fulvestrant or letrozole. In an embodiment, the amount of the CDK4 / 6 inhibitor in the method and use of the present invention further comprises administering a certain amount of letrozole. In an embodiment, the amount of the CDK4 / 6 inhibitor in the method and use of the present invention further comprises administering a certain amount of fulvestrant.

[0274] The KAT6 inhibitors mentioned in the methods, uses and combinations in the previous paragraph include KAT6A inhibitors.

[0275] In its implementation, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a specific amount of a KAT6 inhibitor, a specific amount of a CDK4 inhibitor, and a specific amount of an anti-estrogen, said amounts being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor, a certain amount of a CDK4 inhibitor, and a certain amount of an anti-estrogen, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of KAT6 inhibitors, CDK4 inhibitors, and anti-estrogens for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor, a certain amount of a CDK4 inhibitor, and a certain amount of an anti-estrogen, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor, a CDK4 inhibitor, and an anti-estrogen for the treatment of cancer, particularly breast cancer, ER+ breast cancer, HR+ breast cancer, PR+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the anti-estrogen is fulvestrant or letrozole; fulvestrant; or letrozole.

[0276] The KAT6 inhibitors mentioned in the methods, uses and combinations in the previous paragraph include KAT6A inhibitors.

[0277] In its implementation, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a specific amount of a KAT6 inhibitor, a specific amount of a CDK4 selective inhibitor, and a specific amount of an anti-estrogen, said amounts being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor, a certain amount of a CDK4 selective inhibitor, and a certain amount of an anti-estrogen, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of KAT6 inhibitors, CDK4 inhibitors, and anti-estrogens for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor, a certain amount of a CDK4 selective inhibitor, and a certain amount of an anti-estrogen, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a selective KAT6 inhibitor, a CDK4 inhibitor, and an anti-estrogen for the treatment of cancers, particularly breast cancer, ER+ breast cancer, HR+ breast cancer, PR+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the anti-estrogen is fulvestrant or letrozole; fulvestrant; or letrozole.

[0278] The KAT6 inhibitors mentioned in the methods, uses and combinations in the previous paragraph include KAT6A inhibitors.

[0279] In its implementation, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a specific amount of a KAT6 inhibitor, a specific amount of a CDK4 / 6 inhibitor, and a specific amount of an anti-estrogen, said amounts being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor, a certain amount of a CDK4 / 6 inhibitor, and a certain amount of an anti-estrogen, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of KAT6 inhibitors, CDK4 / 6 inhibitors, and anti-estrogens for the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of a KAT6 inhibitor, a certain amount of a CDK4 / 6 inhibitor, and a certain amount of an anti-estrogen, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor, a CDK4 / 6 inhibitor, and an anti-estrogen for the treatment of cancers, particularly breast cancer, ER+ breast cancer, HR+ breast cancer, PR+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the anti-estrogen is fulvestrant or letrozole; fulvestrant; or letrozole.

[0280] The KAT6 inhibitors mentioned in the methods, uses and combinations in the previous paragraph include KAT6A inhibitors.

[0281] In its embodiments, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a combination of a quantity of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof with a quantity of a CDK4 selective inhibitor, said quantity being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole-1-yl] [Methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, together with a certain amount of CDK4 selective inhibitor, wherein these amounts together are effective in treating cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a CDK4 selective inhibitor for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl]} [Methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, together with a certain amount of a CDK4 selective inhibitor, wherein these amounts together achieve a synergistic effect in the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a CDK4 selective inhibitor for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the amount of the CDK4 selective inhibitor in the method and use of the present invention further comprises administering a certain amount of an anti-estrogen, such as fulvestrant or letrozole. In an embodiment, the amount of the CDK4 selective inhibitor in the method and use of the present invention further comprises administering a certain amount of letrozole.In the implementation scheme, the amount of CDK4 selective inhibitor in the method and use of the present invention further includes administering a certain amount of fulvestrant.

[0282] In the embodiments, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole-1- [[methyl]-1,2-benzoxazole-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof in combination with a certain amount of a CDK4 / 6 inhibitor, said amount being effective in treating cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole-1-yl]... [Methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, together with a certain amount of CDK4 / 6 inhibitor, wherein these amounts together are effective in treating cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a CDK4 / 6 inhibitor for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl]} [Methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, together with a certain amount of CDK4 / 6 inhibitor, wherein these amounts together achieve a synergistic effect in the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a CDK4 / 6 inhibitor for the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the amount of the CDK4 / 6 inhibitor in the method and use of the present invention further comprises administering a certain amount of an anti-estrogen, such as fulvestrant or letrozole. In an embodiment, the amount of the CDK4 / 6 inhibitor in the method and use of the present invention further comprises administering a certain amount of letrozole.In the implementation scheme, the amount of CDK4 / 6 inhibitor in the method and use of the present invention further includes administering a certain amount of fulvestrant.

[0283] In its embodiments, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a combination of a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof with a certain amount of an anti-estrogen, said amount being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole- 1-(1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, together with a certain amount of anti-estrogenic agent, wherein these amounts together are effective in treating cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, in combination with an anti-estrogen, for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole-1 [-methyl]-1,2-benzoxazole-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, and a certain amount of anti-estrogenic agent, wherein these amounts together achieve a synergistic effect in the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and an anti-estrogen for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer, wherein the combination is synergistic.

[0284] In the embodiments, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl...} A combination of 1,2-benzoxazole-3-ylbenzene-1-sulfonamide or a pharmaceutically acceptable salt thereof with a certain amount of palbociclib or a pharmaceutically acceptable salt thereof, said amount being effective in treating cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl...} [1,2-benzoxazole-3-yl]benzyl-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of palbociclib or a pharmaceutically acceptable salt thereof, wherein these amounts together are effective in treating cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer.In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and palbociclib or a pharmaceutically acceptable salt thereof, for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl 1,2-Benzoxazol-3-ylbenzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of palbociclib or a pharmaceutically acceptable salt thereof, wherein these amounts together achieve a synergistic effect in the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and palbociclib or a pharmaceutically acceptable salt thereof for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In an embodiment, the amount of palbociclib or a pharmaceutically acceptable salt thereof in the method and use of the present invention further comprises administering an amount of an anti-estrogen, such as fulvestrant or letrozole. In an embodiment, the amount of palbociclib or a pharmaceutically acceptable salt thereof in the method and use of the present invention further comprises administering an amount of letrozole.In the embodiments, the amount of palbociclib or a pharmaceutically acceptable salt thereof in the methods and uses of the present invention further comprises administering a certain amount of fulvestrant.

[0285] In the embodiments, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a measured amount of 1, 5-Dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof, said amounts being effective in treating cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of 1, 5-Dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threopentanepentol or a pharmaceutically acceptable salt thereof, wherein these amounts together are effective in the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzyl-1-sulfonamide or a pharmaceutically acceptable salt thereof and 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threopentanepentyl alcohol or a pharmaceutically acceptable salt thereof, for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of 1,5- Dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl or a pharmaceutically acceptable salt thereof, wherein these amounts together achieve a synergistic effect in the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzyl-1-sulfonamide or a pharmaceutically acceptable salt thereof and 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threopentanepentyl alcohol or a pharmaceutically acceptable salt thereof, for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer, wherein the combination is synergistic. In embodiments, the amount of 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof in the methods and uses of the present invention further comprises administering an amount of an anti-estrogen, such as fulvestrant or letrozole. In embodiments, the amount of 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof in the methods and uses of the present invention further comprises administering an amount of letrozole. In the embodiments, the amount of 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof in the methods and uses of the present invention further comprises administering a certain amount of fulvestrant.

[0286] In its embodiments, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a combination of a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof with a certain amount of fulvestrant, said amount being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole- 1-(1-yl)methyl]-1,2-benzoxazol-3-yl)benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, together with a certain amount of fulvestrant, wherein these amounts together are effective in the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof in combination with fulvestrant for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, comprising administering to a patient in need a certain amount of fulvestrant and a certain amount of 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2- [(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl or a pharmaceutically acceptable salt thereof, wherein these amounts together achieve a synergistic effect in the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and fulvestrant for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer, wherein the combination is synergistic.

[0287] As used in this article, "endocrine therapy" (also known as hormone therapy) refers to treatments that add, block, or remove hormones. In the treatment of breast cancer, there are two types of endocrine therapy: drugs that stop estrogen and progesterone from promoting breast cancer cell growth, and drugs that stop the ovaries from producing hormones. Endocrine therapy drugs used to treat breast cancer include, but are not limited to, anastrozole, exemestane, fulvestrant, goserelin, letrozole, leuprorelin, megestrol, tamoxifen, and toremifene.

[0288] In its implementation, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6 inhibitor as a single agent, or a combination thereof with a specific amount of a CDK4 inhibitor, said amount being effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a certain amount of a KAT6 inhibitor as a single agent or a combination thereof with a certain amount of a CDK4 inhibitor, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a KAT6 inhibitor as a single agent or a combination thereof with a CDK4 inhibitor for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a certain amount of a KAT6 inhibitor as a single agent or a combination thereof with a certain amount of a CDK4 inhibitor, wherein these amounts together achieve a synergistic effect in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 inhibitor for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is indicated, particularly for breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In embodiments, the method or use of the invention relates to a synergistic combination of targeted therapeutic agents, particularly KAT6 inhibitors and CDK4 inhibitors.

[0289] In its implementation, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6 inhibitor as a single agent or a combination thereof with a specific amount of a CDK4 selective inhibitor, said amount being effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6 inhibitor as a single agent, or a combination thereof with a specific amount of a CDK4 selective inhibitor, wherein these amounts together are effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a KAT6 inhibitor as a single agent or a combination thereof with a CDK4 selective inhibitor, in a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a combination of a KAT6 inhibitor and a CDK4 selective inhibitor, wherein these amounts together achieve a synergistic effect in the method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a selective CDK4 inhibitor for overcoming clinical resistance to endocrine therapy used to treat cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In embodiments, the method or use of the invention relates to a synergistic combination of targeted therapeutic agents, particularly a KAT6 inhibitor and a selective CDK4 inhibitor.

[0290] In its implementation, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6 inhibitor as a single agent or a combination thereof with a specific amount of a CDK4 / 6 inhibitor, said amount being effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6 inhibitor as a single agent or a combination thereof with a specific amount of a CDK4 / 6 inhibitor, wherein these amounts together are effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 / 6 inhibitor for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a specific amount of a KAT6 inhibitor and a specific amount of a CDK4 / 6 inhibitor, wherein these amounts together achieve a synergistic effect in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6 inhibitor and a CDK4 / 6 inhibitor for overcoming clinical resistance to endocrine therapy used to treat cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In embodiments, the method or use of the invention relates to a synergistic combination of targeted therapeutic agents, particularly KAT6 inhibitors and CDK4 / 6 inhibitors.

[0291] In its implementation, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6A inhibitor as a single agent or a combination thereof with a specific amount of a CDK4 inhibitor, said amount being effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6A inhibitor as a single agent or a combination thereof with a specific amount of a CDK4 inhibitor, wherein these amounts together are effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a KAT6A inhibitor as a single agent or in combination with a CDK4 inhibitor for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a specific amount of a KAT6A inhibitor and a specific amount of a CDK4 inhibitor, wherein these amounts together achieve a synergistic effect in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6A inhibitor and a CDK4 inhibitor for overcoming clinical resistance to endocrine therapy used to treat cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In embodiments, the method or use of the invention relates to a synergistic combination of targeted therapeutic agents, particularly KAT6A inhibitors and CDK4 inhibitors.

[0292] In its implementation, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a dose of a KAT6A inhibitor as a single agent or a combination thereof with a dose of a CDK4 selective inhibitor, which is effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, ER+ breast cancer, HR+ breast cancer, PR+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6A inhibitor as a single agent, or a combination thereof with a specific amount of a CDK4 selective inhibitor, wherein these amounts together are effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a KAT6A inhibitor as a single agent or a combination thereof with a CDK4 selective inhibitor for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a specific amount of a KAT6A inhibitor and a specific amount of a CDK4 selective inhibitor, wherein these amounts together achieve a synergistic effect in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6A inhibitor and a selective CDK4 inhibitor for the treatment of overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used for cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In embodiments, the method or use of the invention relates to a synergistic combination of targeted therapeutic agents, particularly a KAT6A inhibitor and a selective CDK4 inhibitor.

[0293] In its implementation, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. The method comprises administering to a patient in need a specific amount of a KAT6A inhibitor as a single agent or a combination thereof with a specific amount of a CDK4 / 6 inhibitor, said amount being effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, ER+ breast cancer, HR+ breast cancer, PR+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a certain amount of a KAT6A inhibitor as a single agent or a combination thereof with a certain amount of a CDK4 / 6 inhibitor, wherein these amounts together are effective in treating cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a KAT6A inhibitor as a single agent or in combination with a CDK4 / 6 inhibitor for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a specific amount of a KAT6A inhibitor and a specific amount of a CDK4 / 6 inhibitor, wherein these amounts together achieve a synergistic effect in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of a KAT6A inhibitor and a CDK4 / 6 inhibitor for overcoming clinical resistance to endocrine therapy used to treat cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic. In embodiments, the method or use of the invention relates to a synergistic combination of targeted therapeutic agents, particularly KAT6A inhibitors and CDK4 / 6 inhibitors.

[0294] In the embodiments, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole- 1-(1-yl)methyl]-1,2-benzoxazol-3-yl)benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof or a combination thereof with a certain amount of a CDK4 selective inhibitor, said amount being effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a specific amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole-1...} as a single agent...} [-(methyl)-1,2-benzoxazol-3-yl)benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof or a combination thereof with a certain amount of a CDK4 selective inhibitor, wherein these amounts together are effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof or a combination thereof with a CDK4 selective inhibitor as a single agent for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl]} [Methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of CDK4 selective inhibitor, wherein these amounts together achieve a synergistic effect in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a CDK4 selective inhibitor for overcoming clinical resistance to endocrine therapy for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer, wherein the combination is synergistic.In embodiments, the methods or uses of the present invention relate to a synergistic combination of a targeted therapeutic agent, particularly 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, and a CDK4 selective inhibitor.

[0295] In the embodiments, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole- 1-(1-yl)methyl]-1,2-benzoxazol-3-yl)benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof or a combination thereof with a certain amount of a CDK4 / 6 inhibitor, said amount being effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a specific amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole-1...} as a single agent...} [-(methyl)-1,2-benzoxazol-3-yl)benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof or a combination thereof with a certain amount of a CDK4 / 6 inhibitor, wherein these amounts together are effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof or a combination thereof with a CDK4 / 6 inhibitor as a single agent for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl]} [Methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of CDK4 / 6 inhibitor, wherein these amounts together achieve a synergistic effect in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a CDK4 / 6 inhibitor for overcoming clinical resistance to endocrine therapy for the treatment of cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer, wherein the combination is synergistic.In embodiments, the methods or uses of the present invention relate to a targeted therapeutic agent, particularly a pharmaceutically acceptable salt thereof, and a synergistic combination of a CDK4 / 6 inhibitor.

[0296] In the embodiments, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a single agent a dose of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazole-1- [[methyl]-1,2-benzoxazole-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, or a combination thereof with a certain amount of palbociclib or a pharmaceutically acceptable salt thereof, said amount being effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In yet another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a specific amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl] as a single agent [Methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, or a combination thereof with a certain amount of palbociclib or a pharmaceutically acceptable salt thereof, wherein these amounts together are effective in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, or a combination thereof with palbociclib or a pharmaceutically acceptable salt thereof, as a single agent, for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a measured amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl 1,2-Benzoxazol-3-ylbenzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a certain amount of palbociclib or a pharmaceutically acceptable salt thereof, wherein these amounts together achieve a synergistic effect in overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer. In another embodiment, the present invention relates to a combination of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and palbociclib or a pharmaceutically acceptable salt thereof, for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer, wherein the combination is synergistic.In embodiments, the methods or uses of the present invention involve a targeted therapeutic agent, particularly a pharmaceutically acceptable salt of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, and a synergistic combination of palbociclib or a pharmaceutically acceptable salt thereof. In embodiments, the amount of palbociclib or a pharmaceutically acceptable salt thereof in the methods and uses of the present invention further comprises administration of an anti-estrogen, such as fulvestrant or letrozole. In embodiments, the amount of palbociclib or a pharmaceutically acceptable salt thereof in the methods and uses of the present invention further comprises administration of a certain amount of letrozole. In embodiments, the amount of palbociclib or a pharmaceutically acceptable salt thereof in the methods and uses of the present invention further comprises administration of a certain amount of fulvestrant.

[0297] In the embodiments, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof or thereof, as a single agent, or a combination thereof with a certain amount of... An amount of 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof, said amount being effective in overcoming clinical resistance to endocrine therapy used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a certain amount of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, or thereof, as a single agent, or a combination thereof with a certain amount of Combinations of 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl or pharmaceutically acceptable salts thereof, wherein these amounts together are effective in overcoming clinical resistance to endocrine therapy used to treat cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the invention relates to 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzyl-1-sulfonamide or a pharmaceutically acceptable salt thereof, or a combination thereof with 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or the thereof as a single agent. Pharmaceutically acceptable combinations of salts used to overcome clinical resistance to endocrine therapy for the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer.In another embodiment, the present invention relates to a method for overcoming clinical resistance to endocrine therapy, wherein the endocrine therapy is used to treat cancer, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, the method comprising administering to a patient in need a dose of 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof and a dose of 1,5- Dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof, wherein these amounts together achieve a synergistic effect in overcoming clinical resistance to endocrine therapy used to treat cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer. In another embodiment, the present invention relates to 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzyl-1-sulfonamide or a pharmaceutically acceptable salt thereof and 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof. A combination of drugs used to overcome clinical resistance to endocrine therapy for the treatment of cancers, particularly breast cancer, HR+ breast cancer, PR+ breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer, or castration-resistant prostate cancer, wherein the combination is synergistic.In embodiments, the methods or uses of the present invention relate to a synergistic combination of a targeted therapeutic agent, particularly 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzyl-1-sulfonamide or a pharmaceutically acceptable salt thereof, and 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof.

[0298] Those skilled in the art, taking into account known methods and factors such as the following, should be able to determine the appropriate amount, dose (or administration) of each compound used in the methods and combinations of the present invention to a patient: age, weight, general health condition, one or more compounds administered, route of administration, the nature and progression of the cancer to be treated, particularly breast cancer, ER+ breast cancer, ER+HER2- breast cancer, ER+HER2+ breast cancer, locally advanced or metastatic ER+ breast cancer, locally advanced or metastatic ER+HER2- breast cancer, locally advanced or metastatic ER+HER2+ breast cancer, non-small cell lung cancer, prostate cancer or castration-resistant prostate cancer, and the presence of other agents.

[0299] In the implementation plan, palbociclib or a pharmaceutically acceptable salt thereof is administered at the following daily doses: about 125 mg once daily, about 100 mg once daily, about 75 mg once daily, or about 50 mg once daily. In the implementation plan, palbociclib or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 125 mg once daily, which is the recommended starting dose. For example, palbociclib or a pharmaceutically acceptable salt thereof is administered at the following doses: about 100 mg once daily, about 75 mg once daily, or about 50 mg once daily. In the implementation plan, palbociclib or a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg once daily. In the implementation plan, palbociclib or a pharmaceutically acceptable salt thereof is administered at a dose of about 75 mg once daily. In the implementation plan, palbociclib or a pharmaceutically acceptable salt thereof is administered at a dose of about 50 mg once daily. The dosages provided herein refer to the dose of palbociclib in its free base form, or are calculated as the free base equivalent of the administered palbociclib salt. For example, the dosage or amount of palbociclib (such as 100 mg, 75 mg or 50 mg) refers to the free base equivalent.

[0300] In some embodiments, 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1H-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanepentyl alcohol or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1 mg to about 1000 mg. In some embodiments, the CDK4 inhibitor is administered at a daily dose of about 10 mg to about 500 mg. In some embodiments, the CDK4 inhibitor is administered at a daily dose of about 25 mg to about 300 mg. In some implementations, CDK4 inhibitors are administered in doses approximately as follows, according to QD, BID, TID, or QID regimens: 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 1 ... 0mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 260mg, 270mg, 275mg, 280mg, 290mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, or 500mg.

[0301] In the embodiments, 2,6-dimethoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, or 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof, is administered in doses that may be administered in the following ranges: about 1 mg to about 1 gram; about 1 mg to about 250 mg; about 1 mg to about 100 mg; about 1 mg to about 50 mg; about 1 mg to about 25 mg; and about 1 mg to about 10 mg.

[0302] The method of the present invention can be practiced through various dosing or administration regimens. The combined compounds of the present invention can be administered intermittently, simultaneously, or sequentially. In embodiments, the combined compounds of the present invention can be administered using a simultaneous dosing regimen.

[0303] Repeated dosing or administration regimens can be performed as needed to achieve the desired reduction or shrinkage of cancer cells. As used herein, a “continuous dosing regimen” is a dosing or administration regimen without dose interruption, such as treatment without rest days. Repeating a 28-day treatment cycle without dose interruption between treatment cycles is an example of a continuous dosing regimen. In embodiments, the compounds of the combination of the present invention can be administered in a continuous dosing regimen. In embodiments, the compounds of the combination of the present invention can be administered simultaneously in a continuous dosing regimen.

[0304] In one embodiment, 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof is administered once daily to constitute a complete 28-day cycle. This 28-day cycle is repeated continuously during combination therapy using the invention.

[0305] The standard recommended dosing regimen for palbociclib or its pharmaceutically acceptable salts (including the standard dosing regimen) is once daily for 21 consecutive days, followed by 7 days of no treatment to constitute a complete 28-day cycle. This 28-day cycle is repeated continuously during combination therapy using the present invention.

[0306] The standard clinical dosing regimen for palbociclib or its pharmaceutically acceptable salts is 125 mg once daily for 21 consecutive days, followed by 7 days of no treatment to constitute a complete 28-day cycle. This 28-day cycle is repeated continuously during combination therapy using the present invention.

[0307] In other embodiments of the invention, 2-methoxy-N-{4-methoxy-6-[(1H-pyrazol-1-yl)methyl]-1,2-benzoxazol-3-yl}benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof is administered in combination with palbociclib and letrozole, wherein palbociclib is administered orally at 125 mg once daily for 21 days, followed by a 7-day break, and wherein letrozole is administered orally at 2.5 mg daily.

[0308] Administration of the compounds in the combination of the present invention can be achieved by any method that enables delivery of the compounds to the site of action. These methods include oral administration, intraduodenal administration, parenteral administration (including intravenous, subcutaneous, intramuscular, intravascular, or infusion), local administration, and rectal administration. Each compound in the combination can be administered via the same or different routes of administration.

[0309] The compounds of the methods or combinations of the present invention can be formulated prior to administration. The formulation will preferably be adapted to a specific mode of administration. These compounds can be formulated with pharmaceutically acceptable carriers known in the art and administered in a variety of dosage forms as known in the art. In preparing the pharmaceutical compositions of the present invention, the active ingredient is typically mixed with, diluted by, or encapsulated in a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, solid diluents or fillers, excipients, sterile aqueous media, and various non-toxic organic solvents. Dosage units or pharmaceutical compositions include tablets, capsules (such as gelatin capsules), pills, powders, granules, aqueous and non-aqueous oral solutions and suspensions, lozenges, troche tablets, hard candies, sprays, creams, salves, suppositories, jelly, gel, paste, lotion, ointment, injectable solutions, elixirs, syrups, and parenteral solutions packaged in containers suitable for dispensing into individual doses.

[0310] Parenteral formulations include pharmaceutically acceptable aqueous or non-aqueous solutions, dispersants, suspensions, emulsions, and sterile powders used to prepare them. Examples of carriers include water, ethanol, polyols (propylene glycol, polyethylene glycol), vegetable oils, and injectable organic esters (such as ethyl oleate). Flowability can be maintained by using coatings (such as lecithin), surfactants, or maintaining an appropriate particle size. Exemplary parenteral formulations include solutions or suspensions of the compounds of the present invention in sterile aqueous solutions (e.g., propylene glycol or dextrose aqueous solutions). These dosage forms may be suitably buffered if desired.

[0311] Additionally, lubricants (such as magnesium stearate, sodium lauryl sulfate, and talc) are commonly used for tableting purposes. Similar types of solid compositions can also be used in soft and hard-filled gelatin capsules. Preferred materials for use include lactose (or milk sugar) and high molecular weight polyethylene glycol. When oral administration of aqueous suspensions or elixirs is desired, the active compounds therein can be combined with various sweeteners or flavoring agents, coloring substances or dyes, and (if desired) emulsifiers or suspending agents, as well as diluents (such as water, ethanol, propylene glycol, glycerin, or combinations thereof).

[0312] Those skilled in the art will know or understand methods for preparing various pharmaceutical compositions using specific amounts of the active compound. For example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th edition (1975).

[0313] This invention also relates to a kit containing a therapeutic agent comprising the combination of the present invention and written instructions regarding the administration of the therapeutic agent. In one embodiment, the written instructions detail and define the administration pattern of the therapeutic agent, such as simultaneous or sequential administration of the therapeutic agents of the present invention. In another embodiment, the written instructions detail and define the administration pattern of the therapeutic agent, such as by specifying the administration day of each of the therapeutic agents during a 28-day cycle.

Example

[0314] Example 1: Palbociclib reversibly arrests ER+ breast cancer cells

[0315] Overview:

[0316] Complete senescence is the terminal state in which cells undergo irreversible cell cycle arrest and fail to divide. This provides an obstacle to tumor growth. In tissue culture, senescent cells arrest in the cell cycle and acquire specific hallmarks, including a large, flattened shape and high senescence-associated β-galactosidase activity (SA-β-gal). Palbociclib-treated ER+ breast cancer cell lines arrest in the G1 phase of the cell cycle and acquire these senescence characteristics.

[0317] Palbociclib treatment induces senescence signatures in ER+ breast cancer cells, but does not induce complete senescence in ER+ breast cancer cells.

[0318] Materials and methods:

[0319] T47D and MCF7 were confirmed as ER+ breast cancer cell lines using the short tandem repeat (STR) test (ATCC STR analysis service). The growth medium used for T47D cells was DMEM (Gibco catalog number 11995-065) + 10% FBS (Gibco catalog number 10082-147), and the growth medium used for MCF7 cells was RPMI (Gibco catalog number 11875-093) + 10% FBS (Gibco catalog number 10082-147).

[0320] T47D cells were seeded at 10% confluence into 6-well tissue culture plates (Falcon catalog number 353046). After 18–24 hours of cell attachment, 500 nM palbociclib was added to each culture. The growth medium and palbociclib were replaced twice weekly (every 3–4 days). This dose of palbociclib caused complete cell cycle arrest in T47D cells. For T47D cultures, cells were resuspended from triplicate cultures by trypsinization on days 1, 14, and 24 of palbociclib treatment, and cell density in each suspension was measured using a Vi-CellXR cell counter. In separate experiments, three additional T47D cultures were treated with 500 nM palbociclib for 14 days, followed by washing off the palbociclib and allowing the cells to recover for 10 days. After 10 days of recovery, cells were resuspended by trypsinization, and cell density in each suspension was measured using a cell counter. T47D cultures treated with 500 nM palbociclib and continuously treated for up to 24 days, followed by T47D cultures allowed to recover for 10 days after palbociclib removal, showed significant cell proliferation. Figure 1A ).

[0321] In similar experiments, MCF7 cells were seeded at 10% confluence into 6-well tissue culture plates and allowed to attach for 18–24 hours, followed by treatment with 500 nM palbociclib for 6 or 14 days. This dose of palbociclib caused complete cell cycle arrest in MCF7 cells. After 6 and 14 days of palbociclib treatment, cells in each well were resuspended by trypsinization, and cell density was measured using a cell counter. For additional MCF7 cultures treated in parallel with 500 nM palbociclib for 6 or 14 days, palbociclib was washed off the cells, and the cells were allowed to recover for 7–8 days. After this recovery period, cells were resuspended by trypsinization, and cell density in each suspension was measured. Similar to T47D cells, palbociclib treatment stably arrested MCF7 cells for up to 14 days. Figure 1B However, after removing palbociclib from MCF7 cultures treated for 6 or 14 days, cells readily re-enter the cell cycle and proliferate.

[0322] result:

[0323] The growth arrest of T47D induced by 500 nM palbociclib treatment was reversible upon palbociclib discontinuation and therefore did not induce complete senescence. Similarly, the growth arrest of MCF7 induced by 500 nM palbociclib treatment was reversible upon palbociclib discontinuation and therefore did not induce complete senescence.

[0324] Although 500 nM palbociclib induces senescence signatures in ER+ breast cancer cells, palbociclib treatment does not induce complete senescence because growth arrest is reversible after palbociclib removal.

[0325] Example 2: Identification of KAT6A as an epigenetic enzyme required for reversible proliferative arrest of ER+ breast cancer cells by palbociclib

[0326] Overview

[0327] Based on the results of Example 1, an analysis was developed for pooled RNAi screening to identify genes required for ER+ breast cancer cells to resume cell division from palbociclib-induced growth arrest. This analysis measures whether knockdown of a specific protein blocks the ability of cells to recover from palbociclib-induced growth arrest.

[0328] The pooled RNAi screening identified KAT6A as an epigenetic enzyme required for the recovery and survival of ER+ breast cancer cells from palbociclib-induced proliferation arrest after palbociclib treatment.

[0329] Materials and Methods

[0330] miR30 shRNA design (Jose M Silva et al., Second-generation shRNA libraries covering the mouse and human genomes. 2005. Nature Genetics 37(11):1281-1288) is an shRNA platform for screening and downstream genetic studies. A summary of the pooled RNAi screening implemented to identify genes required for reversible proliferative arrest (e.g., survival) in ER+ breast cancer cells during palbociclib treatment is shown in [the table / document / etc.]. Figure 2 middle.

[0331] T47D cells were infected with a lentivirus encoding a collection of 4106 different doxycycline-inducible ("Tet-ON") shRNAs targeting 418 different epigenetic enzymes. These shRNAs were selected from a library of shRNAs targeting different epigenetic enzymes. Infection, selection, and expansion of T47D cells infected with viruses encoding different shRNAs were performed in doxycycline-free growth medium, thus ensuring that no different shRNAs were expressed in these cells and that no epigenetic enzymes were knocked down.

[0332] After expanding sufficient cells for selection, cells were seeded at 10-20% confluence in T150 tissue culture flasks in growth medium containing 2 μg / mL puromycin (to maintain selection for cells containing shRNA) and 0.2 μg / mL doxycycline (Sigma catalog number D9891). The addition of doxycycline to the growth medium induced the expression of Tet-on regulated shRNAs. Following shRNA expression induction, the shRNAs were processed by cell treatment to target the knockdown of 418 different epigenetic enzymes. Throughout the selection process, at least 1000 cells per shRNA per T47D culture (4,106,000 cells) were maintained to ensure sufficient representativeness of each shRNA in the T47D culture. shRNA expression was induced for 3 days to knock down different epigenetic enzymes, after which cells were seeded in T150 flasks for selection analysis.

[0333] In parallel, T47D cultures were maintained in doxycycline-free medium that did not induce shRNA expression. These cultures served as control cultures for comparison with shRNA-induced cultures. After T47D-shRNA cells were expanded in growth medium with + / - 0.2 μg / mL doxycycline for 3 days, the cells were suspended and seeded for selection. Under any of four different growth conditions, at least 1000 cells per shRNA were seeded into triplicate cultures. These growth conditions included (1) growth medium without palbociclib and doxycycline (proliferation selection - shRNA off), (2) growth medium without palbociclib + 0.2 μg / mL doxycycline (proliferation selection - shRNA on), (3) growth medium + 500 nM palbociclib, without doxycycline (palbociclib recovery selection - shRNA off), and (4) growth medium + 500 nM palbociclib + 0.2 μg / mL doxycycline (palbociclib recovery selection - shRNA on). The growth medium used for each of these four growth conditions was changed every 3–4 days (twice a week). When the proliferation selection culture was close to confluence, it was splittered to maintain a sub-convergence culture, and at least 1000 cells per shRNA were seeded into a new culture in growth medium + / - 0.2 μg / mL doxycycline. Since the palbociclib-treated cultures experienced growth arrest, there was no need to split these cultures, as they remained subconvergent during palbociclib treatment. After 14 days of treatment, cells were collected from each of three replicates of proliferation selection cultures (+doxycycline, shRNA on and without doxycycline, shRNA off) and stored for downstream analysis. Palbociclib and doxycycline were washed out from the palbociclib recovery selection cultures, and these cultures were allowed to recover for 14 days in the absence of palbociclib and epigenetic knockdown. It was necessary to restore epigenetic expression during this recovery period of selection to minimize the depletion of cells with shRNAs containing genes essential for cell proliferation. Therefore, cells in the recovery period that depleted shRNAs included those with knocked-down genes, which, in combination with palbociclib treatment, blocked recovery from proliferation arrest. After palbociclib removal, these cultures began to expand, and when they were nearing confluence, the cells were suspended and split into new T150 tissue culture flasks, maintaining at least 1000 cells per shRNA. After 14 days of recovery, cells from each of the triplicate palbociclib recovery selection cultures (recovered from +palbociclib shRNA on and +palbociclib shRNA off conditions) were collected and stored for downstream analysis.

[0334] Cells were lysed in triplicate (12 samples) for each of four different growth conditions used for self-proliferation and palbociclib recovery screening, and genomic DNA was purified using the Qiagen DNeasy Genomic DNA Kit (catalog number 69504) and the manufacturer's protocol. shRNA from the genomic DNA samples was amplified using nested PCR and indexed with Illumina for downstream MiSeq analysis (Illumina).

[0335] After completing MiSeq, the generated FASTQ files were processed to obtain the following gene-level scores. The shRNA reads from each growth condition and repeated measurement were summed, and shRNAs with fewer than 10 reads in any shRNA-blocking sample (n = 45 / 4, 106) were excluded. The scores were then evaluated using MAGeCK-MLE (Li W, J, Xu H, Chen CH, Xiao T, Liu JS, Brown M, Liu XS. Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. Genome Biol. 2015 Dec 16; 16:281.) Guided abundance changes (enrichment and depletion) at the gene level were assessed using 8 threads and 10 permutation rounds, comparing shRNA-on (+doxycycline) samples with shRNA-off (doxycycline-free) samples for each proliferation and palbociclib recovery screening. Gene level β values ​​(effect size) for the recovery and proliferation groups of shRNA screening were plotted (data not shown). Negative gene level β values ​​indicate that cells with the specified gene knocked down (shRNA on) were depleted in self-culture under specific growth conditions compared to cells without the gene knocked down (shRNA off). The gene level β score of KAT6A in the proliferation selection was -0.86, indicating that KAT6A knockdown inhibited the proliferation of cells in the selection and that these cells were depleted in the proliferation selection culture.

[0336] result:

[0337] The gene-level β score for KAT6A in palbociclib recovery screening was -1.11, indicating that KAT6A knockdown inhibited cell recovery from palbociclib-induced proliferation arrest, and that these cells were depleted in the palbociclib recovery screening culture. Importantly, among all the selected genes, KAT6A had the most negative gene-level β score in palbociclib recovery screening, suggesting that KAT6A knockdown caused the strongest inhibition of T47D cell recovery from palbociclib-induced proliferation arrest compared to any of the other selected genes.

[0338] In the screening for proliferation and palbociclib recovery RNAi, several epigenetic enzymes required for: 1) proliferation of breast cancer cell lines; 2) recovery from palbociclib arrest; and 3) both proliferation and recovery from palbociclib arrest in breast cancer cell lines were identified. KAT6A was identified as an epigenetic enzyme required for both breast cancer cell proliferation and recovery from palbociclib arrest. KAT6A knockdown was the strongest hit in the palbociclib recovery screening of the ER+ breast cancer cell line T47D. This confirms that KAT6A is not only important for the proliferation of T47D cells, but also required for the survival and resumption of cell division of T47D cells after palbociclib removal. These results indicate that KAT6A depletion during palbociclib treatment leads to irreversible growth arrest in breast cancer cells.

[0339] Example 3: Validation of KAT6A as an epigenetic enzyme required for reversible proliferative arrest of ER+ breast cancer cells via palbociclib

[0340] Overview

[0341] Based on the results of Example 2, it was verified that KAT6A is an epigenetic enzyme required for the proliferation of ER+ breast cancer cells and their recovery from palbociclib arrest.

[0342] Materials and Methods

[0343] Based on the differences in KAT6A expression, ER+ breast cancer cell lines T47D, ZR-75-1, MCF7, and CAMA1 were selected for testing. The KAT6A gene was amplified and overexpressed in ZR-75-1 and CAMA1 cells. KAT6A was overexpressed in T47D cells. The KAT6A gene was neither amplified nor overexpressed in MCF7 cells. The growth medium used for T47D and MCF7 was as described in Example 1. The growth medium used for ZR-75-1 and CAMA1 was the same as that used for MCF7. Tetracycline-free FBS (Takara catalog number 631106) was used in the growth medium to integrate the Tet-opening shRNA into these cell lines. As described below, Western blot analysis confirmed that these shRNAs knocked down ≥75% of KAT6A protein in T47D, ZR-75-1, and MCF7 cells, and Q-PCR confirmed that these shRNAs knocked down >80% of KAT6A mRNA in CAMA1 cells.

[0344] Western blot analysis of whole-cell extracts was performed on ER+ breast cancer cell lines T47D, ZR-75-1, MCF7, and CAMA1.

[0345] Western blot analysis of KAT6A was performed on whole-cell lysates. Q-PCR was used to measure KAT6A expression in CAMA1 cells with Tet-activated KAT6A shRNA.

[0346] use Figure 3A The workflow described herein was used to test the effects of KAT6A knockdown on cell proliferation and recovery from palbociclib arrest in T47D, ZR-75-1, and MCF7 cells. KAT6A knockdown was induced in these cell lines by adding 0.2 μg / mL doxycycline to the growth medium, thereby triggering different shRNAs in these cell lines to knock down KAT6A. After 3 days of shRNA induction, cells were seeded into 6-well plates to achieve approximately 10% confluence. All cultures were seeded into growth medium + 0.2 μg / mL doxycycline to maintain shRNA expression and KAT6A knockdown. Cells were seeded into 6-well plates for colony formation analysis (“CFA”). After 18–24 hours of cell attachment to the wells, 500 nM palbociclib was added to half of the cell culture.

[0347] Proliferation analysis: After 10–14 days, untreated cultures expressing the non-targeted negative control shRNA (shRenilla) were confluent, and KAT6A RNAi and shRenilla RNAi cultures were stained to visualize cell density. To stain the cultures, growth medium was aspirated from each cell culture and discarded. Cells attached to the wells were washed once with 2 mL of phosphate-buffered saline (Hyclone, catalog number SH30256.01), followed by staining each well with 1 mL of 0.4% sulforhodamine B sodium solution dissolved in 1% acetic acid (SRB staining agent, Sigma, catalog number S1402). The cultures were incubated in the staining agent at room temperature for 10 minutes. The staining agent was then aspirated from each culture and discarded. Excess staining agent was washed away from each well three times with 2 mL of 1% acetic acid.

[0348] Palbociclib Recovery Analysis: For cell cultures incubated in growth medium + 0.2 μg / mL doxycycline + 500 nM palbociclib, after 14 days of palbociclib treatment, each cell culture was washed from the palbociclib-containing growth medium and then added back to growth medium free of doxycycline and palbociclib to allow KAT6A re-expression and recovery from palbociclib-induced cell cycle arrest. After 10–14 days of recovery from palbociclib arrest, the cultures were stained with SRB staining agent as described above.

[0349] result

[0350] The results of proliferation and colony formation analysis confirmed that KAT6A is required for the proliferation of T47D and ZR-75-1 cells, but not for MCF7 cells. Figure 3B and Figure 4A Weak SRB staining of T47D and ZR-75-1 cultures induced to express different KAT6A shRNAs and non-targeted shRenilla shRNAs indicated poor cell proliferation at the timeline of the proliferation assay. In contrast, knockdown of KAT6A by more than 90% of the most potent KAT6A_6 shRNA in MCF7 cells did not significantly inhibit the proliferation of this cell line, as reflected by equivalent staining of KAT6A_6 MCF7 cultures compared to shRenilla negative control cultures. The KAT6A dependence of T47D cell proliferation is consistent with the RNAi screening described above in Example 2.

[0351] Analysis of palbociclib-induced colony formation recovery confirmed that KAT6A expression during palbociclib treatment was essential for the recovery of cell division in T47D, ZR-75-1, and MCF7 cells after palbociclib withdrawal in their own cells. Figure 3B and Figure 4B For each of these cell lines, the SRB staining of cultures recovered from the KAT6A knockdown + palbociclib combination was much weaker compared to the non-targeted control shRNA + palbociclib combination. These results validate the dropout of KAT6A shRNA in T47D observed in palbociclib recovery screening and confirm the broad dependence of ER+ breast cancer cells on KAT6A to recover from palbociclib growth arrest.

[0352] Unlike the results above, the ability of CAMA1 cells to recover their dependence on KAT6A from palbociclib growth arrest could not be measured. Analysis of the CAMA1 cell line's response to palbociclib confirmed that, unlike most ER+ breast cancer cells, single-agent palbociclib treatment led to irreversible proliferative arrest in CAMA1 cells.

[0353] Compared to non-targeting CB3 shRNA, SRB staining results of CAMA1 cultures induced to express two different potent KAT6A shRNAs (KAT6A_5 and KAT6A_6) did not indicate a significant difference in culture growth. Figure 5A However, rigorous examination of the cells using an optical microscope revealed that KAT6A knockdown in CAMA1 cells resulted in significant changes in cell morphology, including enlarged and flattened cell morphology indicative of cellular senescence. Figure 6These large cells covered the surface of the tissue culture plate and ingested SRB staining agent, indicating equivalent staining, even though there were far fewer cells in these cultures compared to the negative control cultures without KAT6A knockdown. With this in mind, as a separate analysis to assess the effect of KAT6A knockdown on cell proliferation, the number of cells in KAT6A knockdown-affected CAMA1 cultures was measured. Indeed, when cell numbers were measured in CAMA1 cultures expanded for 14 days by knocking down KAT6A via KAT6A_5, KAT6A_6, or KAT6A_10_1u shRNA, there were consistently and significantly fewer CAMA1 cells compared to cells expressing the non-targeted negative control shCB3 shRNA. Figure 5B These results are consistent with those of T47D and ZR-75-1, and confirm that CAMA1 cells depend on KAT6A for proliferation. Furthermore, visual observation of CAMA1 cells with KAT6A knockdown indicates that KAT6A depletion in this cell line is inducing senescence.

[0354] Example 4: KAT6A RNAi knockdown confirms palbociclib combo activity against ER+ breast cancer cell lines

[0355] Overview

[0356] This example demonstrates that the combination of KAT6A inhibition and palbociclib is broadly effective against ER+ breast cancer cell lines.

[0357] Materials and methods:

[0358] As described in Example 1, a group of luminal ER+ breast cancer cell models with KAT6A expression levels ranging from low to high were evaluated in proliferation and palbociclib recovery analyses (Table 1).

[0359] Table 1

[0360]

[0361]

[0362] Step 1: Generation of stable cell lines expressing various non-targeting control shRNAs or shRNAs targeting KAT6A.

[0363] EFM19 and EFM192A cells were obtained from the German Collection of Microorganisms and Cell Cultures at the Leibniz Institute (DSMZ). MDAMB175VII cells were obtained from the American Type Culture Collection (ATCC).

[0364] The generation and characterization of stable cell lines derived from T47D, ZR75-1, CAMA1, and MCF7 are described in Example 3.

[0365] Stable cell lines derived from EFM19, EFM192A, and MDAMB175 VII were generated by infecting each cell line with lentiviruses carrying puromycin resistance genes and Tet-shutdown non-target control shRNAs (shRenilla or shCB3) or KAT6A shRNAs (shRNA5 and shRNA6), sequences described in Example 3. For the Tet-shutdown shRNA system, the addition of doxycycline to the culture medium silences shRNA expression, while the removal of doxycycline from the culture medium induces shRNA expression. Cells were infected with lentiviruses at a low MOI, followed by puromycin selection of infected cells with single-copy integration of Tet-shutdown shRNA. The entire process was performed in the presence of 0.2 μg / mL doxycycline to silence shRNA expression during cell line generation.

[0366] On day 1, cells from each cell line were plated in 6-well tissue culture plates in DMEM or RPMI tissue medium supplemented with 10% tetracycline-free fetal bovine serum (Takara, catalog number 631367) and penicillin / streptomycin (Gibco catalog number 15140122). On the afternoon of day 2, the medium was removed, and 700 μL of serum-free DMEM (Gibco catalog number 11995-065) + 0.2 μg / mL doxycycline was added back to each well, followed by 2–20 μL of lentivirus carrying each shRNA. After a brief mixing, the cells were returned to the plate and incubated overnight at 37°C and 5% CO2. On the morning of day 3, 2 mL of fresh tissue medium + 10% FBS (tetracycline-free) + penicillin / streptomycin + 0.2 μg / mL doxycycline was added to each well, and the plate was returned to the plate and incubated at 37°C and 5% CO2 for 48 hours. On day 4, remove the culture medium from each well and add fresh medium + 10% FBS (tetracycline-free) + P / S + 0.2 μg / mL doxycycline + 2 μg / mL puromycin (InvivoGen, catalog number ant-pr-1). In the following days, change the medium every 2–3 days to maintain puromycin selection and allow cell expansion.

[0367] Step 2: Settlement Formation Analysis

[0368] As described in Example 3, colony formation analysis was performed in two groups (proliferation group and palbociclib recovery group). Cell lines carrying shRNA were cultured at low densities (2-5 × 10⁻⁶ cells / year) in both the presence and absence of doxycycline. 4 Cells (1 / well) were seeded into 6-well culture dishes and allowed to adhere overnight. The following day, the plates were refreshed with medium containing the carrier or palbociclib (100-500 nM) (with or without doxycycline shRNA induction). Carrier-treated cells were refreshed every three or four days and monitored until the non-targeted control cells confluent. For the palbociclib recovery group, palbociclib-treated cells were treated for 14 days, with the medium refreshed every three to four days. At the end of treatment, the cells were washed three times with complete medium supplemented with 0.2 μg / mL doxycycline to simultaneously shut down shRNA expression and allow the cells to recover from palbociclib-induced cell cycle arrest. When the non-targeted control cells confluent, the cells in each well were fixed and stained with sulforhodamine B (SRB) for visualization, as described in Example 3. For quantitative staining, the stained cells were dissolved in 2 mL of 10 mM Tris-HCl (pH 7.5) and shaken on a shaker for 10 min. The sample was then diluted with 10 mM Tris-HCl (pH 7.5), transferred to a 96-well microtiter plate (100 μL), and read at 565 nm on a SpetraMax.

[0369] result:

[0370] KAT6A depletion affected the proliferation of multiple ER+ breast cancer cell models, as summarized in Table 1. Furthermore, in all cell lines tested, KAT6A knockdown was consistent with synergistic inhibition of palbociclib, as confirmed by the palbociclib recovery group via colony formation analysis.

[0371] KAT6A knockdown had the least effect on MCF7 cell proliferation. Figure 4A However, when combined with palbociclib, KAT6A depletion significantly blocked the recovery of cells from palbociclib-induced cell cycle arrest. Figure 4B ).

[0372] These results confirm that KAT6A inhibition works synergistically with palbociclib to block arrested cancer cells from re-entering the cell cycle.

[0373] EFM192A is an ER+HER2+ mammary cell line with moderate KAT6A expression levels. KAT6A knockdown showed a moderate effect on proliferation, and, when combined with 100 nM palbociclib, a substantial synthetic lethal effect in blocking cell recovery from palbociclib-induced arrest. Figure 7A ). In mRNA ( Figure 7B ) and protein ( Figure 7C ) Horizontal verification of the effectiveness of shKAT6A_5 and shKAT6A_6 in knocking down KAT6A.

[0374] In these cell models, the inhibitory effect on proliferation and palbociclib recovery was independent of KAT6A expression levels, demonstrating broad palbociclib / KAT6A inhibitor combination activity against ER+ luminal breast cancer in both KAT6A-high and KAT6A-low models.

[0375] Example 5: Small molecule KAT6 inhibitors inhibit ER+ breast cancer cell proliferation and recovery from palbociclib arrest

[0376] To evaluate small molecule KAT6 inhibitors in order to determine the proliferation of ER+ breast cancer cells and their dependence on the recovery of KAT6A catalytic function from palbociclib stasis.

[0377] Materials and methods:

[0378] Small molecule KAT6 inhibitor compounds A and B were evaluated for ER+ breast cancer cell lines T47D, ZR-75-1, and MCF7. The growth medium used for the cell lines was the same as described in Examples 1 and 3 above.

[0379] T47D, ZR-75-1, or MCF7 cells were seeded at a confluence of 10% (100,000-150,000 cells per well) into 6-well tissue culture plates. After 18-24 hours of cell attachment, the growth medium was aspirated and discarded, and fresh growth medium containing 0-20 μM of compound A or compound B was added to the cells in two-fold increments (proliferation assay), decreasing from 20 μM. In parallel, seed cultures with the same titration of KAT6 inhibitor were added to the cells. For each concentration of KAT6 inhibitor, 500 nM palbociclib was also added (palbociclib recovery assay). The cultures were incubated for 12-14 days and treated with these inhibitors / inhibitor combinations. The growth medium containing the inhibitors was replaced on the cell cultures every 3-4 days (twice a week).

[0380] For T47D cultures treated with the KAT6 inhibitor (proliferation analysis), after 12 days, the carrier (DMSO)-treated cultures showed >90% confluence, and the cell cultures were stained with SRB as described in Example 3. The stained 6-well plates were scanned using an Epson Perfection V600 photogrammeter and Epson Scan version 3.9.2.0 software. To quantify SRB staining in each 6-well culture, the cultures were destained with 2 mL of 10 mM Tris at pH 7.5 per well, and the absorbance (OD565) of the released staining agent at 565 nm was measured using a SpectraMax Plus plate reader and SoftMaxPro 5.4.3 software. The inhibition of T47D cell proliferation by compounds A and B was comparable to the 50% inhibition observed with 10 μM KAT6 inhibitor. Figure 8A and Figure 8C ).

[0381] For T47D cultures treated with 500 nM palbociclib stasis and then with an increased concentration of KAT6 inhibitor (palbociclib recovery assay), after 14 days of combined treatment, the growth medium containing the inhibitor was aspirated from the cultures and discarded. Each culture was washed three times with 2 mL of inhibitor-free growth medium. The cultures were then allowed to recover in inhibitor-free growth medium. After 14 days of recovery, cultures recovering from 500 nM palbociclib stasis were >90% confluent, and all palbociclib-recovered cultures were subjected to SRB staining, scanning, and destaining / measurement as described above. Palbociclib in combination with compound A or compound B inhibited T47D cell recovery, with inhibition of 50% when using 5 μM and 2.5 μM KAT6 inhibitors, respectively. Figure 8B and Figure 8D Compounds A and B, both KAT6 inhibitors, exhibit comparable activity against T47D cell proliferation and recovery from palbociclib arrest.

[0382] For ZR-75-1 and MCF7 experiments, only compound A was tested. For ZR-75-1 cultures treated with a single agent, the carrier-treated cultures showed >90% confluence after 12 days of treatment. As described above, these cultures were subjected to SRB staining, scanning, and destaining / measurement. A 50% inhibition of proliferation was observed with 0.3 μM compound A. Figure 9 For ZR-75-1 cultures that had stalled after treatment with a combination of 500 nM palbociclib and an increased concentration of compound A, similar to T47D cells, after 14 days of combined inhibitor treatment, both inhibitors were aspirated from the cell cultures. Each cell culture was then washed three times with 2 mL of inhibitor-free growth medium, allowing time for recovery from inhibitor treatment in inhibitor-free medium. After 22 days of recovery, ZR-75-1 cultures treated with 500 nM palbociclib alone showed >90% confluence, and these palbociclib recovery analysis cultures were subjected to SRB staining, scanning, and destaining / measurement as described above. The combination of compound A and palbociclib treatment inhibited the recovery of ZR-75-1 cells, with 50% inhibition observed with 2.5 μM compound A. Figure 9 ).

[0383] MCF7 cells were tested using compound A. For single-agent-treated MCF7 cultures, the carrier-treated cultures showed >90% confluence after 10 days of treatment. As described above, these cultures were subjected to SRB staining, scanning, and destaining / measurement. Compared to T47D and ZR-75-1 cell lines, 50% inhibition of MCF7 proliferation was not achieved in this analysis using up to 20 μM of compound A. Figure 10 For MCF7 cultures that had stalled after treatment with a combination of 500 nM palbociclib and an increased concentration of compound A, similar to T47D cells, after 14 days of combined inhibitor treatment, both inhibitors were aspirated from the cell cultures. Each cell culture was then washed three times with 2 mL of inhibitor-free growth medium, allowing time for recovery from inhibitor treatment in inhibitor-free medium. After 10 days of recovery, MCF7 cultures treated with 500 nM palbociclib alone showed >90% confluence, and these palbociclib recovery analysis cultures were subjected to SRB staining, scanning, and destaining / measurement as described above. Compound A synergistically inhibited MCF7 cell recovery with palbociclib treatment, with 50% inhibition observed with 5 μM compound A. Figure 10 ).

[0384] result

[0385] Treatment of T47D, ZR-75-1, and MCF7ER+ breast cancer cell lines with a small molecule KAT6 inhibitor (compound A) yielded results consistent with those of KAT6A knockdown in these cell lines. Specifically, the combination of KAT6A inhibition and palbociclib strongly delayed the recovery of cell cycle re-entry and cell division after palbociclib removal and KAT6A restoration. Compound A inhibited the proliferation of T47D and ZR-75-1 cells but not MCF7 cells. The combination of compound A and 500 nM palbociclib inhibited / delayed the recovery of cell proliferation in all three breast cancer cell lines after the cessation of inhibitor treatment.

[0386] The catalytic function of KAT6A is important for its ability to drive the proliferation of ER+ breast cancer cell lines with KAT6A gene amplification / overexpression, and for its ability to enable cells to recover from palbociclib-induced proliferation arrest in ER+ breast cancer cell lines, regardless of the level of KAT6A gene amplification / expression.

[0387] The direct anti-cell proliferation activity of KAT6 inhibitors (compounds A and B) and their ability to resist the recovery of cell proliferation from palbociclib-induced arrest when combined with palbociclib are similar to the effects of KAT6A knockdown on breast cancer cell lines T47D, ZR-75-1, and MCF7.

[0388] These results confirm that KAT6 inhibitors synergistically inhibit the proliferation of ER+ breast cancer cells with palbociclib and overcome clinically relevant resistance to endocrine therapy.

[0389] Example 6: Treatment with a KAT6 inhibitor (compound A) that downregulated estrogen receptor α expression and estrogen receptor regulators

[0390] Overview:

[0391] To evaluate transcriptional changes induced in T47D cells by treatment with a small molecule KAT6 inhibitor (compound A) as a single agent and in combination with palbociclib, in order to determine the response of breast cancer cells and to compare with KAT6A knockdown.

[0392] Materials and Methods

[0393] As described in Example 3, parental T47D cells in which no shRNA was introduced were evaluated, or T47D cells in which Tet-activated regulated KAT6A shRNA (KAT6A_5 or KAT6A_6) or non-targeted shRNA (shRenilla) was stably integrated.

[0394] T47D cell lines containing Tet-activated KAT6A_5, KAT6A_6, or shRenilla shRNA were treated with 0.2 μg / mL doxycycline to induce expression of each shRNA in T47D cells and trigger KAT6A knockdown (KAT6A_5 and KAT6A_6 cells only). Cells were treated with doxycycline for 4 days, providing sufficient time for KAT6A protein depletion in the cells. On day 3 of doxycycline treatment, KAT6A_5, KAT6A_6, and shRenilla T47D cells were suspended and seeded at 10% confluence in 6-well tissue culture plates. Parental T47D cells (without shRNA) were also seeded at 10% confluence in 6-well tissue culture plates. On day 4 after shRNA induction, half of the T47D KAT6A_5, KAT6A_6, and shRenilla cultures were treated with 0.5 μM palbociclib, while the other half remained untreated. All cultures were maintained in growth medium containing 0.2 μg / mL doxycycline to maintain KAT6A knockdown in cells throughout the experiment. Parental T47D cells were divided into four groups: (1) carrier treatment (DMSO), (2) carrier + 0.5 μM palbociclib, (3) 10 μM compound A treatment, and (4) 10 μM compound A + 0.5 μM palbociclib. Parental T47D and T47D-shRNA cell lines were then treated for 6 days, with the growth medium changed on day 3 of treatment to contain inhibitors representing the different growth conditions. On day 6 of treatment, cells were collected for GeneChip analysis (Table 2).

[0395] Table 2

[0396]

[0397]

[0398] RNA was purified from each sample using the Qiagen RNeasy kit (Qiagen catalog number 74136) according to the manufacturer's instructions. cDNA synthesis was performed using the Ovation Pico WTA System (NuGEN) and Ribo-SPIA technology. The Ovation Pico WTA products were fragmented and biotin-labeled using the Encore Biotin Module (NuGEN). For each sample, 5 μg of biotin-labeled cDNA was hybridized to the human genome U133+2.0 oligonucleotide array (Affymetrix) using the buffers and conditions recommended by the manufacturer. GeneChips were then washed and stained with streptavidin R-phycoerythrin (Molecular Probes) using a GeneChip FluidicsStation 450, and scanned using an AffymetrixGeneChip Scanner 3000. Expression data were processed using the Microarray Suite 5.0 (MAS5) algorithm.

[0399] Differential gene expression analysis confirmed a high degree of overlap in the expression changes of the following cells: (1) KAT6A knockdown in T47D cells by either of two independent KAT6A shRNAs (KAT6A_5 vs. KAT6A_6; p = 5.4 × 10⁻⁶). -197 (2) KAT6A knockdown relative to T47D cells treated with compound A was achieved by KAT6A_5shRNA (p = 3.7 × 10⁻⁶). -176 (2) KAT6A knockdown relative to T47D cells treated with compound A by KAT6A_6shRNA (p = 1.98 × 10-122).

[0400] result:

[0401] The expression changes in T47D cells treated with a small-molecule KAT6 inhibitor were similar to those in T47D cells with KAT6A knockdown. Gene set enrichment analysis of differentially expressed genes in T47D cells treated with KAT6A knockdown or compound A confirmed significant enrichment of estradiol-responsive genes and stem cell signatures. Furthermore, both KAT6A knockdown and compound A treatment led to significant downregulation of ESR1 (encoding estrogen receptor α (ERα) expression and downstream ERα regulatory genes (such as CCND1)). These results demonstrate the consistent effect of KAT6A knockdown / catalytic inhibition on gene expression in the ER+ breast cancer cell line T47D and confirm the important function of KAT6A in regulating estrogen receptor expression and stem cell expression.

[0402] Example 7: Treatment with a KAT6 inhibitor (compound C) that downregulated estrogen receptor α expression and estrogen receptor regulators

[0403] Overview:

[0404] Expression profiling studies in T47D cells using the KAT6 inhibitor (compound C) and subsequent RNA-Seq confirmed that the KAT6 inhibitor maintained its function of downregulating ESR1 transcription in ER+ breast cancer cells.

[0405] Materials and methods:

[0406] T47D cells were seeded at 1 million cells per 10 cm culture dish and incubated overnight in RPMI-1640 medium containing 10% FBS (Takara, catalog number 631106) and 1× penicillin / streptomycin. The following day, cells were treated with either a 20 nM load control (DMSO) or compound C. The medium and compound were replaced after three days, with a total treatment period of 6 days. At the end of the 6-day treatment, cells were harvested by trypsinization and lysed in RLT buffer (Qiagen, catalog number / ID79216) + β-mercaptoethanol. The RNA lysates were submitted for RNA sequencing (“RNA-Seq”) (WuXi). ).

[0407] RNA-Seq data analysis: Using Trimmomatic (v0.36) for paired-end readings, free WuXi Adapter sequences were removed from the provided Illumina raw reads, and low-quality reads were filtered out. The remaining reads after the preprocessing steps were plotted to the reference gene body hg38 using STAR (v2.6.0c). Gene expression at the gene level was quantified as expected counts and transcripts per million (TPM) using RSEM (v1.3.0) with preset parameters' "rsem-compute-expression". Differential expression analysis of robustly expressed genes (maximum expression >= 10 across samples) was performed using the DESeq2 (v1.18.1) software package. Gene set enrichment analysis was performed using the GseaPreranked option (GSEA, v3.0). Genes were graded based on signed log10 pval. Gene sets (c2.all.v6.1) from the Molecular Signatures Database (MSigDB) were used in this analysis.

[0408] result:

[0409] Gene set enrichment analysis confirmed that estradiol-induced genes were downregulated in cells treated with compound C (NES: -9.92, FDR: <0.0001), while estradiol-suppressed genes were upregulated in cells treated with compound C (NES: 6.27, FDR: <0.0001). These results using compound C are consistent with gene set enrichment analysis of transcriptional profiling data using compound A as described in Example 6, further supporting the important role of KAT6A in regulating estrogen receptor-dependent response genes and the role of KAT6A catalytic function in regulating this process in ER+ breast cancer cells.

[0410] Example 8: KAT6A knockdown leads to depletion of estrogen receptor α in ER+ breast cancer cell lines

[0411] In Examples 6 and 7, KAT6A inhibition downregulated the expression of the ESR1 gene and estrogen receptor regulator in T47D cells, as confirmed by expression analysis in T47D cells. In this example, KAT6A depletion led to downregulation of ESR1 in other ER+ breast cancer cell lines.

[0412] Materials and methods:

[0413] All T47D, ZR75-1, and CAMA1 cell lines prepared as described in Example 3 with high levels of KAT6A gene expression were selected for testing. Specifically, T47D and ZR75-1 cells infected with Tet-shutdown non-targeted shRenilla, shKAT6A_5, or shKAT6A_6 (through expression induced by the absence of doxycycline) and CAMA1 cells infected with the Tet-on form of the same set of shRNAs (through expression induced by the presence of doxycycline) were selected for testing.

[0414] Cells were seeded at a density of 200 kJ / well in T-25 culture flasks in RPMI + 10% FBS (tetracycline-free) + penicillin / streptomycin (with and without doxycycline). The medium was changed every 3 days. After 9 days of culture, cells were harvested by trypsinization for protein and RNA. Total RNA was isolated using the RNeasy Plus microkit (Qiagen, catalog number 74134). cDNA was synthesized by reverse transcription using the iScript cDNA synthesis kit (BIO-RAD, catalog number 1708890). Quantitative PCR was performed using a Quantifast Probe PCR+ROX vial kit (Qiagen, catalog number 204354) on a CFX real-time system C1000 cycler. This cycler contained the following probes from ThermoFisher Scientific: KAT6A (Hs00198899_m1), ESR1 (Hs01046816_m1), and GAPDH (Hs99999905_m1). The cycling conditions were: 95℃ × 3 min, (95℃ × 3 sec, 60℃ × 30 sec) × 40 cycles.

[0415] Protein lysates were collected in RIPA lysis buffer supplemented with HALT (Thermo Fisher Scientific). 40 μg of protein extract was heated in sample buffer at 70°C and separated via Tris-Bis protein gel (NuPAGE Noves 10% gel, Thermo Fisher), then transferred to a nitrocellulose membrane (iBlot2, Life Technologies). The membrane was blocked and incubated overnight at 4°C with the following antibodies: anti-ERα (8644, Cell Signaling) and anti-β-actin (4970, Cell Signaling). The membrane was then blotted with horseradish peroxidase-conjugated anti-rabbit secondary antibody (7074P2, Cell Signaling), and the bands were visualized using an enhanced chemiluminescence system (Thermo Scientific) according to the manufacturer's instructions.

[0416] result:

[0417] ESR1 mRNA ( Figure 11B ) and ERα protein ( Figure 11C The reduction in ) is associated with KAT6A knockdown ( Figure 11A This indicates that KAT6A regulates ESR1 transcription in ER+ breast cancer cell lines with high KAT6A expression.

[0418] Example 9: ESR1 reexpression rescued T47D cells from growth inhibition induced by KAT6A knockdown and partially restored cell recovery from palbociclib-induced cell cycle arrest.

[0419] This example illustrates that ESR1 reexpression rescued the proliferative phenotype induced by KAT6A knockdown.

[0420] Step 1: Generation of ESR1 rescue cell lines

[0421] The ESR1 open reading frame was cloned downstream of the CMV promoter in a pLenti7.3 / V5-TOPO (Invitrogen)-based vector carrying a blasticidin selection marker. The construct and plenti7 vector were then packaged into lentiviral particles. T47D cells carrying Tet-closed shRenilla or shKAT6A_6 were transduced at low MOI using the pLenti7 vector or lentivirus expressing the ESR1 construct. Selection was performed with 20 μg / mL blasticidin (InvivoGen, catalog number ant-bl-05), followed by cell expansion to generate stable cell lines.

[0422] Step 2: Confirmation of ESR1 re-expression from the CMV promoter in the presence of KAT6A depletion

[0423] To confirm that ESR1 expression is restored via the CMV promoter, KAT6A was knocked down in the absence of doxycycline in the following cell lines generated in step 1:

[0424] 1. T47D / Tet - Shut down shRenilla / plenti7

[0425] 2.T47D / Tet - Shut down shRenilla / pLenti7-CMV-ESR1

[0426] 3. T47D / Tet - Disable shKAT6A_6 / plenti7

[0427] 4.T47D / Tet-shut down shKAT6A_6 / pLenti7-CMV-ESR1

[0428] Cells were seeded at a density of 25,000 cells / well in 12-well plates with RPMI + 10% FBS (tetracycline-free) + penicillin / streptomycin + 20 μg / mL blastomycin (with or without doxycycline). The medium was replaced every 3 days. After 7 days of culture, cells were harvested by trypsinization for protein and RNA samples. Protein lysates were prepared, and total RNA was isolated from each sample. SDS-PAGE, Western blotting, and qPCR were performed as described in Example 8.

[0429] Step 3: Salvage of proliferation and palbociclib recovery analysis

[0430] As described in Example 4, colony formation analysis was performed to assess direct proliferation and recovery from palbociclib-induced cell cycle arrest.

[0431] result:

[0432] KAT6A knockdown leads to ESR1 transcript reduction ( Figure 12B ) and ERα protein ( Figure 12A ) and cell growth ( Figure 12C The number of cells showed a significant decrease. However, ectopic expression of ESR1 was sufficient to partially restore cell growth in the presence of KAT6A depletion. Furthermore, reexpression of ESR1 also partially restored the recovery of T47D cells from cell cycle arrest induced by the combination of 300 nM palbociclib and KAT6A depletion. Figure 12D These data confirm that ERα alone or in combination with palbociclib plays a crucial role in KAT6A-mediated growth inhibition of T47D cells.

[0433] Example 10: The combination of a KAT6 small molecule inhibitor and palbociclib affects the proliferation and recovery of ER+ breast cancer cell lines.

[0434] Dosage titration with a small molecule KAT6 inhibitor confirmed that the catalytic function of KAT6A is required for the proliferation of ER+ breast cancer cells and recovery from palbociclib arrest, similar to the results obtained by KAT6A RNAi knockdown.

[0435] Materials and methods:

[0436] Based on the proliferation and palbociclib-restored colony formation analysis as described in Example 4, dosage titration was performed in T47D and ZR-75-1 cells (ATCC) using compounds C and D.

[0437] Cells were seeded at low density in 6-well plates and incubated overnight at 37°C and 5% CO2. The following day, in parallel with the proliferation group, palbociclib was added to another plate at a final concentration of 300 nM, which served as the palbociclib recovery group. DMSO or KAT6 inhibitor was added to each well of both the proliferation and palbociclib recovery plates at increasing doses. The medium and KAT6 inhibitor were replaced twice weekly. Cells were cultured with the drug for approximately two weeks until the wells treated with DMSO reached confluence. At the end of treatment, cells were washed once with phosphate-buffered saline, fixed, and stained with SRB for visualization, followed by quantification. For palbociclib recovery analysis with co-treatment with KAT6 inhibitor, cells were treated with both palbociclib and KAT6 inhibitor for two weeks. At the end of treatment, cells were washed three times with complete medium to remove palbociclib and KAT6 inhibitor and to allow recovery until the DMSO-treated cells reached at least 80% confluence. The wells were fixed, stained with SRB for visualization, and then quantified. For quantitative staining, stained cells were dissolved in 2 mL of 10 mM Tris-HCl (pH 7.5) and shaken for 10 min. The sample was then diluted with 10 mM Tris-HCl (pH 7.5), transferred to a 96-well microtiter plate (100 μL), and read at 565 nm on a SpetraMax.

[0438] When used as a single agent, compound C exhibits inhibitory effects on T47D cell growth, achieving a maximum growth reduction of approximately 40%. Figure 13A However, co-treatment with compound C and 300 nM palbociclib significantly enhanced its ability to block cell recovery from palbociclib-induced cell cycle arrest. In contrast, compound C exhibited a strong dose-dependent cytotoxic effect on the growth of ZR75-1 cells, achieving 50% growth inhibition at approximately 3.9 nM. The antiproliferative effect was significantly enhanced when ZR75-1 cells were treated with increased concentrations of compound C in the presence of 300 nM palbociclib. Figure 13B Similar cytotoxic effects on T47D cells and ZR75-1 cells were observed with compound D in the presence or absence of palbociclib, but its potency was reduced compared to compound C. Figure 13C and Figure 13D ).

[0439] result:

[0440] Compared with treatment with KAT6 small molecule inhibitors alone, the combination treatment with KAT6 small molecule inhibitors and palbociclib demonstrated a significantly enhanced efficacy in inhibiting the growth of these ER+ breast cancer cells, indicating a strong dependence on KAT6A catalytic function in cell proliferation. More importantly, these KAT6 inhibitors exhibited a synergistic effect with palbociclib in blocking the resumption of cell cycle arrest.

[0441] Example 11: KAT6 inhibitor treatment leads to depletion of estrogen receptor α in ER+ breast cancer cells

[0442] Dosage titration with a small molecule KAT6 inhibitor confirmed that KAT6 inhibitors downregulate ERα expression in ER+ breast cancer cells, similar to the results obtained by RNAi knockdown of KAT6A.

[0443] Materials and methods:

[0444] In T47D cells (ATCC), dose titration was performed using compounds C and D.

[0445] Cells were seeded at 10,000 cells per well in 6-well culture dishes and incubated overnight at 37°C and 5% CO2. The following day, in addition to DMSO as a load control, compound C or compound D was added to the wells at increased concentrations. Cells were cultured for 14 days, with the medium and KAT6 inhibitor being replaced twice a week (every 3-4 days). At the end of drug treatment, one million cells were collected via trypsinization to obtain protein lysates. Cells were lysed in 100 μL of 1×LDS buffer diluted from 4×LDS buffer (ThermoFisher catalog number NP0009) with a reducing agent (ThermoFisher catalog number NP0007). The lysates were then ultrasonically treated and loaded onto NuPAGE 4–12% Bis-Tris gels (ThermoFisher, catalog number NP0321BOX) for electrophoresis. The gels were then transferred to nitrocellulose membranes (iBlot2, ThermoFisher). The membrane was blocked and incubated overnight at 4°C with the following antibodies: anti-Erα (D8H8) rabbit mAb (Cell Signaling, No. 8644) and anti-β-actin (8H10D10) mouse mAb (Cell Signaling, No. 3700S). The next day, after thorough washing with 1×TBST (containing 0.1% Tween 20) buffer (prepared by diluting 10×Tris-buffered saline (TBS) (BIO-RAD No. 1706435) 10-fold and 10% Tween 20 (BIO-RAD No. 1610781) 100-fold), the membrane was blotted with horseradish peroxidase-conjugated anti-rabbit secondary antibody for ERα (Cell Signaling No. 7074P2) and HRP-linked anti-mouse IgG secondary antibody for β-actin (Cell Signaling No. 7076). Following the manufacturer's instructions, the bands were developed using an enhanced chemiluminescence (ECL) system (Thermo Scientific). The membrane was then washed three times with 1×TBST + 0.1% Tween 20, removing the portion detected by the anti-β-actin antibody, to completely remove the ECL. The membrane was then incubated on a shaker at room temperature for 2 hours with an anti-cyclin D1 rabbit mAb (Cell Signaling, Serial No. 2922S), followed by washing and detection with an HRP-linked anti-rabbit secondary antibody (Cell Signaling, Serial No. 7074P2). ECL was then performed to visualize the bands on the membrane.

[0446] result:

[0447] Using compound C ( Figure 14A ) or compound D ( Figure 14B Treatment with compound C resulted in dose-dependent depletion of both ERα and cyclin D1, with compound C being more potent than compound D. Consistent with the decrease in both proteins, ESR1 and CCND1 (encoding cyclin D1) mRNA, as measured by qPCR, also showed a dose-dependent decrease induced by treatment with compound D. Figure 14C and Figure 14D This data demonstrates that regulating ERα expression requires the catalytic function of KAT6A, which is similar to the results obtained by knocking down KAT6A.

[0448] Example 12: Synergistic effect of KAT6 inhibitor and palbociclib against ER+ breast cancer cells

[0449] To evaluate the in vitro antiproliferative effect of the combination of a KAT6 small molecule inhibitor and palbociclib.

[0450] Materials and methods:

[0451] The following cell lines were obtained from ATCC (Manassas, VA) and grown in cryogenic stock solutions prepared within six months of their initial purchase: T47D, ZR75-1, MCF7, and CAMA1. Cells were maintained according to the supplier's guidelines.

[0452] Cells were seeded at 1000 cells per well (for T47D, CAMA1, or MCF7) and 2000 cells per well (for ZR75-1) into 96-well plates and incubated overnight in RPMI-1640 medium containing 10% FBS and 1× penicillin / streptomycin. The following day, the plates were treated with compound C, compound E, or fulvestrant + palbociclib at concentrations present in the matrix. Except for two wells containing a control treated with DMSO, in the dilution series, each KAT6 inhibitor was horizontally diluted four-fold from 1000 nM (for T47D, CAMA1, and MCF7) and from 250 nM (for ZR75-1) across the entire plate, for a total of eight doses. In addition to the DMSO control, fulvestrant was horizontally diluted three-fold at 100 nM (for T47D and CAMA1), 300 nM (for ZR75-1), and 1.235 nM (for MCF7) across the entire plate, for a total of 8 spots. In addition to the DMSO control, palbociclib was vertically diluted three-fold at 500 nM (for T47D, CAMA1, and ZR75-1) and 167 nM (for MCF7) across the entire plate, for a total of 5 spots. The medium and compounds were refreshed every three days for a total of 10 days.

[0453] At the end of treatment, cell nuclei were stained using CyQUANT Direct (ThermoFisher, catalog number C35011) according to the manufacturer's protocol, and direct cell counting was performed using a Celigo S imaging cell counter (Nexcelom BioScience). The percentage of growth inhibition was calculated by normalizing cell counts from the compound-treated wells to cell counts from the DMSO / DMSO control wells. The Loewe additivity score (Loewe, 1926) was calculated using Chalice Bioinformatics software (Horizon Discovery) for synergistic analysis. The synergistic score was derived from the excess inhibition 2D matrix calculated in Chalice software (Lehar, 2009) based on the Loewe additivity model. Values ​​greater than 1 indicated synergistic effects of the combination, while values ​​less than 1 indicated antagonistic effects.

[0454] result:

[0455] Treatment with either compound C or compound E in combination with palbociclib exhibited synergistic inhibition of cell growth in T47D, ZR-75-1, CAMA1, and MCF7 cells (Table 3). Analysis of the combination data using the Loewe Additive Distributive (ADD) model showed that the levels of growth inhibition achieved by these combinations were synergistic. The synergistic effect of compound C or compound E with palbociclib was equivalent to the synergistic effect of the palbociclib plus fulvestrant combination observed in T47D, CAMA1, or MCF7 cells, as well as in ZR75-1 cells (where fulvestrant and palbociclib were not synergistic) (Table 3).

[0456] Table 3

[0457]

[0458] Example 13: Synergistic effect of KAT6 inhibitor and fulvestrant against ER+ breast cancer cells

[0459] To evaluate the in vitro antiproliferative effect of the combination of a KAT6 small molecule inhibitor and fulvestrant.

[0460] Materials and methods:

[0461] The experimental design provided in Example 12 differs in that fulvestrant is used instead of palbociclib. In the dilution series, compound E was horizontally diluted four-fold from 1000 nM across the entire plate, for a total of eight doses, except for two wells containing a control treated with DMSO. In addition to the DMSO control, fulvestrant was vertically diluted four-fold from 100 nM (for T47D, CAMA1, and ZR75-1) and from 1.563 nM (for MCF7) across the entire plate, for a total of five spots. The medium and compound were refreshed every three days for 13 days.

[0462] At the end of treatment, cell nuclei were stained using CyQUANT Direct (ThermoFisher, catalog number C35011) according to the manufacturer's protocol, and direct cell counting was performed using a Celigo S imaging cell counter (Nexcelom BioScience). The percentage of growth inhibition was calculated by normalizing cell counts from the compound-treated wells to cell counts from the DMSO / DMSO control wells. The Loewe additivity score (Loewe, 1926) was calculated using Chalice Bioinformatics software (Horizon Discovery) for synergistic analysis. The synergistic score was derived from the over-inhibition 2D matrix calculated in Chalice software (Lehar, 2009) based on the Loewe additivity model. Values ​​greater than 1 indicated synergistic effects of the combination, while values ​​less than 1 indicated antagonistic effects.

[0463] result:

[0464] The combined treatment of compound E with fulvestrant showed a synergistic effect in inhibiting the proliferation of all four cell lines (T47D, ZR-75-1, CAMA1, and MCF7) (Table 3).

[0465] Example 14: Synergistic effect of KAT6 inhibitors and selective CDK4 inhibitors against ER+ breast cancer cells

[0466] To evaluate the in vitro antiproliferative effect of the combination of a KAT6 small molecule inhibitor and a selective CDK4 inhibitor (compound F).

[0467] Materials and methods:

[0468] Following the procedure in Example 12, cells were seeded into 96-well plates at 1000 cells per well (for T47D, CAMA1, or MCF7) and 2000 cells per well (for ZR75-1), and incubated overnight in RPMI-1640 medium containing 10% FBS and 1× penicillin / streptomycin. The following day, the plates were treated with fulvestrant, compound C, or compound E and compound F, which were present in the matrix, at specific concentrations. Except for two wells containing a control treated with DMSO, in the dilution series, each KAT6 inhibitor was horizontally diluted four-fold from 1000 nM (for T47D, CAMA1, and MCF7) and from 250 nM (for ZR75-1) across the entire plate, for a total of eight doses. In addition to the DMSO control, fulvestrant was horizontally diluted three-fold at 300 nM (for ZR75-1), 100 nM (for T47D and CAMA1), and 1.235 nM (for MCF7) across the entire plate, for a total of 8 spots. In addition to the DMSO control, compound F was vertically diluted three-fold at 500 nM (for T47D, CAMA1, and ZR75-1) and 167 nM (for MCF7) across the entire plate, for a total of 5 spots.

[0469] The culture medium and compounds were changed every three days for 10 days. At the end of treatment, cell nuclei were stained using CyQUANT Direct (ThermoFisher, catalog number C35011) according to the manufacturer's protocol, and direct cell counting was performed using a Celigo S imaging cell counter (Nexcelom BioScience). The percentage of growth inhibition was calculated by normalizing cell counts from the compound-treated wells to cell counts from the DMSO / DMSO control wells. The Loewe additivity score (Loewe, 1926) was calculated using Chalice Bioinformatics software (Horizon Discovery) for synergy analysis. The synergy score (Table 4) was derived from the over-inhibition 2D matrix calculated using the Loewe additivity model in Chalice software (Lehar, 2009). Values ​​greater than 1 indicated synergistic effects of the combination, while values ​​less than 1 indicated antagonistic effects.

[0470] result:

[0471] Combination treatment with compound C or compound E and the selective CDK4 inhibitor (compound F) synergistically inhibited the growth of T47D, ZR-75-1, CAMA1 and MCF7 cells (Table 4).

[0472] Table 4

[0473] cell lines Fluvestrant + compound F Compound C + Compound F Compound E + Compound F T47D 5.77 3.00 2.82 ZR75-1 0.14 2.83 2.80 CAMA1 2.50 2.64 2.69 MCF7 3.20 2.73 2.80

[0474] Example 15: The mechanism by which the combination of KAT6 inhibitor and palbociclib overcomes drug resistance caused by ESR1 mutations in ER+ breast cancer cells.

[0475] The study investigated the direct inhibition of ESR1 transcription and ER-dependent genes to determine whether KAT6 inhibitors, as single agents or in combination with palbociclib, could overcome mutations in estrogen receptor-α (ERα), a major mechanism of resistance to endocrine therapy.

[0476] Materials and methods:

[0477] Using gene editing via CRISPR / CAS9, clinically relevant ESR1 mutations (Y537S and D538G) were introduced individually and together into the endogenous ESR1 gene under the control of the natural promoter in T47D cells (ATCC).

[0478] Step 3: Characterization of T47D ESR1 mutant clones during in vitro drug treatment

[0479] Each mutant clone was evaluated by its response to fulvestrant and KAT6 inhibitors (Table 5). Clones with Y537S, D538G, or Y537S / D538G exhibited resistance to fulvestrant, with their cell proliferation IC50 increasing by 3-17, 3-11, and 22-100 times relative to the parental cell lines, respectively. Figure 15A and Figure 15B In contrast, clones with mutations in Y537S, D538G, or Y537S / D538G showed resistance to palbociclib when compared to parental cell lines. Figure 15A and Figure 15B Equivalent sensitivity to KAT6 inhibitor compound C ( Figure 15A ) or compound D ( Figure 15B Enhanced sensitivity to palbociclib and compound C. Furthermore, these mutant clones maintained enhanced sensitivity to the combination of palbociclib and compound C. Figure 15A ) and the combination of palbociclib and compound D ( Figure 15B )sensitive.

[0480] Table 5

[0481]

[0482]

[0483] result:

[0484] The T47D cells with ESR1 mutations studied above exhibited reduced sensitivity to fulvestrant, but similar to their parental T47D cells, they remained sensitive to KAT6 inhibitors, both as single agents and in combination with palbociclib, in vitro. These results confirm that KAT6 inhibitors, as single agents or in combination with palbociclib, overcome the resistance mechanism induced by ESR1 mutations.

[0485] Example 16: Synergistic effect of KAT6 inhibitor and palbociclib against ER+ breast cancer cells with clinically relevant ESR1 mutations

[0486] A palbociclib combination study was conducted to determine whether knocking in clinically relevant ESR1 mutations alters the synergistic effect between KAT6 inhibitors and palbociclib in ER+ breast cancer cells.

[0487] Materials and methods:

[0488] Palbociclib combination studies were conducted using ESR1 mutant clones (Y537S, D538G, and dual Y537S_D538G) to evaluate the synergistic effect between palbociclib and KAT6 inhibitors, as described in Example 12.

[0489] Cells were seeded at 1000 cells per well into 96-well plates and incubated overnight in RPMI-1640 medium containing 10% FBS and 1× penicillin / streptomycin. The following day, the plates were treated with palbociclib at concentrations of fulvestrant, compound C, or compound E present in the matrix. In the dilution series, except for two wells with a control treated with DMSO, each KAT6 inhibitor was horizontally diluted four times from 1000 nM across the entire plate, for a total of eight doses. Except for the DMSO control, fulvestrant was horizontally diluted three times from 100 nM across the entire plate, for a total of eight spots. Except for the DMSO control, palbociclib was vertically diluted three times from 500 nM across the entire plate, for a total of five spots. The medium and compounds were refreshed every three days for a total of 10 days.

[0490] At the end of treatment, cell nuclei were stained using CyQUANT Direct (ThermoFisher, catalog number C35011) according to the manufacturer's protocol, and direct cell counting was performed using a Celigo S imaging cell counter (Nexcelom BioScience). The percentage of growth inhibition was calculated by normalizing cell counts from the compound-treated wells to cell counts from the DMSO / DMSO control wells. The Loewe additivity score (Loewe, 1926) was calculated using Chalice Bioinformatics software (Horizon Discovery) for synergistic analysis. The synergistic score was derived from the over-inhibition 2D matrix calculated in Chalice software (Lehar, 2009) based on the Loewe additivity model. Values ​​greater than 1 indicated synergistic effects of the combination, while values ​​less than 1 indicated antagonistic effects.

[0491] result:

[0492] When compared with the parental wild type, the synergistic effect between fulvestrant and palbociclib is reduced due to the decreased sensitivity of the mutant clone to fulvestrant. However, the ESR1 mutation does not affect the synergistic effect between palbociclib and the KAT6 inhibitor, as shown by the synergistic score (Table 6).

[0493] These data confirm that the combination of KAT6 inhibitors and palbociclib provides a mechanism to overcome clinically relevant resistance to endocrine therapies used to treat breast cancer.

[0494] Table 6

[0495]

[0496] Example 17: ESR1 expression downregulated by palbociclib in ER+ breast cancer cells

[0497] Further investigation into ESR1 expression is needed to explore the potential molecular mechanism of the synergistic effect between KAT6 inhibitors and palbociclib.

[0498] Materials and methods:

[0499] As described in Examples 6 and 7, through a microarray ( Figure 16A ) and RNA-Seq Figure 16B Expression profiling was performed in T47D cells.

[0500] T47D(ATCC) and MCF7 cells were treated with palbociclib as a single agent and in combination with compound C, respectively. Cells were seeded into 6-well plates and allowed to attach overnight at 37°C and 5% CO2. The following day, palbociclib was added to the culture medium at 500 nM with DMSO or 20 nM with compound C. The culture medium was replaced every 3 days. Cells were harvested for protein and RNA lysates at 3, 6, and 8 days of treatment.

[0501] Protein samples were subjected to SDS-PAGE electrophoresis and Western blotting to detect phosphate Rb (Ser780) (CellSignaling catalog number 9307S), Rb (Cell Signaling catalog number 9309S), ERα (Cell Signaling catalog number 2922S) and β-actin (Cell Signaling catalog number 3700).

[0502] result:

[0503] Palbociclib effectively inhibited the phosphorylation of Rb protein in both cell lines at all three time points. Figure 16C Furthermore, consistent with gene profiling studies conducted via microarrays or RNA-Seq, palbociclib treatment resulted in a gradual decrease in ERα protein levels from day 3 to day 8, with this decrease reaching approximately 50% after 8 days of treatment in both cell lines.

[0504] Combined treatment with palbociclib and compound C resulted in a significant decrease in ERα levels. Figure 16C The decrease in ERα protein was consistent with the decrease in ESR1 mRNA levels measured by qPCR. Figure 16D ).

[0505] The results confirmed that palbociclib as a single agent and the KAT6 inhibitor as a single agent both reduced ESR1 mRNA, with the KAT6 inhibitor showing a greater reduction. The combination of palbociclib and the KAT6 inhibitor resulted in a significant reduction in ESR1 transcripts. Figure 16A This is related to its synergistic anti-tumor combination activity.

[0506] Example 18: KAT6 inhibitors combined with palbociclib and fulvestrant demonstrate in vivo combination benefits in an ER+ breast cancer patient-derived xenograft (PDX) model.

[0507] In vivo studies were conducted in two clinically relevant, patient-derived xenograft (PDX) models of ER+ breast cancer to determine whether KAT6 inhibitors, when used in combination with palbociclib and fulvestrant, could provide additional antitumor efficacy benefits.

[0508] Materials and methods:

[0509] A combination study using palbociclib, fulvestrant, and the KAT6 inhibitor (compound E) (KAT6i) was conducted in two ER+ breast cancer PDX models (ST340 and ST941). ST340 was wild-type for ESR1. ST941 had an activating mutation Y537S in the ligand-binding domain (LBD) of ESR1, a mutation associated with clinically recognized resistance to endocrine therapy. Both studies were conducted by XenoSTART (San Antonio, Texas), in which approximately 70 mg of tumor fragment was subcutaneously implanted into 6-12 week old female athymic nude mice (JAX, stock number 007850). Exogenous estradiol was supplemented ad hocly via drinking water throughout the study period. Tumor measurements and animal weight were obtained twice weekly, and mean tumor volume (TV, formula: width) was measured. 2 (×length×0.5) to reach 150-300mm 3 At that time, the animals were stratified and included in the study.

[0510] Each study contained eight treatment groups, with n = 10 animals per group. The groups were as follows: 1) carrier (5% DMSO / 40% PEG300 / 55% 1×PBS), 2) compound E, 3) palbociclib, 4) fulvestrant, 5) palbociclib + fulvestrant, 6) compound E + fulvestrant, 7) compound E + palbociclib, and 8) compound E + palbociclib + fulvestrant. Details of dosage levels, routes of administration, and regimens are listed in Table 7. Details of compounds and formulations are listed in Table 8. Animals remained in the study and continued treatment until the mean tumor volume in each group reached approximately 1500 mm. 3 At this point, the final dose was administered, the animals were euthanized, and the final sample was collected. Micro-blood samples were collected from each group on day 14 and again on the last day of administration at hours 0, 3, and 7 (n = 3 / time point) for pharmacokinetic (PK) analysis.

[0511] Tumor growth inhibition (TGI) associated with the loading group was calculated for ST340 and ST941 on days 21 and 17, respectively, where TGIδ = 1 – (treatment) t –Treatment 0) / (Reference) t – Reference 0). Statistical significance of TGI percentages between comparison groups was determined using analysis of covariance (ANCOVA). Weight change was calculated relative to starting weight at the start of the study (day 0 of treatment).

[0512] Table 7. Study Design and Dosing Details

[0513]

[0514] *LD = Loading dose to be administered on day 3 of each new dosing cycle (dosing cycle = 1 month).

[0515] Table 8. Test Products and Formulations

[0516] Test product Carrier Compound E 5% DMSO / 40% PEG300 / 55% 1×PBS Fulvestrant Peanut oil Pabosini 0.5% MC A4M in water

[0517] result:

[0518] TGI analysis showed that fulvestrant produced only about 20% growth inhibition compared to the load, and this was not significant in either ST340 or ST941 (Tables 9 and 12), indicating that these models are largely insensitive to ER antagonism. The lack of response to fulvestrant is consistent with the previous molecular characteristics of these models, as ST340 was derived from patients previously treated with HER2 inhibitors and ST941 contained an ESR1 activating mutation. Both models also showed only moderate responses to CDK4 / 6 inhibition induced by palbociclib, producing about 40% TGI compared to the load. Monotherapy with compound E also had moderate efficacy, with the efficacy seen in ST340 (55%, Table 9) slightly better than that seen in ST941 (35%, Table 12).

[0519] Although each of the three agents showed only low to moderate efficacy when used as a single agent, greater tumor growth inhibition and growth delay were observed in the combination group. Figure 17A , Figure 18A In the dual-combination groups, compound E + palbociclib exhibited the strongest antitumor activity, producing 75% and 67% TGI in ST340 and ST941, respectively (Tables 9 and 12). Both studies also indicated that compound E (KAT6i) + fulvestrant was the least effective combination, and fulvestrant did not provide significant additional efficacy in either model. Figure 17A , Figure 18A However, when fulvestrant was administered as part of the triple combination of compound E + palbociclib + fulvestrant, the efficacy was further enhanced, demonstrating the strongest antitumor efficacy with 78% inhibition in ST340 and 72% inhibition in ST941. Despite the excellent triple combination, it did not reach statistical significance when compared with compound E + palbociclib or palbociclib + fulvestrant (Tables 10 and 13).

[0520] Health and weight monitoring in both studies indicated that the average weight loss in either treatment group did not exceed 10%. Figure 17B , Figure 18B(Tables 11 and 14). All combinations of compound E, palbociclib, and fulvestrant were well tolerated, and no drug-related deaths were observed. In summary, these results confirm that combination therapy with the KAT6 inhibitor compound E is tolerable and effective in models with relative resistance to ER or CDK4 / 6 inhibition compared to the load agent and to standard single-agent therapy.

[0521] Table 9. Results of PDXST340TGI compared to the carrier (Group 1)

[0522]

[0523] Table 10. Statistical significance of PDX ST340 for cross-group comparisons

[0524]

[0525] Table 11. Changes in PDX ST340 based on body weight

[0526]

[0527] Table 12. PDX ST941 TGI results compared to the carrier (Group 1)

[0528]

[0529]

[0530] Table 13. Statistical significance of PDX ST941 for cross-group comparisons

[0531]

[0532] Table 14. Changes in PDX ST94 based on body weight

[0533]

Claims

1. Use of lysine acetyltransferase 6 (KAT6) inhibitors in the preparation of medicaments for use in combination with the following to treat patients with cancer: a) Cyclin-dependent kinase 4 (CDK4) inhibitors; or b) Anti-estrogens; or c) CDK4 inhibitors and anti-estrogens; The cancer mentioned is breast cancer; The KAT6 inhibitor mentioned above is 2-methoxy- N -{4-methoxy-6-[(1 H [-pyrazol-1-yl]methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof; The anti-estrogen mentioned above is fulvestrant; and The CDK4 inhibitor is 1,5-dehydr-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1-yl)-1-yl H -benzimidazole-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanediol or a pharmaceutically acceptable salt thereof, or palbociclib or a pharmaceutically acceptable salt thereof.

2. The use as described in claim 1, wherein the CDK4 inhibitor is 1,5-dehydr-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1- H -benzimidazole-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanediol or a pharmaceutically acceptable salt thereof.

3. The use as described in claim 1, wherein the CDK4 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

4. The use as described in claim 1, wherein the breast cancer is estrogen receptor-positive (ER+) breast cancer.

5. The use as claimed in claim 1, wherein the breast cancer is locally advanced or metastatic estrogen receptor-positive (ER+) breast cancer.

6. The use as claimed in claim 1, wherein the breast cancer is locally advanced or metastatic estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer.

7. The use as claimed in any one of claims 1-6, wherein the patient is a human.

8. Use of a KAT6 inhibitor in the preparation of a medicament for overcoming clinical resistance to endocrine therapy in patients, wherein the medicament is combined with a CDK4 inhibitor, wherein the endocrine therapy is used to treat breast cancer; The KAT6 inhibitor mentioned above is 2-methoxy- N -{4-methoxy-6-[(1 H [-pyrazol-1-yl]methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof; and The CDK4 inhibitor is 1,5-dehydr-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1-yl)-1-yl H -benzimidazole-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanediol or a pharmaceutically acceptable salt thereof, or palbociclib or a pharmaceutically acceptable salt thereof.

9. The use as claimed in claim 8, wherein the CDK4 inhibitor is 1,5-dehydr-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1-yl)-1-yl H -benzimidazole-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanediol or a pharmaceutically acceptable salt thereof.

10. The use as described in claim 8, wherein the CDK4 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

11. The use as claimed in claim 8, wherein the breast cancer is estrogen receptor-positive (ER+) breast cancer.

12. The use as claimed in claim 8, wherein the breast cancer is locally advanced or metastatic estrogen receptor-positive (ER+) breast cancer.

13. The use as claimed in claim 8, wherein the breast cancer is locally advanced or metastatic estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer.

14. The use as claimed in any one of claims 8 to 13, wherein the patient is a human.

15. A pharmaceutical composition comprising a lysine acetyltransferase 6 (KAT6) inhibitor and a) Cyclin-dependent kinase 4 (CDK4) inhibitors; or b) Anti-estrogens; or c) CDK4 inhibitors and anti-estrogens; and pharmaceutically acceptable carriers. The KAT6 inhibitor mentioned above is 2-methoxy- N -{4-methoxy-6-[(1 H [-pyrazol-1-yl]methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof; The anti-estrogen mentioned above is fulvestrant; and The CDK4 inhibitor is 1,5-dehydr-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1-yl)-1-yl H -benzimidazole-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanediol or a pharmaceutically acceptable salt thereof, or palbociclib or a pharmaceutically acceptable salt thereof.

16. The pharmaceutical composition of claim 15, wherein the CDK4 inhibitor is 1,5-dehydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1- H -benzimidazole-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanediol or a pharmaceutically acceptable salt thereof.

17. The pharmaceutical composition of claim 15, wherein the CDK4 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical kit comprising a pharmaceutical composition comprising a lysine acetyltransferase 6 (KAT6) inhibitor and a pharmaceutically acceptable carrier, and: a) A pharmaceutical composition comprising a cyclin-dependent kinase 4 (CDK4) inhibitor and a pharmaceutically acceptable carrier; and / or b) A pharmaceutical composition comprising an anti-estrogen and a pharmaceutically acceptable carrier. The KAT6 inhibitor mentioned above is 2-methoxy- N -{4-methoxy-6-[(1 H [-pyrazol-1-yl]methyl]-1,2-benzoxazol-3-yl]benzene-1-sulfonamide or a pharmaceutically acceptable salt thereof; The CDK4 inhibitor is 1,5-dehydr-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1-yl)-1-yl H -benzimidazole-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanediol or a pharmaceutically acceptable salt thereof, or palbociclib or a pharmaceutically acceptable salt thereof; and The anti-estrogen mentioned therein is fulvestrant.

19. The kit of claim 18, wherein the CDK4 inhibitor is 1,5-dehydr-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropyl-2-yl)-1-(propyl-2-yl)-1-yl)-1-yl H -benzimidazole-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-threo-pentanediol or a pharmaceutically acceptable salt thereof.

20. The kit of claim 18, wherein the CDK4 inhibitor is palbociclib or a pharmaceutically acceptable salt thereof.

Images