Treatment of Cancer with KDM4 Inhibitors

Abstract

Compositions and methods for the treatment of cancer are provided herein, the compositions comprising a KDM4 inhibitor.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 173,219, filed April 9, 2021; U.S. Provisional Patent Application No. 63 / 252,542, filed October 5, 2021; and U.S. Provisional Patent Application No. 63 / 297,960, filed January 10, 2022, all of which are incorporated by reference in their entireties into this specification. Summary of the Invention

[0002]

[0002] Provided herein are compositions and methods for the treatment of cancer.The types of cancer suitable for the methods disclosed herein include, but are not limited to, colorectal cancer, triple-negative breast cancer, gastric adenocarcinoma and diffuse large B-cell non-Hodgkin's lymphoma.

[0003]

[0003] Compositions useful in the methods of treating cancer disclosed herein include the heterocyclic KDM4 inhibitors described herein.

[0004] One embodiment provides a method of treating cancer in a cancer patient in need thereof, comprising administering to the individual a compound having the following structure:

[0004] [ka]

[0005] Another aspect provides the method, wherein the cancer patient has been diagnosed with a cancer selected from colorectal cancer, esophageal cancer, triple negative breast cancer, gastric cancer, lymphoma, gastric adenocarcinoma, diffuse large B-cell non-Hodgkin's lymphoma, acute T-cell leukemia, esophageal squamous cell carcinoma, multiple myeloma, acute myeloid leukemia, colorectal adenocarcinoma, colorectal cancer, pancreatic cancer, pancreatic carcinoma, breast cancer, or T-cell acute lymphoblastic leukemia.

[0006]

[0005] Another aspect provides a method of reducing a tumorigenic cell population in a cancer patient in need thereof, comprising administering to the individual a compound having the following structure, or a pharma- ceutically acceptable salt or solvate thereof:

[0007] [ka]

[0008] . Another aspect provides a method of reducing tumor initiating cell frequency in a cancer patient in need thereof, comprising administering to the individual a compound having the following structure, or a pharma- ceutically acceptable salt or solvate thereof:

[0009] [ka]

[0010] .

[0007] Another aspect provides a method of inhibiting cancer stem cells in a cancer patient in need thereof, comprising administering to the individual a compound having the following structure, or a pharma- ceutically acceptable salt or solvate thereof:

[0011] [ka]

[0012] . [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 illustrates the biochemical activity of compound 1, a potent pan-KDM4 inhibitor. [Diagram 2]

[0009] Figure 2 illustrates the reversible and competitive inhibition of H3K9me3 demethylation. Inhibition of H3K3me3 demethylation was measured at various compound 1 concentrations in the presence of α-KG using time-resolved fluorescence-based (TR-FRET) LANCE® detection. [Diagram 3]

[0010] Figure 3 illustrates that Compound 1 induces cell cycle arrest in S phase. Compound 1 increased the percentage of cells in S phase in a dose-dependent manner. Cell cycle analysis was performed on MDA-MB-231 cells using MultiCycle AV DNA analysis software. [Figure 4] FIG. 4 demonstrates that compound 1 induces apoptosis in human cancer cell lines. [Diagram 5]

[0012] Figure 5 demonstrates that compound 1 exhibits potent antiproliferative activity across a large panel of cancer cell lines. Evaluation of compound 1 in a panel of PDX / organoid models confirms the sensitivity of compound 1 in gastric cancer and demonstrates sensitivity against MSI CRC vs. MSS CRC. [Figure 6]

[0013] Figure 6 illustrates the dose-dependent inhibition of histone demethylase by compound 1. Left panel: Representative immunoblot showing the change in staining intensity for actin (red band) and H3K36me3 (green band) in response to various concentrations of compound 1. Right panel: Normalized ratio of band intensity for H3K36me3 and actin versus concentration of compound 1 (μM) (mean IC50±SD value -0.085±0.004μM). [Figure 7]

[0014] Figure 7 illustrates the reduction of tumorigenic cells upon treatment of tumors with Compound 1. Compound 1 reduced tumor initiating cell (TIC) frequency by 4.4-fold. [Figure 8]

[0015] FIG. 8 illustrates the in vivo efficacy of Compound 1 in multiple cancer models. [Figure 9]

[0016] FIG. 9 illustrates inhibition of cell proliferation in the SU60 organoid model. [Figure 10]

[0017] Figure 10. In vivo efficacy of compound 1 in the SU60 patient-derived colorectal cancer xenograft model. [Figure 11]

[0018] FIG. 11. In vivo efficacy of Compound 1 in the KYSE-150 human esophageal cancer xenograft model. [Figure 12]

[0019] FIG. 12. In vivo efficacy of Compound 1 in Study No. 2 in the KYSE-150 human esophageal cancer xenograft model. [Figure 13]

[0020] FIG. 13. In vivo efficacy of Compound 1 in a COH70 patient-derived triple-negative breast cancer xenograft model. [Figure 14]

[0021] FIG. 14. In vivo efficacy of Compound 1 in the GXA-3036 gastric adenocarcinoma patient-derived xenograft model. [Figure 15]

[0022] FIG. 15. In vivo efficacy of Compound 1 in the OCI-LY19 human diffuse large B-cell non-Hodgkin's lymphoma xenograft model. [Figure 16]

[0023] FIG. 16 illustrates flow cytometry analysis of tumorigenic cell populations following Compound 1 treatment. [Figure 17]

[0024] FIG. 17 illustrates the results of an in vivo tumorigenicity functional assay following Compound 1 treatment. [Figure 18]

[0025] Figure 18 illustrates a heat map of the mutation status of genes of various pathways in different colorectal cancer cell lines. The respective microsatellite instability (MSI-H), CpG island methylator phenotype (CIMP) and MLH1 methylation status as well as the sensitivity of these cell lines to Compound 1 are also shown. [Figure 19]

[0026] FIG. 19 illustrates a heatmap showing the MSI-H status and respective MMR pathway gene mutations of the various colorectal cancer cell lines studied. [Figure 20-1]

[0027] FIG. 20 illustrates that the genes PNUTS (PPP1R10), ANK1, IBA57, SOWAHD, MF12-AS1 and CECR1 showed dose-dependent changes following treatment with Compound 1 in vivo. [Figure 20-2] FIG. 20 illustrates that the genes PNUTS (PPP1R10), ANK1, IBA57, SOWAHD, MF12-AS1 and CECR1 showed dose-dependent changes following treatment with Compound 1 in vivo. [Figure 21]

[0028] FIG. 21 illustrates that ChIP-seq analysis in MDA-MB-231 TNBC cells shows occupancy of KDM4 in the PNUTS (PPP1R10) promoter. [Figure 22A]

[0029] FIG. 22 illustrates a heatmap representation of single cell gene expression analysis (RT-qPCR) from vehicle control or compound 1 treated SU60 xenograft tumors. A) Heatmap representation of single cell gene expression analysis (RT-qPCR) from vehicle control or compound 1 treated SU60 xenograft tumors. Gene expression levels are color coded with red high expression, green low expression, and gray no expression. Three cell clusters were identified based on shared patterns of gene expression using a gene set of immature and mature cell markers. On the far right of the heatmap, the distribution of 335 vehicle treated (blue) and 332 compound 1 treated (red) single cells is displayed in red / blue ribbons. The top dendrogram clusters genes with similar expression patterns across all cells. Gene expression (at Ct values) was mean centered at 0 and divided by 2.5 times the standard deviation. [Figure 22B] B) The graph represents the total number of vehicle-treated and Compound 1-treated cells in each cluster. P values ​​(Fisher's exact test) are indicated at the top of the bars.

[0014] Incorporation by Reference

[0030] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference for the specific purposes identified herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Specific Terminology

[0031] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "an agent" includes a plurality of such agents, a reference to "the cell" includes a reference to one or more cells (or cells) and equivalents thereof known to those of skill in the art, etc. When ranges are used herein with respect to physical properties such as molecular weight or chemical properties such as chemical formulas, all combinations and subcombinations of the ranges and specific embodiments therein are intended to be included. The term "about" when referring to a numerical value or numerical range means that the numerical value or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the numerical value or numerical range will vary, in some cases, by 1% to 15% of the numerical value or numerical range stated. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude other specific embodiments, such as "consisting of" or "consisting essentially of" the recited features in one embodiment of any composition of matter, composition, method, or process described herein.

[0016]

[0032] As used in this specification and the appended claims, unless specified to the contrary, the following terms have the meanings indicated below.

[0033] The compounds disclosed herein may, in certain embodiments, be present in different enriched isotopically forms, e.g. 2 H, 3 H,11 C. 13 C and / or 14 In one particular embodiment, the compound is deuterated at at least one position. Such deuterated form can be prepared by the procedure described in U.S. Patent Nos. 5,846,514 and 6,334,997. As described in U.S. Patent Nos. 5,846,514 and 6,334,997, deuteration can improve metabolic stability and / or efficacy, thus increasing the duration of action of the drug.

[0017]

[0034] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of hydrogen by deuterium or tritium, or 13 C- or 14 Compounds having this structure except for the replacement of a carbon with a C-enriched carbon are within the scope of this disclosure.

[0018]

[0035] The compounds of the present disclosure may optionally contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain, for example, deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I) or carbon-14( 14 C) can be labeled with an isotope. 2 H, 11 C. 13 C. 14 C. 15 C. 12 N, 13 N, 15 N, 16 N, 16 O. 17 O. 14 F, 15 F, 16 F, 17 F, 18 F, 33 S, 34 S,35 S, 36 S, 35 Cl, 37 Cl, 79 Br, 81 Br, 125 All isotopic substitutions with I are contemplated. 18 Isotopic substitution with F is contemplated. All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0019]

[0036] In certain embodiments, the compounds disclosed herein are 1 Some or all of the H atoms 2 H atom is substituted. Synthetic methods for deuterium-containing compounds are known in the art and include, by way of non-limiting example only, the following synthetic methods:

[0020]

[0037] Deuterium substituted compounds are synthesized using a variety of methods such as those described in: Dean, Dennis C.; Editor. Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development. [Curr., Pharm. Des., 2000; 6(10)] 2000, 110 pp; George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans, E. Anthony. Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.

[0021]

[0038] Deuterated starting materials are readily available and are subject to the synthetic methods described herein to provide for the synthesis of deuterium-containing compounds. Many deuterated reagents and building blocks are commercially available from chemical vendors such as Aldrich Chemical Co.

[0022]

[0039] Deuterium transfer reagents suitable for use in nucleophilic substitution reactions, such as iodomethane-d3 (CD3I), are readily available and can be employed to transfer deuterium-substituted carbon atoms to reaction substrates under nucleophilic substitution reaction conditions. The use of CD3I is illustrated, by way of example only, in the following reaction scheme.

[0023] [ka]

[0024]

[0040] Deuterium transfer reagents, such as lithium aluminum deuteride (LiAlD4), are utilized to transfer deuterium to reaction substrates under reducing conditions. The use of LiAlD4 is illustrated, by way of example only, in the following reaction scheme.

[0025] [ka]

[0026]

[0041] Deuterium gas and a palladium catalyst are utilized to reduce unsaturated carbon-carbon bonds and effect reductive displacement of aryl carbon-halogen bonds, as illustrated, by way of example only, in the reaction scheme below.

[0027] [ka]

[0028]

[0042] In one embodiment, the compounds disclosed herein contain one deuterium atom. In another embodiment, the compounds disclosed herein contain two deuterium atoms. In another embodiment, the compounds disclosed herein contain three deuterium atoms. In another embodiment, the compounds disclosed herein contain four deuterium atoms. In another embodiment, the compounds disclosed herein contain five deuterium atoms. In another embodiment, the compounds disclosed herein contain six deuterium atoms. In another embodiment, the compounds disclosed herein contain more than six deuterium atoms. In another embodiment, the compounds disclosed herein are fully substituted with deuterium atoms and are non-exchangeable. 1 In one embodiment, the level of deuterium incorporation is determined by the synthetic method in which deuterated synthetic building blocks are used as starting materials.

[0029]

[0043] "Pharmaceutically acceptable salts" include both acid and base addition salts. Pharmaceutically acceptable salts of the heterocyclic KDM4 inhibitors described herein are intended to include any and all pharma- ceutically suitable salt forms. Preferred pharma-ceutically acceptable salts of the compounds described herein are pharma-ceutically acceptable acid addition salts and pharma-ceutically acceptable base addition salts.

[0030]

[0044] "Pharmaceutically acceptable acid addition salts" refer to salts which retain the biological effectiveness and properties of the free base, which are not biologically or otherwise undesirable, and which are formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts formed with organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like, including, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Thus, exemplary salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, malate, tartrate, methanesulfonate, and the like. Also contemplated are salts of amino acids such as arginate, gluconate and galacturonate (see, e.g., Berge SM et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66:1-19 (1997)). Acid addition salts of basic compounds are prepared in some embodiments by contacting the free base form with a sufficient amount of the desired acid to produce the salt according to methods and techniques familiar to those of ordinary skill in the art.

[0031]

[0045] "Pharmaceutically acceptable base addition salt" refers to salts which retain the biological effectiveness and properties of the free acids and which are not biologically or otherwise undesirable. These salts are prepared from the addition of an inorganic or organic base to the free acid. Pharmaceutically acceptable base addition salts are formed in some embodiments with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. See Berge et al., supra.

[0032]

[0046] "Pharmaceutically acceptable solvates" refer to compositions of matter that are in a solvent addition form. In some embodiments, solvates contain stoichiometric or non-stoichiometric amounts of a solvent and are formed during the process of preparation with a pharma- ceutical acceptable solvent, such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of the compounds described herein are conveniently prepared or formed during the processes described herein. The compounds provided herein optionally exist in either unsolvated form as well as solvated form.

[0033]

[0047] The term "subject" or "patient" includes mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, guinea pigs, and the like. In one aspect, the mammal is a human.

[0034]

[0048] As used herein, "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms refer to an approach to obtain beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. "Therapeutic benefit" refers to the eradication or amelioration of the underlying disorder being treated. Therapeutic benefit can also be achieved by eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient even though the patient is still suffering from the underlying disorder. With regard to prophylactic benefit, the composition is administered in some embodiments to a patient at risk of developing a particular disease, or to a patient who reports one or more of the physiological symptoms of a disease, even if the diagnosis of this disease has not yet been made. The term "treating" as used herein, unless otherwise indicated, means to reverse, alleviate, inhibit the progression of, or prevent the disorder or disease to which such term applies, or one or more symptoms of such disorder or disease. In certain embodiments, the term "treating" includes slowing or retarding the progression of the disease or disorder to which the term applies. In addition, in certain embodiments, the term "treating" applies to one or more of the complications resulting from the disease or disorder to which the term applies. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating, as "treating" is defined immediately above.

[0035]

[0049] The term "tumor" or "cancer", as used herein, unless otherwise specified, refers to neoplastic cell growth and includes precancerous and cancerous cells and tissues. A tumor usually appears as a lesion or lump. As used herein, "treating" a tumor means that one or more symptoms of the disease, such as the tumor itself, the tumor's angiogenesis, or other parameters by which the disease is characterized, are reduced, alleviated, inhibited, placed in remission, or maintained in remission. "Treating" a tumor also means that one or more characteristics of the tumor may be eliminated, reduced, or prevented by treatment. Non-limiting examples of such characteristics include uncontrolled degradation of the basement membrane and proximal extracellular matrix, migration, division, and organization of endothelial cells into new functioning capillaries, and persistence of such functioning capillaries.

[0036]

[0050] The terms "refractory" or "therapy-resistant" indicate that a patient has never responded to therapy.

[0051] The term "relapsed" or "relapsed after therapy" indicates that a patient, after initially responding to therapy, has progressive disease due to acquired resistance and / or intolerance.

[0037]

[0052] The term "therapy resistance" or "acquired therapy resistance" refers to a patient who, after an initial response to a therapy, has progressive disease due to clinical or molecular resistance to the therapy. Acquired resistance can result from the emergence of resistance mutations in the molecular target of the therapy or in the expression of a physiological function such as an efflux pump.

[0038]

[0053] The phrase "therapeutically effective amount," as used herein, refers to an amount of a drug or pharmaceutical agent that will elicit the biological or medical response in a tissue, system, animal or human that is desired by a researcher, veterinarian, physician, or the like.

[0039]

[0054] The term "tumorigenic" refers to cells that are capable of or tend to form tumors.

[0055] The term "tumorigenic cell population" refers to a population of cells that have neoplastic properties and are capable of forming a tumor.

[0040]

[0056] The term "tumor-initiating cell" or "cancer stem cell" refers to the cell or cells that give rise to the diverse differentiated progeny that make up the bulk of a rapidly dividing tumor.

[0057] Other aspects, advantages and features of the present invention will become apparent from the following detailed description.

[0041] Histone demethylases

[0058] Chromatin is the complex of DNA and proteins that make up chromosomes. Histones are the major protein components of chromatin and act as spools around which DNA is wound. Changes in chromatin structure are influenced by the covalent modification of histone proteins and by non-histone binding proteins. Several classes of enzymes are known that can covalently modify histones at various sites.

[0042]

[0059] Proteins can be post-translationally modified by methylation on the amino group of lysine and the guanidino group of arginine, or carboxymethylated on aspartic acid, glutamic acid, or on the C-terminus of proteins. Post-translational protein methylation has been shown to be involved in various cellular processes, such as RNA processing, receptor-mediated signal transduction, and cell differentiation. Post-translational protein methylation is widely known to occur on histones, and such reactions are known to be catalyzed by histone methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM) to histones. Histone methylation is known to participate in diverse biological processes, including heterochromatin formation, X-chromosome inactivation, and transcriptional regulation (Lachner et al., (2003) J. Cell Sci. 116:2117-2124; Margueron et al., (2005) Curr. Opin. Genet. Dev. 15:163-176).

[0043]

[0060] Unlike acetylation, which generally correlates with transcriptional activation, whether histone methylation leads to transcriptional activation or repression depends on the specific site of methylation and the degree of methylation (e.g., whether a particular histone lysine residue is mono-, di-, or tri-methylated). However, in general, methylation on H3K9, H3K27, and H4K20 is associated with gene silencing, while methylation on H3K4, H3K36, and H3K79 is generally associated with active gene expression. In addition, tri- and di-methylation of H3K4 generally marks the transcription start sites of actively transcribed genes, while mono-methylation of H3K4 is associated with enhancer sequences.

[0044]

[0061] "Demethylase" or "protein demethylase", as referred to herein, refers to an enzyme that removes at least one methyl group from an amino acid side chain. Some demethylases act on histones, for example as histone H3 or H4 demethylases. For example, H3 demethylases can demethylate one or more of H3K4, H3K9, H3K27, H3K36 and / or H3K79. Alternatively, H4 demethylases can demethylate histone H4K20. It is known that demethylases can demethylate either mono-, di- and / or trimethylated substrates. Furthermore, histone demethylases can act on methylated core histone substrates, mononucleosomal substrates, dinucleosomal substrates and / or oligonucleosomal substrates, peptide substrates and / or chromatin (e.g., in cell-based assays).

[0045]

[0062] The first lysine demethylase discovered was lysine-specific demethylase 1 (LSD1 / KDM1), which uses flavin as a cofactor to demethylate both mono- and di-methylated H3K4 or H3K9. A second class of JumonjiC (JmjC) domain-containing histone demethylases was predicted and confirmed when an H3K36 demethylase was discovered using a formaldehyde release assay, which was named JmjC domain-containing histone demethylase 1 (JHDM1 / KDM2A).

[0046]

[0063] Subsequently, many more JmjC domain-containing proteins have been identified, which can be phylogenetically clustered into seven subfamilies: JHDM1, JHDM2, JHDM3, JMJD2, JARID, PHF2 / PHF8, UTX / UTY, and JmjC domain only.

[0047] JMJD2 Family

[0064] The JMJD2 family of proteins is a family of histone demethylases known to demethylate tri- and di-methylated H3-K9 and was the first histone trimethyl demethylase to be identified. Notably, ectopic expression of JMJD2 family members was found to dramatically reduce the levels of tri- and di-methylated H3-K9 while increasing the levels of monomethylated H3-K9, which delocalizes heterochromatin protein 1 (HP1) and globally reduces the levels of heterochromatin in vivo. Members of the JMJD2 subfamily of jumonji proteins include JMJD2C and its homologs JMJD2A, JMJD2B, JMJD2D and JMJD2E. Common structural features found in the JMJD2 subfamily of jumonji proteins include JmjN, JmjC, PHD and Tdr sequences.

[0048]

[0065] JMJD2C, also known as GASC1 and KDM4C, is known to demethylate trimethylated H3K9 and H3K36. Histone demethylation by JMJD2C occurs via an iron- and α-ketoglutarate-dependent hydroxylation reaction, in which oxidative decarboxylation of α-ketoglutarate by JMJD2C produces carbon dioxide, succinate, and ferryl, which subsequently hydroxylates the methyl group of lysine H3K9 and releases formaldehyde. JMJD2C is known to regulate the control of adipogenesis by the nuclear receptor PPARγ and is known to be involved in the control of self-renewal in embryonic stem cells.

[0049]

[0066] KDM4 histone lysine demethylase is an epigenetic regulator and key oncogenic driver across multiple tumor types. The KDM4 family is composed of four major isoforms (KDM4A, B, C, D); all have been shown to be involved in epigenetic dysregulation in various cancers (Zack et al., 2013). KDM4 controls the transition between transcriptionally repressed and active chromatin states by removing methyl marks on histone H3K9 and histone H3K36. KDM4 is also required for embryonic stem cell self-renewal and generation of induced pluripotent stem cells (Das et al. 2014; Kim et al. 2010; Loh et al. 2007; Wang et al. 2010). Overexpression of KDM4 is associated with more aggressive disease and poorer clinical outcomes (Jia et al. 2020; Bur et al. 2016; Soini et al. 2015). Functional redundancy and cross-activity between KDM4 isoforms has been observed, and selective inhibition of one isoform does not appear to be effective (Agger et al. 2016; Pedersen et al. 2016).

[0050] Heterocyclic KDM4 inhibitors

[0067] The heterocyclic KDM4 inhibitors described herein refer to compound 1 having the following structure and chemical name 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid:

[0051] [ka]

[0052] .

[0068] Compound 1 has been previously disclosed in PCT Patent Publication WO 2015 / 200709 and related patent applications and granted patents, such as U.S. Patent No. 9,242,968, which are incorporated by reference in their entirety. Compound 1 is a pan-inhibitor of KDM4 that simultaneously targets multiple isoforms of KDM4. Throughout this disclosure, when reference is made to a heterocyclic KDM4 inhibitor or its pharma-ceutically acceptable salt or solvate, the reference is to Compound 1.

[0053] Cancer and methods of treatment

[0069] In one embodiment, described herein is a method for inhibiting histone demethylase, comprising contacting the enzyme with compound 1 disclosed herein, wherein the histone demethylase comprises KDM4. In certain aspects, disclosed herein is a method for treating cancer in an individual in need thereof, comprising administering to the individual an effective amount of a heterocyclic KDM4 inhibitor described herein. In certain aspects, disclosed herein is a heterocyclic KDM4 inhibitor described herein for use in treating cancer. In certain aspects, disclosed herein is a heterocyclic KDM4 inhibitor described herein for use in preparing a medicament for treating cancer.

[0054]

[0070] One embodiment provides a method of treating cancer in a cancer patient in need thereof, comprising administering to the individual a compound having the following structure, or a pharma- ceutical acceptable salt or solvate thereof:

[0055] [ka]

[0056] Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from colorectal cancer, esophageal cancer, triple negative breast cancer, gastric cancer, lymphoma, gastric adenocarcinoma, diffuse large B-cell non-Hodgkin's lymphoma, acute T-cell leukemia, esophageal squamous cell carcinoma, multiple myeloma, acute myeloid leukemia, colorectal adenocarcinoma, colorectal cancer, pancreatic cancer, pancreatic cancer, breast cancer, or T-cell acute lymphoblastic leukemia.

[0057]

[0071] Another aspect provides a method in which the cancer patient is diagnosed with colorectal cancer. Another aspect provides a method in which the cancer patient is diagnosed with esophageal cancer. Another aspect provides a method in which the cancer patient is diagnosed with triple negative breast cancer. Another aspect provides a method in which the cancer patient is diagnosed with gastric cancer. Another aspect provides a method in which the cancer patient is diagnosed with lymphoma. Another aspect provides a method in which the cancer patient is diagnosed with gastric adenocarcinoma. Another aspect provides a method in which the cancer patient is diagnosed with diffuse large B-cell non-Hodgkin's lymphoma. Another aspect provides a method in which the cancer patient is diagnosed with acute T-cell leukemia. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from esophageal squamous cell carcinoma. Another aspect provides a method in which the cancer patient is diagnosed with multiple myeloma. Another aspect provides a method in which the cancer patient is diagnosed with acute myeloid leukemia. Another aspect provides this method in which the cancer patient is diagnosed with colorectal adenocarcinoma. Another aspect provides a method in which the cancer patient is diagnosed with colorectal cancer. Another embodiment provides a method in which the cancer patient is diagnosed with pancreatic cancer. Another embodiment provides a method in which the cancer patient is diagnosed with pancreatic cancer. Another embodiment provides a method in which the cancer patient is diagnosed with breast cancer. Another embodiment provides a method in which the cancer patient is diagnosed with T-cell acute lymphoblastic leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from lung cancer, small cell lung cancer, non-small cell lung cancer, large cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung carcinoma, large cell lung carcinoma, or bronchoalveolar adenocarcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from acute lymphoblastic B-cell leukemia, mantle cell lymphoma, plasma cell myeloma, diffuse large B-cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, and blast phase chronic myelogenous leukemia.

[0058]

[0072] Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from intestinal cancer, intestinal adenocarcinoma, squamous cell carcinoma of the upper gastrointestinal tract. Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from gastric cancer, gastric signet ring adenocarcinoma, gastric adenocarcinoma, or adenosquamous carcinoma. Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from ovarian cancer, ovarian endometrioid carcinoma, ovarian clear cell carcinoma, ovarian adenocarcinoma, endometrial cancer, endometrial adenocarcinoma, prostate cancer, or prostate adenocarcinoma.

[0059]

[0073] Another embodiment provides a method in which the cancer patient is diagnosed with skin cancer. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from thyroid cancer or follicular thyroid cancer. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from breast cancer or ductal breast cancer. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from liver cancer or hepatocellular carcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from central nervous system cancer, astrocytoma grade IV or gliosarcoma. Another embodiment provides a method in which the cancer patient is diagnosed with bone cancer. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from kidney cancer, clear cell renal cell carcinoma, renal cell carcinoma, urinary tract cancer or urinary tract transitional cell carcinoma. Another embodiment provides a method in which the cancer is recurrent after prior therapy, resistant to prior therapy or acquired resistance to prior therapy.

[0060]

[0074] Another aspect provides a method of reducing a tumorigenic cell population in a cancer patient in need thereof, comprising administering to the individual a compound having the following structure, or a pharma- ceutical acceptable salt or solvate thereof:

[0061] [ka]

[0062] Another embodiment provides a method in which the population of tumorigenic cells is reduced by about 1.5-fold to about 100-fold. Another embodiment provides a method in which the population of tumorigenic cells is reduced by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3.0-fold, about 3.5-fold, about 4.0-fold, about 4.5-fold, about 5.0-fold, about 6.0-fold, about 7.0-fold, about 8.0-fold, about 9.0-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold. Another embodiment provides a method in which the population of tumorigenic cells is reduced by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3.0-fold, about 3.5-fold, about 4.0-fold, about 4.5-fold, or about 5.0-fold. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from colorectal cancer, esophageal cancer, triple-negative breast cancer, gastric cancer, lymphoma, gastric adenocarcinoma, diffuse large B-cell non-Hodgkin's lymphoma, acute T-cell leukemia, esophageal squamous cell carcinoma, multiple myeloma, acute myeloid leukemia, colorectal adenocarcinoma, colorectal cancer, pancreatic cancer, breast cancer, or T-cell acute lymphoblastic leukemia. Another aspect provides a method in which the cancer patient is diagnosed with colorectal cancer. Another aspect provides a method in which the cancer patient is diagnosed with esophageal cancer. Another aspect provides a method in which the cancer patient is diagnosed with triple-negative breast cancer. Another aspect provides a method in which the cancer patient is diagnosed with gastric cancer. Another aspect provides a method in which the cancer patient is diagnosed with lymphoma. Another aspect provides a method in which the cancer patient is diagnosed with gastric adenocarcinoma. Another aspect provides a method in which the cancer patient is diagnosed with diffuse large B-cell non-Hodgkin's lymphoma. Another embodiment provides a method in which the cancer patient is diagnosed with acute T-cell leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with esophageal squamous cell carcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with multiple myeloma. Another embodiment provides a method in which the cancer patient is diagnosed with acute myeloid leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with colorectal adenocarcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with colorectal cancer. Another embodiment provides a method in which the cancer patient is diagnosed with pancreatic cancer. Another embodiment provides a method in which the cancer patient is diagnosed with pancreatic cancer. Another embodiment provides a method in which the cancer patient is diagnosed with breast cancer.Another embodiment provides a method in which the cancer patient is diagnosed with T-cell acute lymphoblastic leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from lung cancer, small cell lung cancer, non-small cell lung cancer, large cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, large cell lung carcinoma, or bronchioloalveolar adenocarcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from acute lymphoblastic B-cell leukemia, mantle cell lymphoma, plasma cell myeloma, diffuse large B-cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, and blast phase chronic myeloid leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from intestinal cancer, intestinal adenocarcinoma, and squamous cell carcinoma of the upper gastrointestinal tract. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from gastric cancer, gastric signet adenocarcinoma, gastric adenocarcinoma, or adenosquamous carcinoma. Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from ovarian cancer, ovarian endometrioid cancer, ovarian clear cell carcinoma, ovarian adenocarcinoma, endometrial cancer, endometrial adenocarcinoma, prostate cancer, or prostate adenocarcinoma.

[0063]

[0075] Another aspect provides a method in which the cancer patient is diagnosed with skin cancer. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from thyroid cancer or follicular thyroid cancer. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from breast cancer or ductal breast cancer. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from liver cancer or hepatocellular carcinoma. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from central nervous system cancer, astrocytoma grade IV or gliosarcoma. Another aspect provides a method in which the cancer patient is diagnosed with bone cancer. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from kidney cancer, clear cell renal cell carcinoma, renal cell carcinoma, urinary tract cancer or urinary tract transitional cell carcinoma. Another aspect provides a method in which the cancer recurred after a prior treatment, is resistant to a prior therapy, or is acquiredly resistant to a prior therapy.

[0064]

[0076] Another aspect provides a method of reducing tumor initiating cell frequency in a cancer patient in need thereof, comprising administering to the individual a compound having the following structure, or a pharma- ceutical acceptable salt or solvate thereof:

[0065] [ka]

[0066] Another embodiment provides a method in which the tumor-initiating cell frequency is reduced by about 1.5-fold to about 100-fold. Another embodiment provides a method in which the tumor-initiating cell frequency is reduced by about 1.5-fold, about 2.0-fold, about 2.5-fold, about 3.0-fold, about 3.5-fold, about 4.0-fold, about 4.5-fold, about 5.0-fold, about 6.0-fold, about 7.0-fold, about 8.0-fold, about 9.0-fold, or about 10-fold. Another embodiment provides a method in which the tumor-initiating cell frequency is reduced to a frequency of about 1 in 100 cells to about 1 in 10,000 cells. Another embodiment is a tumor-initiating cell frequency of about 1 in 100 cells, about 1 in 200 cells, about 1 in 300 cells, about 1 in 400 cells, about 1 in 500 cells, about 1 in 600 cells, about 1 in 700 cells, about 1 in 800 cells, about 1 in 900 cells, about 1 in 1,000 cells, about 1 in 1,200 cells, about 1 in 1,400 cells, about 1 in 1,600 cells, about 1 in 1,800 cells, about 1 in 1,900 cells, about 1 in 1,100 cells, about 1 in 1,200 cells, about 1 in 1,400 cells, about 1 in 1,500 cells, about 1 in 1,600 cells, about 1 in 1,800 cells, about 1 in 1,900 cells, about 1 in 1,100 cells, about 1 in 1,200 cells, about 1 in 1,3 ...100 cells, about 1 in 1,200 cells, about 1 in 1,300 cells, about 1 in 1,400 cells, about 1 in 1,500 cells, about 1 in 1,600 cells, about 1 in 1,100 cells, about In one embodiment, the method reduces the tumor-initiating cell frequency to a frequency of about 1 in 200 cells, about 1 in 300 cells, about 1 in 4000 cells, about 1 in 5000 cells, about 1 in 6000 cells, about 1 in 7000 cells, about 1 in 8000 cells, about 1 in 9000 cells, or about 1 in 10,000 cells. Another embodiment provides a method reduces the tumor-initiating cell frequency to a frequency of about 1 in 200 cells, about 1 in 300 cells, about 1 in 400 cells, about 1 in 500 cells, about 1 in 600 cells, about 1 in 700 cells, about 1 in 800 cells, about 1 in 900 cells, about 1 in 1000 cells, or about 1 in 1200 cells. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from colorectal cancer, esophageal cancer, triple-negative breast cancer, gastric cancer, lymphoma, gastric adenocarcinoma, diffuse large B-cell non-Hodgkin's lymphoma, acute T-cell leukemia, esophageal squamous cell carcinoma, multiple myeloma, acute myeloid leukemia, colorectal adenocarcinoma, colorectal cancer, pancreatic cancer, pancreatic cancer, breast cancer, or T-cell acute lymphoblastic leukemia. Another aspect provides a method in which the cancer patient is diagnosed with colorectal cancer. Another aspect provides a method in which the cancer patient is diagnosed with esophageal cancer. Another aspect provides a method in which the cancer patient is diagnosed with triple-negative breast cancer. Another aspect provides a method in which the cancer patient is diagnosed with gastric cancer. Another aspect provides a method in which the cancer patient is diagnosed with lymphoma. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from gastric adenocarcinoma.Another embodiment provides a method in which the cancer patient is diagnosed with diffuse large B-cell non-Hodgkin's lymphoma. Another embodiment provides a method in which the cancer patient is diagnosed with acute T-cell leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with esophageal squamous cell carcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with multiple myeloma. Another embodiment provides a method in which the cancer patient is diagnosed with acute myeloid leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with colorectal adenocarcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with colorectal cancer. Another embodiment provides a method in which the cancer patient is diagnosed with pancreatic cancer. Another embodiment provides a method in which the cancer patient is diagnosed with pancreatic cancer. Another embodiment provides a method in which the cancer patient is diagnosed with breast cancer. Another embodiment provides a method in which the cancer patient is diagnosed with T-cell acute lymphoblastic leukemia. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from lung cancer, small cell lung cancer, non-small cell lung cancer, large cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, large cell lung carcinoma, or bronchioloalveolar adenocarcinoma. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from acute lymphoblastic B-cell leukemia, mantle cell lymphoma, plasma cell myeloma, diffuse large B-cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, and blast phase chronic myeloid leukemia. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from intestinal cancer, intestinal adenocarcinoma, and squamous cell carcinoma of the upper gastrointestinal tract. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from gastric cancer, gastric signet adenocarcinoma, gastric adenocarcinoma, or adenosquamous carcinoma. Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from ovarian cancer, ovarian endometrioid cancer, ovarian clear cell carcinoma, ovarian adenocarcinoma, endometrial cancer, endometrial adenocarcinoma, prostate cancer, or prostate adenocarcinoma.

[0067]

[0077] Another aspect provides a method in which the cancer patient is diagnosed with skin cancer. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from thyroid cancer or follicular thyroid cancer. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from breast cancer or ductal breast cancer. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from liver cancer or hepatocellular carcinoma. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from central nervous system cancer, astrocytoma grade IV, or gliosarcoma. Another aspect provides a method in which the cancer patient is diagnosed with bone cancer. Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from kidney cancer, clear cell renal cell carcinoma, renal cell carcinoma, urinary tract cancer, or urinary tract transitional cell carcinoma. Another aspect provides a method in which the cancer recurs after a prior therapy, is refractory to a prior therapy, or is acquired resistant to a prior therapy.

[0068]

[0078] Another aspect provides a method of inhibiting cancer stem cells in a cancer patient in need thereof, comprising administering to the individual a compound having the following structure, or a pharma- ceutical acceptable salt or solvate thereof:

[0069] [ka]

[0070] Another aspect provides a method in which the cancer patient is diagnosed with a cancer selected from colorectal cancer, esophageal cancer, triple-negative breast cancer, gastric cancer, lymphoma, gastric adenocarcinoma, diffuse large B-cell non-Hodgkin's lymphoma, acute T-cell leukemia, esophageal squamous cell carcinoma, multiple myeloma, acute myeloid leukemia, colorectal adenocarcinoma, colorectal cancer, pancreatic cancer, breast cancer, or T-cell acute lymphoblastic leukemia. Another aspect provides a method in which the cancer patient is diagnosed with colorectal cancer. Another aspect provides a method in which the cancer patient is diagnosed with esophageal cancer. Another aspect provides a method in which the cancer patient is diagnosed with triple-negative breast cancer. Another aspect provides a method in which the cancer patient is diagnosed with gastric cancer. Another aspect provides a method in which the cancer patient is diagnosed with lymphoma. Another aspect provides a method in which the cancer patient is diagnosed with gastric adenocarcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with diffuse large B-cell non-Hodgkin's lymphoma. Another embodiment provides a method in which the cancer patient is diagnosed with acute T-cell leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with esophageal squamous cell carcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with multiple myeloma. Another embodiment provides a method in which the cancer patient is diagnosed with acute myeloid leukemia. Another embodiment provides a method in which the cancer patient is diagnosed with colorectal adenocarcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with colorectal cancer. Another embodiment provides a method in which the cancer patient is diagnosed with pancreatic cancer. Another embodiment provides a method in which the cancer patient is diagnosed with pancreatic cancer. Another embodiment provides a method in which the cancer patient is diagnosed with breast cancer. Another embodiment provides a method in which the cancer patient is diagnosed with T-cell acute lymphoblastic leukemia. Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from lung cancer, small cell lung cancer, non-small cell lung cancer, large cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, small cell lung carcinoma, large cell lung carcinoma, or bronchioloalveolar adenocarcinoma.Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from acute lymphoblastic B-cell leukemia, mantle cell lymphoma, plasma cell myeloma, diffuse large B-cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, blast phase chronic myelogenous leukemia.

[0071] Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from intestinal cancer, intestinal adenocarcinoma, squamous cell carcinoma of the upper gastrointestinal tract. Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from gastric cancer, gastric signet mark adenocarcinoma, gastric adenocarcinoma, or adenosquamous carcinoma. Another aspect provides a method, wherein the cancer patient has been diagnosed with a cancer selected from ovarian cancer, ovarian endometrioid carcinoma, ovarian clear cell carcinoma, ovarian adenocarcinoma, endometrial cancer, endometrial adenocarcinoma, prostate cancer, or prostate adenocarcinoma.

[0079] Another embodiment provides a method in which the cancer patient is diagnosed with skin cancer. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from thyroid cancer or follicular thyroid cancer. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from breast cancer or ductal carcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from liver cancer or hepatocellular carcinoma. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from central nervous system cancer, astrocytoma grade IV or gliosarcoma. Another embodiment provides a method in which the cancer patient is diagnosed with bone cancer. Another embodiment provides a method in which the cancer patient is diagnosed with a cancer selected from kidney cancer, clear cell renal cell carcinoma, renal cell carcinoma, urinary tract cancer or urinary tract transitional cell carcinoma. Another embodiment provides a method in which the cancer recurred after a prior therapy, is resistant to a prior therapy, or is acquired resistant to a prior therapy.

[0072] Microsatellite instability (MSI-H) status

[0080] Microsatellites (MS), also called short tandem repeats (STRs) or simple sequence repeats (SSRs), consist of repeated sequences of 1–6 nucleotides (see, for example, Garrido-Ramos (2017) Genes 8(9):230). The distribution characteristics of MS are different from tandem repeats of small satellite DNA of 15–65 nucleotides. Depending on the frequency of MSI, it can be differentiated into three types: microsatellite instability-high (MSI-H), microsatellite instability-low (MSI-L) and microsatellite stable (MSS) (see, for example, Li, et al., (2020) Cancer Cell International 20:16). Patients with tumors with high microsatellite instability (MSI-H), due to defects in the mismatch repair (MMR) genes or in the process of replication repair in tumor cells, show higher sensitivity to treatment and may benefit from immunotherapy.

[0073]

[0081] In certain embodiments of the methods provided herein, the methods further comprise determining the microsatellite instability high (MSI-H) status of the cancer patient. In certain embodiments of the methods provided herein, the methods further comprise determining whether the cancer patient is diagnosed with microsatellite instability high (MSI-H).

[0074]

[0082] In certain embodiments of the methods provided herein, the cancer patient has been determined to have microsatellite instability high (MSI-H) status. In certain embodiments of the methods provided herein, the cancer patient has been diagnosed with microsatellite instability high (MSI-H) cancer.

[0075]

[0083] Another embodiment provides a method for treating cancer in a cancer patient in need thereof, comprising: (a) determining the microsatellite instability-high (MSI-H) status of the cancer patient; and (b) administering a therapeutically effective amount of compound 1, or a pharma- ceutically acceptable salt or solvate thereof, to the cancer patient if the cancer patient is determined to be an MSI-H cancer patient. In some embodiments, the cancer patient is diagnosed with colorectal cancer. In some embodiments, the cancer patient is diagnosed with colorectal cancer with MSI-H status.

[0076]

[0084] Another embodiment provides a method of treating a cancer with high microsatellite instability (MSI-H) status in a cancer patient in need thereof, comprising administering to the patient determined to be an MSI-H cancer patient a therapeutically effective amount of Compound 1, or a pharma- ceutical acceptable salt or solvate thereof. In some embodiments, the cancer patient has been diagnosed with colorectal cancer.

[0077]

[0085] Another embodiment provides a method of treating a patient with microsatellite instability-high (MSI-H) cancer, comprising administering a therapeutically effective amount of Compound 1, or a pharma- ceutical acceptable salt or solvate thereof, to a patient determined to be a MSI-H cancer patient. In some embodiments, the cancer patient has been diagnosed with colorectal cancer.

[0078]

[0086] Another embodiment provides a method for reducing tumorigenic cell population in a cancer patient in need thereof, comprising: (a) determining the microsatellite instability-high (MSI-H) status of the cancer patient; and (b) administering to the patient determined to be an MSI-H cancer patient a therapeutically effective amount of Compound 1 or a pharma-ceutically acceptable salt or solvate thereof. In some embodiments, the cancer patient is diagnosed with colorectal cancer. In some embodiments, the cancer patient is diagnosed with colorectal cancer with MSI-H status.

[0079]

[0087] Another embodiment provides a method for reducing tumor-initiating cell frequency in a cancer patient in need thereof, comprising: (a) determining the microsatellite instability-high (MSI-H) status of the cancer patient; and (b) administering a therapeutically effective amount of compound 1 or a pharma- ceutically acceptable salt or solvate thereof to the patient determined to be an MSI-H cancer patient. In some embodiments, the cancer patient is diagnosed with colorectal cancer. In some embodiments, the cancer patient is diagnosed with colorectal cancer with MSI-H status.

[0080]

[0088] Another embodiment provides a method for inhibiting cancer stem cells in a cancer patient in need thereof, comprising: (a) determining the microsatellite instability-high (MSI-H) status of the cancer patient; and (b) administering to the patient determined to be an MSI-H cancer patient a therapeutically effective amount of compound 1 or a pharma-ceutically acceptable salt or solvate thereof. In some embodiments, the cancer patient is diagnosed with colorectal cancer. In some embodiments, the cancer patient is diagnosed with colorectal cancer with MSI-H status.

[0089] Microsatellite instability (MSI-H) status can be detected or determined using methods known to those skilled in the art, including, but not limited to, next-generation sequencing (NGS), fluorescent multiplex polymerase chain reaction (PCR), capillary electrophoresis (CE), immunohistochemistry (IHC), single molecule molecular inversion probes (smMIPs), and MSI calculation (e.g., MANTIS). In one embodiment, the MSI-H status is determined by next-generation sequencing (NGS). In another embodiment, the MSI-H status is determined by fluorescent multiplex PCR. In yet another embodiment, the MSI-H status is determined by capillary electrophoresis (CE). In yet another embodiment, the MSI-H status is determined by immunohistochemistry (IHC). In yet another embodiment, the MSI-H status is determined by single molecule molecular inversion probes (smMIPs). In yet another embodiment, the MSI-H status is determined by MSI calculation.

[0081]

[0090] In certain embodiments, MSI-H has two or more repeat locus microsatellite markers selected from the group consisting of BAT-25, BAT-26, D2S123, D5S346 and D17S250. In certain embodiments, a cancer patient is determined to have MSI-H status when the cancer patient is determined to have two or more repeat locus microsatellite markers selected from the group consisting of BAT-25, BAT-26, D2S123, D5S346 and D17S250.

[0082] Biomarkers and methods of use thereof

[0091] The methods provided herein, in part, involve the observation that a detectable increase or decrease in a particular biomarker upon compound treatment is observed in cancer (e.g., colorectal cancer) cell cultures and organoids that are responsive to a given treatment, e.g., Compound 1, having the following structure, or a pharma- ceutically acceptable salt or solvate thereof:

[0083] [ka]

[0084] And it is based on the observation that the levels of these biomarkers can be used to predict a patient's response to treatment.

[0092] A "biological marker" or "biomarker" is a substance whose change and / or detection is indicative of a particular biological state. In some embodiments, the indication is the responsiveness of a disease, e.g., cancer (e.g., colorectal cancer), to a given treatment (e.g., Compound 1 or a pharma- ceutical acceptable salt or solvate thereof).

[0085]

[0093] As described in the Examples and shown in the Figures, the levels of certain proteins and / or mRNAs change in response to Compound 1. These biomarkers include PNUTS (PPP1R10), ANK1, IBA57, SOWAHD, MF12-AS1 and CECR1. Each of the biomarkers provided herein includes their various isoforms, phosphorylated forms, truncated forms, modified forms and splicing variants. In some embodiments, the levels of the isoforms, phosphorylated forms, truncated forms, modified forms and / or splicing variants of these biomarkers increase or decrease in response to compound treatment, and thus these isoforms, phosphorylated forms, truncated forms, modified forms and / or splicing variants of the biomarkers can be used to predict patient response.

[0086]

[0094] In one aspect, provided herein is a method for identifying a cancer patient having a cancer that is likely to respond to a treatment compound, comprising the steps of: (a) administering a treatment compound to the patient; (b) obtaining a sample from the patient; (c) determining the level of a biomarker in the sample; and (d) diagnosing the patient as likely to respond to the treatment compound if the level of the biomarker in the sample differs from a reference level of the biomarker; wherein the treatment compound is Compound 1 or a pharma- ceutically acceptable salt or solvate thereof. In one embodiment, the biomarker is PNUTS.

[0087]

[0095] In another aspect, provided herein is a method of identifying a cancer patient having a cancer likely to respond to a treatment compound, comprising the steps of: (a) obtaining a sample from the patient; (b) administering a treatment compound to the sample; (c) determining the level of a biomarker in the sample; and (d) diagnosing the patient as likely to respond to the treatment compound if the level of the biomarker in the sample differs from a reference level of the biomarker; wherein the treatment compound is Compound 1 or a pharma- ceutically acceptable salt or solvate thereof. In one embodiment, the biomarker is PNUTS.

[0088]

[0096] In some embodiments of the method provided herein, administering the treatment compound to the sample from a cancer patient is carried out in vitro.In other embodiments, administering the treatment compound to the sample from a patient with cancer is carried out in vivo.In certain embodiments, the sample is a cell.In one embodiment, the cell is contacted with the treatment compound for a period of time, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2, 3, or more days.In other embodiments, the cell is obtained from another cancer patient.

[0089]

[0097] In some embodiments, the level of the biomarker in the patient's sample is higher than the reference level of the biomarker, hi other embodiments, the level of the biomarker in the patient's sample is lower than the reference level of the biomarker.

[0090]

[0098] In yet another aspect, provided herein is a method for predicting the responsiveness of cancer patients to a treatment compound, comprising the steps of: (a) administering a treatment compound to the patient; (b) obtaining a sample from the patient; (c) determining the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1 or CECR1 in the sample; (d) diagnosing the patient as likely to respond to the treatment of cancer with the treatment compound if the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1 or CECR1 in the sample is different from the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1 or CECR1 obtained from reference sample; wherein the treatment compound is compound 1 or its pharma-ceutically acceptable salt or solvate.In one embodiment, the biomarker is PNUTS.

[0091]

[0099] In yet another aspect, provided herein is a method for predicting the responsiveness of a cancer patient to a treatment compound, comprising the steps of: (a) obtaining a sample from the patient; (b) administering a treatment compound to the sample; (c) determining the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1 or CECR1 in the sample; and (d) diagnosing the patient as likely to be responsive to the treatment of cancer with the treatment compound if the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1 or CECR1 in the sample is different from the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1 or CECR1 obtained from a reference sample; wherein the treatment compound is Compound 1 or a pharma-ceutically acceptable salt or solvate thereof.In one embodiment, the level of PNUTS is determined and compared.

[0092]

[0100] In yet another aspect, provided herein is a method for monitoring the effectiveness of a treatment compound for a cancer in a subject, comprising the steps of: (a) administering the treatment compound to the patient; (b) obtaining a sample from the patient; (c) determining the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1, or CECR1 in the sample; (d) diagnosing the patient as likely to respond to treatment of cancer with the treatment compound if the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1, or CECR1 in the sample is different from the level of PNUTS, ANK1, IBA57, SOWAHD, MF12-AS1, or CECR1 obtained from a reference sample, wherein the treatment compound is Compound 1 or a pharma- ceutically acceptable salt or solvate thereof.In an embodiment, a change in the level of a biomarker compared to the reference indicates the effectiveness of the treatment compound in treating cancer in the patient.In one embodiment, an increase in the level of a biomarker indicates the effectiveness of the treatment compound in treating cancer in the patient. In another embodiment, a decrease in the level of the biomarker indicates the effectiveness of the treatment compound in treating cancer in the patient. In one embodiment, the levels of PNUTS are determined and compared.

[0093]

[0101] In certain embodiments, if the cancer patient is diagnosed as likely to be responsive to a treatment compound, the methods provided herein further include administering a therapeutically effective amount of the treatment compound to the patient.

[0094]

[0102] In another aspect, provided herein is a method comprising the steps of: (a) obtaining a sample from a patient having cancer; (b) determining the level of a biomarker in the sample; (c) diagnosing the patient as likely to respond to a treatment compound if the level of the biomarker in the sample differs from a reference level of the biomarker; and (d) administering a therapeutically effective amount of a treatment compound to the patient; wherein the treatment compound is Compound 1 or a pharma- ceutical acceptable salt or solvate thereof.

[0095]

[0103] In some embodiments, the level of the biomarker in the patient's sample is higher than the reference level of the biomarker, hi other embodiments, the level of the biomarker in the patient's sample is lower than the reference level of the biomarker.

[0096]

[0104] In some embodiments of the various methods provided herein, the treatment compound is administered to a patient who is likely to respond to the treatment compound.Also provided herein are methods for treating patients who have been previously treated for cancer but are non-responsive to standard therapy, as well as patients who have not been previously treated.The present invention also includes methods for treating patients regardless of the age of the patient, although some diseases or disorders are more prevalent in certain age groups.The present invention further includes methods for treating patients who have undergone surgery to treat the disease or illness in question, as well as patients who have not.Since patients with cancer have heterogeneous clinical symptoms and various clinical outcomes, the treatment given to a patient may vary depending on his / her prognosis.A skilled clinician will be able to easily determine the specific secondary agent, type of surgery, and type of non-drug-based standard therapy that can be effectively used to treat an individual patient with cancer without undue experimentation.

[0097]

[0105] As shown in the Examples, the levels of certain biomarkers, such as PNUTS (PPP1R10), ANK1, IBA57, SOWAHD, MF12-AS1 and CECR1, change in response to Compound 1 treatment. Thus, in some embodiments, the biomarker is selected from the group consisting of PNUTS (PPP1R10), ANK1, IBA57, SOWAHD, MF12-AS1 and CECR1. In one embodiment, the biomarker is PNUTS (PPP1R10). In another embodiment, the biomarker is ANK1. In yet another embodiment, the biomarker is IBA57. In yet another embodiment, the biomarker is SOWAHD. In yet another embodiment, the biomarker is MF12-AS1. In yet another embodiment, the biomarker is CECR1.

[0098]

[0106] In some embodiments of the various methods provided herein, the biomarker is PNUTS, ANK1 or IBA57, wherein the level of PNUTS, ANK1 or IBA57 is decreased compared to the reference in response to Compound 1 treatment. In some embodiments of the various methods provided herein, the biomarker is SOWAHD, MF12-AS1 or CECR1, wherein the level of SOWAHD, MF12-AS1 or CECR1 is increased compared to the reference in response to Compound 1 treatment.

[0099]

[0107] In certain embodiments, various methods provided herein use a sample (e.g., a biological sample) from a subject or individual (e.g., a patient). The patient can be a patient, such as a patient with cancer (e.g., colorectal cancer). The patient can be male or female, and can be an adult, child, or infant. The sample can be analyzed during the active stage of the cancer (e.g., colorectal cancer) or when the cancer (e.g., colorectal cancer) is inactive. In certain embodiments, more than one sample can be obtained from a patient.

[0108] In some embodiments, the sample used in the method comprises biopsy (e.g., tumor biopsy).Biopsy can be from any organ or tissue, such as skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph node, hair, spleen, brain, breast or other organ.Any biopsy technique known to those skilled in the art, such as open biopsy, closed biopsy, core biopsy, incision biopsy, excision biopsy or fine needle aspiration biopsy, can be used to isolate sample from subject.

[0100]

[0109] In one embodiment, the sample used in the methods provided herein is obtained from a patient before the patient undergoes treatment for disease or disorder.In another embodiment, the sample is obtained from a patient while the patient undergoes treatment for disease or disorder.In another embodiment, the sample is obtained from a patient after the patient undergoes treatment for disease or disorder.In various embodiments, the treatment comprises administering Compound 1 to the patient.

[0101]

[0110] In certain embodiments, the sample used in the methods provided herein comprises a plurality of cells, such as cancer (e.g., colorectal cancer) cells. Such cells can include any type of cell, such as stem cells, blood cells (e.g., peripheral blood mononuclear cells (PBMCs)), lymphocytes, B cells, T cells, monocytes, granulocytes, immune cells, or cancer cells.

[0102] Pharmaceutical Compositions

[0111] In certain embodiments, the heterocyclic KDM4 inhibitors described herein are administered as pure chemicals.In other embodiments, the heterocyclic KDM4 inhibitors described herein are combined with a pharma- ceutically suitable or acceptable carrier (also referred to herein as a pharma- ceutically suitable or acceptable excipient, a physiologically suitable or acceptable excipient, or a physiologically suitable or acceptable carrier), which is selected based on the selected route of administration and standard pharmaceutical practice.

[0103]

[0112] Provided herein is a pharmaceutical composition comprising the heterocyclic KDM4 inhibitor described herein or its stereoisomer, pharma- ceutically acceptable salt, hydrate or solvate together with one or more pharma- ceutical acceptable carriers.Carrier(s) (or excipient(s)) is acceptable or suitable if it is compatible with other components of the composition and is not harmful to the recipient (i.e., subject or patient) of the composition.

[0104]

[0113] One embodiment provides a method for preparing a pharmaceutical composition comprising mixing a heterocyclic KDM4 inhibitor described herein or a stereoisomer, a pharma- ceutically acceptable salt, hydrate or solvate thereof, and a pharma- ceutically acceptable carrier.

[0105]

[0114] Provided herein is a method for orally administering pharmaceutical compositions.Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose, or other suitable materials that dissolve easily in the digestive tract.In some embodiments, suitable non-toxic solid carriers are used, including, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. (see, for example, Remington: The Science and Practice of Pharmacy (Gennaro, 21st Edition Mack Pub. Co., Easton, PA (2005)).

[0106]

[0115] Provided herein is a method in which pharmaceutical composition is administered by injection.In some embodiments, the heterocyclic KDM4 inhibitor described herein or its pharma-ceutically acceptable salt or solvate is formulated for administration by injection.In some cases, the injection formulation is an aqueous formulation.In some cases, the injection formulation is a non-aqueous formulation.In some cases, the injection formulation is an oil-based formulation, such as sesame oil.

[0107]

[0116] The dosage of the composition comprising the heterocyclic KDM4 inhibitors described herein, or a stereoisomer, pharma- ceutically acceptable salt, hydrate or solvate thereof, varies depending on the condition of the subject or patient (e.g., human). In some embodiments, such factors include general health, age and other factors. The pharmaceutical composition is administered in a manner appropriate to the disease to be treated (or prevented). The appropriate dosage and the appropriate duration and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, the specific form of the active ingredient and the method of administration. In general, an appropriate dosage and treatment regimen provides a sufficient amount of the composition(s) to provide a therapeutic and / or prophylactic benefit (e.g., improved clinical outcomes such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or reduced symptom severity). The optimal dosage is generally determined using experimental models and / or clinical trials. The optimal dosage depends on the patient's body weight, weight or blood volume.

[0108] Working Example EXAMPLES

[0109]

[0117] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. background

[0118] The KDM4 family of histone lysine demethylases consists of four major isoforms (KDM4A, B, C, D), all of which have been identified as important oncogenic drivers. They function as epigenetic regulators, controlling the transition between transcriptionally repressed and active chromatin states via removal of methyl marks on histone H3K9 and histone H3K36. KDM4 isoforms play a key role in epigenetic dysregulation in various cancers and are associated with more aggressive disease and poorer clinical outcomes. Functional redundancy and cross-activity have been observed across KDM4 family members, and selective inhibition of one isoform does not appear to be effective. Compound 1 is a pan-inhibitor of KDM4 that simultaneously targets multiple isoforms of KDM4.

[0110] Example 1: KDM4 inhibitory activity of compound 1

[0119] Figure 1 illustrates the biochemical activity of Compound 1, a potent pan-KDM4 inhibitor. Figure 2 illustrates the reversible and competitive inhibition of H3K9me3 demethylation. Figure 3 illustrates that Compound 1 induces cell cycle arrest in S phase. Figure 4 demonstrates that Compound 1 induces apoptosis in human cancer cell lines. See also PCT Patent Publication WO 2015 / 200709 and related patent applications and granted patents, such as U.S. Patent No. 9,242,968, which are incorporated by reference in their entirety.

[0111] Example 2a: Compound 1 inhibits cell proliferation in cell culture and organoid models

[0120] Compound 1 was evaluated in in vitro cell proliferation assays (BrdU assay, MTS assay, or Cell Titer Glo (CTG) assay) in multiple cancer cell lines and patient-derived organoid models to determine the half-maximal inhibitory concentration (IC50).

[0112]

[0121] Viability and BrdU thymidine incorporation assays were performed in multiwell plates seeded with cells in growth medium. After seeding, the multiwell plates were cultured for 24 hours in a humidified incubator at 37°C to promote adhesion. The assay was initiated in individual test wells by adding either DMSO as a negative control or ≥100xIC50 concentration of the inhibitor panobinostat or serially diluted compound 1 as a positive control. Cultures were incubated for 168 hours, after which the number of viable cells or the amount of thymidine incorporation in each test well was assessed by the methods indicated above. Readouts were performed using an EnVision® multilabel reader (PerkinElmer, Waltham, MA) and results were expressed as a percentage of the negative control. The percentage of control values ​​were plotted against the corresponding compound 1 concentrations, and relative IC50 values ​​were determined from the nonlinear regression curve as the concentration at which inhibition was half maximal. For cell lines with more than one IC50 determination, IC50 was expressed as the mean IC50 ± SD.

[0113]

[0122] Half-maximal inhibitory concentration (IC50) values ​​for compound 1 inhibition of cell proliferation were determined for nine cancer cell lines and one normal human fibroblast cell line. As shown in Table 1, except for IMR-90, a normal human fibroblast cell line, and a human breast cancer line, ZR75-1, compound 1 treatment for 168 hours showed potent antiproliferative activity across the panel, which included both solid and hematological cancer cell lines. Compound 1 showed potent antiproliferative activity (<0.040 μM) for eight solid and hematological cancer cell lines: Jurkat, MDA-MB-231, KYSE-150, MM.1s, HL-60, HT-29, MCF-7, and Loucy.

[0114] [Table 1]

[0115]

[0123] IC of Compound 1 for inhibition of colony formation and viability 50Values ​​were determined for patient-derived colon cancer organoid models: SU60, T002C, SU62, SU34, SU103, T035C and SU106, patient-derived pancreatic cancer organoid models PA165F, PA0143F, T016P and T028P, and patient-derived breast cancer organoid model FS53.

[0116]

[0124] Colony formation and viability assays were performed in organoid-seeded multiwell plates using an in vitro culture system consisting of a Matrigel layer, medium containing a defined set of growth factors, and co-culture with Wnt3A-secreting mouse embryonic fibroblasts. The assay was initiated by adding either DMSO as a negative control or ≧100×IC50 concentration of the inhibitor panobinostat or serially diluted Compound 1 as a positive control. Cultures were incubated for 168 or 144 hours, after which the number of viable organoids in each test well was assessed using one of two detection methods: staining with CTG or calcein-AM, followed by imaging and colony counting, respectively. Colony number or viability of cells treated with Compound 1 was expressed as a percentage of the negative control. Aggregated percentages of control values ​​were plotted against the corresponding Compound 1 concentrations, and absolute IC50 values ​​were determined from the nonlinear regression curve as the concentration at which inhibition was 50% of the control.

[0117]

[0125] As shown in Table 2, compound 1 treatment showed submicromolar antiproliferative activity for colorectal cancer models SU60, T002C, and SU62, resulting in IC50 values ​​<0.15M. Compound 1 treatment also showed potent antiproliferative activity for PA165F pancreatic cancer model, resulting in IC50 values ​​<0.03M. SU34, SU103, T035C, and SU106 colorectal cancer models and pancreatic cancer models PA143F, T016P, and T028P were unresponsive to compound 1 in these colony formation and viability assays, with IC50 values ​​>10M. An IC50 value of 13.6M was determined for the FS53 human breast cancer model after compound 1 treatment. A representative titration curve for data aggregated across the two SU60 assays was constructed and shown as FIG. 9.

[0118] [Table 2]

[0119]

[0126] As shown in Figure 9, percent of control = colony number of compound 1 treated organoids normalized to negative control colony number (expressed as a percentage); data of combined curves from two SU60 experiments; error bars are ± SD for two independent experiments. Colony formation inhibition assays were performed in multi-well plates seeded with SU60 organoids. Cultures were incubated for 168 hours and then stained with calcein AM, followed by assessment of colony number by imaging and colony counting. Colony numbers were expressed as a percentage of the negative control, and the percentage of tabulated control values ​​was plotted against the corresponding compound 1 concentration. Curves were fitted using nonlinear regression.

[0120] Example 2b: Evaluation of Compound 1 in a panel of cancer cell lines

[0127] In a larger screening study, compound 1 was evaluated across an extensive cancer cell line panel (OncoPanel; HD Biosciences) consisting of 301 cancer cell lines from different tumor types.

[0121]

[0128] 2D cell viability inhibition assays were performed in multi-well plates seeded with cells from the panel. After seeding, plates were cultured overnight in a humidified incubator at 37°C to promote adhesion. The assay was initiated in individual wells by adding either DMSO as vehicle control, growth medium as blank or serially diluted Compound 1 (10 μM to 0.0005 μM in 1:3 serial dilutions). Cultures were incubated for 168 hours, after which the number of viable cells in each test well was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. Luminescent readouts were performed using an EnVision® multilabel reader, and Compound 1 readouts were normalized to the DMSO control readout and expressed as a percentage of control. Outliers were flagged out by visual inspection. Percentage of control was plotted against the corresponding Compound 1 concentration, and absolute IC50 values ​​were determined as the concentration at which inhibition was 50% of control using a four-parameter logistic nonlinear regression. For maximum inhibition <50%, absolute IC50 was reported as >10. In addition to EC50 and IC50, AUC was determined and normalized to the area corresponding to theoretical no inhibition (rectangular area defined by compound dose range, and 0-100 in y). Emax was recorded as the minimum value of y over the compound dose range. For cell lines with Emax>40, EC50 was manually set to 10 μM. Cellular potency data for compound 1 was visualized using a hierarchical clustering algorithm based on Euclidean distance between normalized input values ​​and the “Ward.D” method implemented in the R hclust function. Sensitive / Intermediate / Resistant calls for each cell line were determined by visual inspection of the resulting clustered dendrogram. All analyses were performed in the R Core Team.

[0122]

[0129] The results showed that compound 1 potently inhibited the proliferation of cancer cell lines, with absolute IC values ​​ranging from <500 pM to >10 μM. 50 Of the 301 strains, 209 were inhibited by compound 1 with an IC value of less than 1 μM. 50values, 89 strains had IC values ​​greater than 10 μM 50 Table 3 provides the results for all 301 cell lines.

[0123] [Table 3-1]

[0124] [Table 3-2]

[0125] [Table 3-3]

[0126] [Table 3-4]

[0127] [Table 3-5]

[0128] [Table 3-6]

[0129] [Table 3-7]

[0130] [Table 3-8]

[0131] [Table 3-9]

[0132] Example 3: Determination of antiproliferative activity in a xenograft model The therapeutic in vivo efficacy of compound 1 was evaluated in SU60 colorectal cancer, KYSE-150 esophageal cancer, COH70 triple-negative breast cancer, GXA-3036 gastric adenocarcinoma and OCI-LY19 diffuse large B-cell non-Hodgkin's lymphoma xenograft models.In all studies, efficacy was determined based on the statistical evaluation of the percent tumor growth inhibition (TGI), the difference in mean tumor growth and the difference in net tumor volume distribution on the day of TGI analysis between control animals and treated animals.Tolerability was evaluated based on the difference in mean body weight between control animals and treated animals over the course of the study.

[0133] A. In vivo efficacy of Compound 1 in the SU60 patient-derived colorectal cancer xenograft model The in vivo therapeutic efficacy and tolerability of compound 1 orally administered as free acid form in a vehicle composed of 50% polyethylene glycol 400 (PEG 400) and 50% PBS (pH 9) was evaluated in an SU60 CRC PDX model established in female non-obese diabetic / severe combined immunodeficiency (NOD / SCID) gamma (NSG) mice. Each mouse was subcutaneously inoculated with 2×106 SU60 tumor cells (0.1 ml) suspended in Matrigel into the peritoneal cavity. When the average tumor size reached approximately 270 mm3, the test animals were randomized into 6 groups of 8 mice per group and treatment was initiated. Control mice received 50% PEG 400 and 50% PBS (pH 9) vehicle PO QD ×7. Treated mice received Compound 1 in vehicle PO at 20 or 10 mg / kg QD x 7, 20 mg / kg QOD, 23 mg / kg 3 on / 4 off, or 14 mg / kg 5 on / 2 off. TGI was determined on day 19 as (1-Diff treated / Diff control) x 100. Differences in mean net tumor volume on the day of TGI analysis for treated versus control animals were statistically evaluated using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. Adjusted p-values ​​are shown, where a calculated probability (p) ≦ 0.05 was considered statistically significant. Individual tumors were measured in two dimensions twice weekly using calipers, and tumor volume (TV) in mm3 was calculated using the formula: TV=0.5(l×w×h), where l, w, and h are tumor length, width, and height in mm, respectively; mean tumor volume ± standard error of the mean (SEM) was calculated and plotted against days of treatment (Figure 10, left panel). Body weight was measured twice weekly. Body weight ± SEM was calculated and plotted against days of treatment (Figure 10, right panel).

[0134] As shown in Table 4, all five compound 1 regimens were significantly effective (p<0.005) based on statistical evaluation of the difference in mean net tumor volume on the day of TGI analysis for treated versus control animals. Compound 1 at 20 or 10 mg / kg administered once daily for 7 days (QD×7) resulted in dose-dependent TGI of 71% and 48%. To identify the optimal intermittent dosing schedule, four compound 1 dosing regimens were tested at similar weekly dose levels (69-70 mg / kg / week): QD×7, once every 2 days (QOD), once daily for 3 days followed by no dosing for 4 days repeated until the end of the study (3 on / 4 off), and 5 on / 2 off. The 3 on / 4 off and 5 on / 2 off intermittent schedules resulted in similar tumor growth responses as QD×7 (52% and 42% vs. 48% TGI). Compound 1 at 20 mg / kg administered QOD was the least effective regimen resulting in a TGI of 29%.

[0135] As shown in Figure 10 (left panel), tumor growth in compound 1 treatment group was reduced compared to control animals. The relative rates of tumor growth between various compound 1 treatment groups were consistent with the TGI achieved at the end of the study. As shown in Figure 10 (right panel), compound 1 appeared to be well tolerated with nominal mean body weight change over the course of the study.

[0136] [Table 4]

[0137] B. In vivo efficacy of Compound 1 in the KYSE-150 human esophageal cancer cell line xenograft model Test No. 1 - The in vivo therapeutic efficacy and tolerability of compound 1 orally administered as a lysine salt suspended in a vehicle composed of 10% PEG400 and 90% 0.5% methylcellulose (MC) was evaluated in the established KYSE-150 ESCC CDX model in female NSG mice. As shown in Table 5, compound 4 at 5, 15, or 50 mg / kg (free acid equivalent) administered on a 3 on / 4 off schedule resulted in a dose-dependent TGI of 19%, 50%, and 72%, respectively. The difference in mean net tumor volume on the day of TGI analysis for treated versus control animals was significant for compound 1 at 15 and 50 mg / kg (p<0.001), but not at 5 mg / kg (p>0.05). As shown in Figure 11 (left panel), tumor growth in compound 1-treated groups was reduced compared to control animals. The relative rates of tumor growth among the various Compound 1 treatment groups were consistent with the TGI achieved at the end of the study. As shown in Figure 11 (right panel), Compound 1 appeared to be well tolerated with nominal mean weight gain over the course of the study. Each mouse was subcutaneously inoculated with 1x107 KYSE-150 tumor cells (0.1 ml) suspended in PBS on the right flank. When the mean tumor size reached approximately 117 mm3, the test animals were randomized into four groups of nine mice per group and treatment was initiated. Control mice received 10% PEG 400 and 90% 0.5% MC vehicle PO 3 on / 4 off. Treated mice received the lysine salt form of Compound 1 suspended in vehicle at 5, 15, or 50 mg / kg on the same schedule. TGI was determined on day 19 as (1-Diff treated / Diff control) x 100; differences in mean net tumor volume on the day of TGI analysis for treated versus control animals were statistically evaluated using one-way ANOVA followed by Dunnett's multiple comparison test; adjusted p-values ​​are shown, where a calculated probability (p) ≦ 0.05 was considered statistically significant. Individual tumors were measured twice weekly in three dimensions with calipers and tumor volume (TV) in mm3 was calculated using the formula: TV = 0.5 (l x w x h), where l, w and h are the length, width and height of the tumor, respectively, in mm; mean tumor volume ± SEM was calculated and plotted against days of treatment (left panel).Body weight was measured twice weekly and body weight ± SEM was calculated and plotted against days of treatment (right panel).

[0138] [Table 5]

[0139] Study #2 - The in vivo therapeutic efficacy and tolerability of compound 1 orally administered as a lysine salt suspended in a vehicle composed of 10% PEG400 and 90% 0.5% MC was evaluated in the established KYSE-150 ESCC CDX model in female NOD / SCID mice. Each mouse was subcutaneously inoculated with 1x107 KYSE-150 tumor cells (0.1 ml) suspended in 50% Matrigel on the right flank. When the average tumor size reached approximately 153 mm3, the test animals were randomized into 4 groups of 8 mice per group and treatment was initiated. Control mice received 10% PEG 400 and 90% 0.5% MC vehicle PO QDx21. Treated mice received the lysine salt form of compound 1 suspended in vehicle at 10, 15, or 20 mg / kg on the same schedule. TGI was determined on day 21 as (1-Diff treatment / Diff control) x 100. Individual tumors were measured in two dimensions twice weekly with calipers and tumor volume (TV) in mm3 was calculated using the formula: TV = 0.5a x b2, where a and b are the long and short tumor diameters in mm, respectively; mean tumor volume ± SEM was calculated and plotted against days of treatment (Figure 12, left panel). Body weight was measured twice weekly. Body weight ± SEM was calculated and plotted against days of treatment (Figure 12, right panel).

[0140] The difference in mean net tumor volume on the day of TGI analysis for treated versus control animals was statistically evaluated using one-way ANOVA followed by Dunnett's multiple comparison test.Adjusted p-values ​​are shown, where a calculated probability (p)≦0.05 is considered statistically significant.

[0141] As shown in Table 6, Compound 1 administered at 10, 15 or 20 mg / kg (free acid equivalent) on a QD×21 schedule resulted in dose-dependent TGI of 54%, 76% and 84%, respectively. The difference in mean net tumor volume on the day of TGI analysis for treated versus control animals was significant (p<0.02).

[0142] As shown in Figure 12 (left panel), tumor growth in compound 1 treatment group is reduced compared to control animals.The relative rate of tumor growth between various compound 1 treatment groups is consistent with the TGI achieved at the end of the study.As shown in Figure 12 (right panel), compound 1 treatment animals do not experience the weight gain shown by control animals, but appear to be well tolerated with nominal average weight change over the course of the study.

[0143] [Table 6]

[0144] C. In vivo efficacy of Compound 1 in the COH70 patient-derived triple-negative breast cancer xenograft model The in vivo therapeutic efficacy and tolerability of compound 1 orally administered as free acid form in PBS vehicle + 2 equivalents NaOH (final pH 10) was evaluated in a dose-ranging study using the COH70 TNBC PDX model established in female NSG mice. Each mouse was subcutaneously inoculated with 2x106 COH70 tumor cells (0.2ml) suspended in Matrigel into the peritoneal cavity. When the average tumor size reached ~107mm3, the test animals were randomized into 5 groups of 9 mice per group and treatment was initiated. Control mice received PBS (pH 10) vehicle PO QD x 36. Treated mice received compound 1 (dissolved in PBS + 2 equivalents NaOH, final pH 10) PO at 50, 40, 25 or 12.5mg / kg QD x 36. TGI was determined on day 36 as (1-Diff treated / Diff control) x 100. Individual tumors were measured twice weekly in two dimensions with calipers, and tumor volumes (TV) (in mm3) were calculated using the formula: TV=0.5(l×w×h), where l, w, and h are tumor length, width, and height (mm), respectively. Mean tumor volumes ± SEM were calculated and plotted against days of treatment (left panel). Body weights were measured twice weekly. Body weights ± SEM were calculated and plotted against days of treatment (right panel).

[0145]

[0140] Differences in the distribution of net tumor volumes for treated versus control animals on the day of TGI analysis were statistically evaluated using one-way ANOVA followed by Dunnett's multiple comparison test; adjusted p-values ​​are shown, where a calculated probability (p) ≦ 0.05 was considered statistically significant; these are tests of statistical significance and do not provide an estimate of the magnitude of differences between groups, nor are they a measure of clinical or biological significance.

[0146] As shown in Table 7, Compound 1 at 12.5, 20, 40, or 50 mg / kg administered on a QD×36 schedule was significantly effective (p<0.0001) based on the difference in mean net tumor volume on the day of TGI analysis for treated versus control animals, resulting in a TGI of 57%, 68%, 86%, and 86%, respectively. Compound 1 produced a dose-dependent inhibition of tumor growth that reached a plateau at doses of ≧40 mg / kg.

[0147] As shown in Figure 13 (left panel), tumor growth in the compound 1 treatment group was reduced compared to control animals. The relative rates of tumor growth between the various compound 1 treatment groups were consistent with the TGI achieved at the end of the study. As shown in Figure 13 (right panel), compound 1 appeared to be well tolerated with nominal dose-dependent mean body weight changes (≦6.5% mean body weight loss) over the course of the study.

[0148] [Table 7]

[0149] D. In vivo efficacy of Compound 1 in the GXA-3036 gastric adenocarcinoma patient-derived xenograft model The in vivo efficacy and tolerability of compound 1 administered orally as a lysine salt form suspended in a vehicle composed of 5% 2-hydroxypropyl-beta-cyclodextrin (HPBCD) in 50 mM phosphate buffer (pH 7.4) was evaluated in female immunodeficient NMRI-Foxn1nu mice using a subcutaneous gastric adenocarcinoma GXA-3036, a moderately differentiated intestinal type (based on Lauren's criteria) PDX model of Asian origin. Each mouse was inoculated subcutaneously in the right flank with a GXA-3036 tumor fragment (edge ​​length of 3-4 mm). When the average tumor size reached approximately 109 mm3, the test animals were randomized into 5 groups of 8 mice per group and treatment was initiated. Control mice received 5% HPBCD in 50 mM phosphate buffer (pH 7.4) vehicle PO 3 on / 4 off. Treated mice received Compound 1 lysine salt form at 5, 15, or 50 mg / kg in vehicle on the same schedule, or 22.5 mg / kg 2 on / 5 off. One control animal was classified as moribund, euthanized on day 17, and censored from analysis. Individual tumors were measured twice weekly in two dimensions with calipers, and tumor volume (TV) in mm3 was calculated using the formula: TV=0.5a×b2, where a and b are the major and minor diameters in mm, respectively. Mean tumor volume ± SEM was calculated and plotted against days of dosing (Figure 14, left panel). Body weight was measured twice weekly. Body weight ± SEM was calculated and plotted against days of dosing (Figure 14, right panel).

[0150]

[0144] TGI was determined as (1-Diff treated / Diff control) x 100 on days 35 and 38. Differences in median net tumor volumes on TGI analysis days for treated versus control animals were statistically evaluated using the non-parametric Kruskal-Wallis test followed by Dunn's multiple comparison test. The adjusted p-values ​​reported are for both days of TGI analysis where a calculated probability (p) ≦ 0.05 was considered statistically significant.

[0151] As shown in Table 8, compound 1 at 5, 15 or 50 mg / kg (free acid equivalent) administered on a 3 on / 4 off schedule resulted in TGIs of 52%, 71% and 55% on day 35 and 43%, 69% and 52% on day 38, respectively. The lower TGI shown by the 5 mg / kg compound 1 group at D38 was due to a moderate increase in tumor volume for a single animal, which shifted the median. Compound 1 at 22.5 mg / kg (free acid equivalent) administered on a 2 on / 5 off schedule resulted in TGIs of 42% and 41% on days 35 and 38, respectively. The difference in median net tumor volume on both days of TGI analysis for treated versus control animals was significant for compound 1 at 15 mg / kg (p=0.0007), but not for other treatment regimens (p>0.05).

[0152] As shown in Figure 14 (left panel), tumor growth in compound 1 treatment groups was reduced compared to control animals. The relative rates of tumor growth between the various compound 1 treatment groups were consistent with the TGI achieved at the end of the study. As shown in Figure 14 (right panel), compound 1 appeared to be well tolerated with little change in mean body weight over the course of the study.

[0153] [Table 8]

[0154] E. In vivo efficacy of Compound 1 in the OCI-LY19 human diffuse large B-cell non-Hodgkin's lymphoma xenograft model The in vivo efficacy and tolerability of compound 1 administered orally as a lysine salt form suspended in 0.5% MC vehicle was evaluated in female CB-17 severe combined immunodeficiency (scid) mice subcutaneously inoculated with OCI-LY19 tumor cells established from the bone marrow of a female patient with diffuse large B-cell non-Hodgkin's lymphoma (DLBCL) stage 4B at relapse. Each mouse was subcutaneously inoculated with 5x106 OCI-LY19 tumor cells (0.1 ml) suspended in 50% Matrigel in the right flank. When the average tumor size reached approximately 165 mm3, the test animals were randomized into 6 groups of 8 mice per group and treatment was initiated. Test control mice received 0.5% MC vehicle PO QD 3 on / 4 off, and positive control mice received CHOP therapy QD x 5. Mice treated with Compound 1 received the lysine salt form of Compound 1 suspended in vehicle at 5, 15 or 50 mg / kg QD or 25 mg / kg BID on a 3 on / 4 off schedule. TGI was determined on day 21 as (1-Diff treatment / Diff control) x 100. Individual tumors were measured in two dimensions with calipers on the indicated days, and tumor volume (TV) in mm3 was calculated using the formula: TV = 0.5a x b2, where a and b are the long and short tumor diameters (mm), respectively. Mean tumor volume ± SEM was calculated and plotted against days of dosing (Figure 15, left panel). Body weight was measured twice weekly. Body weight ± SEM was calculated and plotted against days of dosing (Figure 15, right panel). Differences in mean net tumor volumes on the day of TGI analysis for treated versus control animals were statistically evaluated using one-way ANOVA followed by Dunnett's multiple comparison test; adjusted p values ​​are shown, where a calculated probability (p) ≦ 0.05 was considered statistically significant.

[0155] As shown in Table 9, Compound 1 administered at 5, 15, or 50 mg / kg QD or 25 mg / kg (free acid equivalent) twice daily (BID) on a 3 on / 4 off schedule resulted in dose-dependent TGIs of 55%, 83%, 90%, and 102%, respectively, on day 21. As shown in Figure 15, the difference in mean net tumor volume on the day of TGI analysis for Compound 1-treated versus control animals was significant (p<0.0001).

[0156] The gold standard first-line chemotherapy regimen CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) recommended in the treatment of non-Hodgkin's lymphoma was included as a positive control. CHOP administered QD×5 resulted in a TGI of 108% on day 21 and appeared to be curative in 7 of 8 test animals that showed no measurable tumor volume for 8 or more consecutive days.

[0157] As shown in Figure 15 (left panel), tumor growth in compound 1 treatment groups was reduced compared to control animals. The relative rates of tumor growth between the various compound 1 treatment groups were consistent with the TGI achieved at the end of the study. As shown in Figure 15 (right panel), compound 1 appeared to be well tolerated, with little change in mean body weight over the course of the study.

[0158] [Table 9]

[0159] Example 4: Determination of Cancer Stem Cell Characteristics Following Treatment with Compound 1

[0151] The ability of Compound 1 to affect tumorigenicity was analyzed by flow cytometric analysis of tumorigenic cell populations and by functional in vivo tumorigenicity assays (limiting dilution assays).

[0160] To evaluate the effect of compound 1 on tumorigenic cell population, compound 1 was used to treat SU60 xenograft tumors. Compound 1 was administered at 40mg / kg PO QD×21 to NSG female mice (7 mice per group) implanted with SU60 cells. The day after the last administration, the three tumors closest to the mean size from each group were dissociated and single cells were used to perform: 1) flow cytometry analysis of tumorigenic cell population, and 2) functional in vivo tumorigenicity assay (limiting dilution assay).

[0161] Flow cytometry analysis of tumorigenic cell populations following compound 1 treatment Tumorigenic CD44 in cell populations High ;EpCAM + The proportion of tumorigenic cells was quantified by flow cytometry. Compound 1 significantly reduced the number of tumorigenic cells (CD44 High ;EpCAM + ) significantly reduced the rate of IL-1 mediated ...

[0162] Functional in vivo tumorigenicity assay following compound 1 treatment To study the effect of Compound 1 on tumor-initiating cell (TIC) frequency in CRC tumors, we evaluated the tumorigenicity of SU60 xenograft cells after treatment with Compound 1 or vehicle. At the end of administration, SU60 dissociated cells from 3 animals per group that are closest to the average size were injected into recipient NSG mice in a limiting dilution format, and tumor volume was measured. The number of cells required for tumor formation was reduced in the Compound 1 treatment group. The reduction in TIC frequency was 4.4-fold (Figure 17, Table 10). Differentiation of progenitor cells is a potential explanation for this reduction.

[0163] [Table 10]

[0164] Example 5: Sensitivity of various colorectal cancer cell lines with MSI-H status to Compound 1 treatment To further study the efficacy of compound 1, the sensitivity of various colon cancer cell lines with specific genes of interest to compound 1 was evaluated. These specific genes of interest are found in frequently mutated pathways such as gene mismatch repair, beta-catenin, MAPK, PI3K, p53, TGF beta, chromatin remodeling and histone modification pathways. Figure 18 shows the heat map of the mutation status of genes in these pathways, together with their respective microsatellite instability (MSI-H), CpG island methylator phenotype (CIMP) and MLH1 methylation status, where cell lines are classified according to their sensitivity to compound 1.

[0165] Further studies showed that colorectal cancer cell lines with MSI-H status have significantly higher sensitivity to Compound 1 than colorectal cancer cell lines with microsatellite stability (MSS). A panel of 13 colorectal cancer PDX models and 6 colorectal cancer organoid PDX models were tested for their sensitivity to Compound 1 in a culture-based viability inhibition assay. The MSI-H status of the colorectal cancer cell lines studied and the respective mutations of MMR pathway genes are shown in Figure 19.

[0166] As summarized in Table 11 below, 100% (5 out of 5) of the PDX models characterized as MSI were sensitive to Compound 1 treatment (IC 50 <1 μM) and IC 50 IC was found to range from 0.001 to 0.014 μM. However, only 50% (4 out of 8) of PDX models characterized as MSS were sensitive to Compound 1 treatment, with IC 50 Similarly, 100% (3 out of 3) of the organoid models characterized as MSI were sensitive to Compound 1 treatment (IC 50ranged from 0.022 to 0.149 μM), none of the organoid models characterized as MSS (0 out of 3) were sensitive to compound 1 treatment (IC 50 exceeded 10 μM).

[0167] [Table 11]

[0168] Example 6: Identification of biomarkers associated with Compound 1 treatment

[0158] RNA-seq and ChIP-seq analyses were used to identify potential clinical pharmacodynamic (PD) biomarkers as molecular indicators for monitoring the effect of treatment with Compound 1.

[0169] In the RNA-seq analysis carried out, 326 genes were initially identified that were up-regulated or down-regulated by compound 1 treatment by more than 2-fold. The tested dose of compound 1 was 37.3mg / kg, which was the highest dose tested in diffuse large B-cell lymphoma xenograft model. In the ChIP-seq analysis, 65 genes were identified that showed direct KDM4C promoter occupancy. Figure 20 shows that genes PNUTS (PPP1R10), ANK1, IBA57, SOWAHD, MF12-AS1 and CECR1 showed dose-dependent changes after treatment with compound 1 in vivo at 2.5mg / kg, 5mg / kg, 7.5mg / kg, 15mg / kg and 50mg / kg. FIG. 20 also shows that PNUTS, ANK1, and IBA57 are downregulated during Compound 1 treatment, while SOWAHD, MF12-AS1, and CECR1 are upregulated.

[0170] Compound 1 treatment for 24 hours showed strong inhibition of PNUTS gene expression. In a study of the inhibitory effect of Compound 1 on PNUTS gene expression, MDA-MB-231 cells were treated for 24 hours and the expression levels of PNUTS mRNA in response to Compound 1 treatment were measured by qPCR. Inhibition of PNUTS mRNA was quantified by calculating the average percent change in PNUTS gene expression after Compound 1 treatment compared to DMSO-treated controls, and the IC 50 Values ​​were calculated as the concentration at which inhibition was half-maximal. IC 50 The value was 0.007 μM.

[0171] Furthermore, ChIP-seq analysis in MDA-MB-231 cells shows KDM4 occupancy at the PNUTS (PPP1R10) promoter marked by H3K4me3 (Figure 21). Example 7: Single cell gene expression analysis of immature and mature cell markers Vehicle control and Compound 1-treated samples were characterized by single-cell transcriptional profiling using a gene set designed to distinguish between immature colon progenitor cells and more mature cell types (Dalerba, 2011). Compound 1-treated tumors showed a shift in cellularity upon KDM4C inhibition (Figure 22, Panel A). Hierarchical clustering analysis was used to identify three clusters based on the expression of a subset of immature and mature cell markers. Progenitor-like cell clusters were identified by the presence of high expression of immature cell markers (Lgr5, Notch1, and Ezh2) and low expression of mature cell markers (Krt20, Ceacam1, and Spink4). Intermediate progenitor-like cells were identified by the presence of high expression of Lgr5, but low expression of other immature cell markers such as Notch1, Ezh2. This cluster also expressed some markers of mature cells at higher levels than the progenitor-like cells, but at lower levels of cell cycle markers, suggesting that these cells are less proliferative than the progenitor-like cells (data not shown). Finally, we identified a cluster of differentiation-like cells that expressed low levels of immature cell markers (Lgr5, Notch1, Ezh2) and high levels of mature cell markers (Krt20 / Ceacam1 and Tff3 / Dll1 / Spink4). Compound 1-treated tumors had a significant reduction in the proportion of progenitor-like cells (p=0.01) and an increase in the number of both intermediate-like and differentiation-like cells, although the increase was not significant (p=0.07; Figure 22, Panel B).

[0172] Example 8: Effect of Compound 1 on hematopoietic progenitor cells Compound 1 was evaluated for its effect on proliferation of human bone marrow-derived erythroid and myeloid hematopoietic progenitor cells at STEMCELL Technologies Inc. (Vancouver, BC, Canada).

[0173]

[0164] Cells were thawed quickly in DNAse I at 37°C, then diluted with 10mL of Iscove's Modified Dulbecco's Medium containing 2% fetal bovine serum (IMDM + 2% FBS) and washed by centrifugation (1200 rpm, 10 min at room temperature). The supernatant was discarded and the cell pellet was resuspended in a known volume of IMDM + 2% FBS. Nucleated cell count (3% glacial acetic acid) and viability assessment (trypan blue exclusion test) were performed.

[0174] Compound 1 and the positive control (panobinostat) were diluted in DMSO and tested at concentrations ranging from 0.002 μM to 5 μM in final medium. These dilutions provided the appropriate final test concentrations required for the test and control substances (5, 1.67, 0.56, 0.19, 0.06, 0.02, 0.07 and 0.002 μM).

[0175]

[0166] Test and control articles were incubated with human bone marrow hematopoietic progenitor cells. After 14 days, cell colonies were evaluated.

[0167] The 50% and 90% inhibition of colony growth (IC 50 and I.C. 90 To calculate the concentration of (LogIC), a dose-response curve was generated using GraphPad Prism plotting the logarithm of the test substance concentration versus the percentage of control colony growth. To generate a curve fit to these data points, the log(inhibitor) versus response-variable slope (four parameters) equation [Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X) * The IC for both test and control substances was used. 50 and I.C. 90 Values ​​were derived from interpolation of curve fits. IC reported by GraphPad Prism 50 / 90 If the value actually exceeds the range of concentrations tested, if this value is more than 4-fold higher than the highest concentration tested, or if GraphPad Prism does not find an IC 50 / 90 If the value cannot be calculated, the notation >5 μM (for test article Compound 1) is used.

[0176] Compound 1 had no significant effect on colony formation of normal human bone marrow-derived progenitor cells compared to 0.1% DMSO (p >0.01). As shown in Table 12 below, the IC50 for inhibition of erythroid and myeloid colony formation was greater than 5 μM. Statistical analysis of photographs taken of representative erythroid and myeloid derived colonies showed that their morphology (size and density) was slightly impaired at compound 1 concentrations of ≧1.67 μM. Panobinostat, the positive control, had an IC50 of less than 0.01 μM. 50 It inhibited erythroid and myeloid colony formation at low levels.

[0177] [Table 12]

Claims

1. 1. A pharmaceutical composition comprising a compound or a pharma- ceutical acceptable salt or solvate thereof for use in a method of treating cancer in a cancer patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound or a pharma- ceutical acceptable salt or solvate thereof, the compound having the following structure: 【Chemistry 1】 1. A pharmaceutical composition comprising:

2. 1. A pharmaceutical composition comprising a compound or a pharma- ceutical acceptable salt or solvate thereof for use in a method of reducing a tumorigenic cell population in a cancer patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound or a pharma- ceutical acceptable salt or solvate thereof, the compound having the following structure: 【Chemistry 2】 1. A pharmaceutical composition comprising:

3. 1. A pharmaceutical composition comprising a compound or a pharma- ceutical acceptable salt or solvate thereof for use in a method of reducing tumor initiating cell frequency in a cancer patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound or a pharma- ceutical acceptable salt or solvate thereof, the compound having the following structure: 【Chemistry 3】 1. A pharmaceutical composition comprising:

4. 1. A pharmaceutical composition comprising a compound or a pharma- ceutical acceptable salt or solvate thereof for use in a method of inhibiting cancer stem cells in a cancer patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound or a pharma- ceutical acceptable salt or solvate thereof, the compound having the following structure: 【Chemistry 4】 1. A pharmaceutical composition comprising:

5. 1. A pharmaceutical composition comprising a compound or a pharma- ceutical acceptable salt or solvate thereof for use in a method of treating cancer in a cancer patient in need thereof, the method comprising the steps of: (a) determining the microsatellite instability-high (MSI-H) status of the cancer patient; and (b) if the cancer patient is determined to be an MSI-H cancer patient, administering to the cancer patient a therapeutically effective amount of a compound, or a pharma- ceutical acceptable salt or solvate thereof, wherein the compound has the following structure: 【Chemistry 5】 1. A pharmaceutical composition comprising:

6. 1. A pharmaceutical composition comprising a compound or a pharma- ceutical acceptable salt or solvate thereof for use in a method of treating cancer, the method comprising the steps of: (a) obtaining a sample from a patient having cancer; (b) determining the level of a biomarker in the sample; (c) diagnosing the patient as likely to respond to a treatment compound if the level of the biomarker in the sample differs from a reference level of the biomarker; and (d) administering to said patient a therapeutically effective amount of said compound; and wherein the compound has the following structure: 【Chemistry 6】 and The pharmaceutical composition wherein the biomarker is PNUTS.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the cancer patient has been diagnosed with a cancer selected from the group consisting of colorectal cancer, esophageal cancer, triple-negative breast cancer, gastric cancer, lymphoma, gastric adenocarcinoma, diffuse large B-cell non-Hodgkin's lymphoma, acute T-cell leukemia, esophageal squamous cell carcinoma, multiple myeloma, acute myeloid leukemia, colorectal adenocarcinoma, colorectal cancer, pancreatic cancer, pancreatic cancer, breast cancer and T-cell acute lymphoblastic leukemia.

8. 3. The pharmaceutical composition of claim 2, wherein the tumorigenic cell population is reduced by about 1.5-fold to about 100-fold.

9. 4. The method of claim 3, wherein said tumor-initiating cell frequency is reduced to a frequency of about 1 in 100 cells to about 1 in 10,000 cells.

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