THERAPEUTIC COMPOUNDS AND METHODS

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/332,967, filed May 6, 2016, which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] A pressing need exists for the discovery and development of new therapies for the treatment of cancer and other neoplastic disorders. In addition to being effective for the treatment of the target diseases and disorders, the new therapies need to be free of harmful side effects.

SUMMARY OF THE INVENTION

[0003] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a truncated-RXRa antagonist, wherein the truncated- RXRa antagonist induces apoptosis in a portion of the cancer cells. Another embodiment provides the method wherein the cancer cells have increased TNFa levels compared to the equivalent non-cancer cells. Another embodiment provides the method further comprising coadministration of TNFa. Another embodiment provides the method wherein the portion of cancer cells in which apoptosis is induced is greater than the portion of apoptotic cancer cells induced by the administration of the truncated-RXRa antagonist alone. Another embodiment provides the method wherein the portion of cancer cells in which apoptosis is induced is greater than the portion of apoptotic cancer cells induced by the administration of the truncated-RXRa antagonist alone. Another embodiment provides the method wherein the portion of cancer cells in which apoptosis is induced is greater than the portion of apoptotic cancer cells induced by the administration of the truncated-RXRa antagonist and TNFa alone. Another embodiment provides the method wherein the cancer cells are colorectal cancer cells. Another embodiment provides the method wherein the colorectal cancer cells harbor KRAS mutation. Another embodiment provides the method wherein the colorectal cells are resistant to cetuximab.

[0004] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a truncated-RXRa antagonist. Another embodiment provides the method wherein the truncated-RXRa antagonist lacks COX-1 or COX-2 activities. Another embodiment provides the method wherein the truncated-RXRa antagonist is free of COX-1 or COX-2 activities. Another embodiment provides the method wherein the truncated- RXRa antagonist is substantially free of COX-1 or COX-2 activities. Another embodiment provides the method wherein the truncated-RXRa antagonist has a COX-1 and COX-2 IC ₅₀ greater than 1000 μΜ. Another embodiment provides the method wherein the truncated-RXRa antagonist is TX803, or a pharmaceutically acceptable salt thereof.

[0005] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a truncated-RXRa antagonist and an inhibitor of the Ras-Raf-Mek-Erk Kinase pathway selected from a MEK inhibitor, a RAF inhibitor, or an Erk 1/2 inhibitor. Another embodiment provides the method wherein the inhibitor of the Ras-Raf- Mek-Erk Kinase pathway is a MEK inhibitor. Another embodiment provides the method wherein the MEK inhibitor is cobimetinib. Another embodiment provides the method wherein the MEK inhibitor is trametinib. Another embodiment provides the method wherein the inhibitor of the Ras-Raf-Mek-Erk Kinase pathway is a RAF inhibitor. Another embodiment provides the method wherein the RAF inhibitor is selected from vemurafenib, dabrafenib, encorafenib (formerly LGX818), PLX-4720, sorafenib, TAK-632, MLN2480, SB90885, XL281, RAF265, or any combination thereof. Another embodiment provides the method wherein the inhibitor of the Ras-Raf-Mek-Erk Kinase pathway is an Erk 1/2 inhibitor. Another embodiment provides the method wherein the Erk 1/2 inhibitor is selected from SCH772984, VTXl le, BIX02189, ERK5- IN-1, FR180204, Pluripotin, TCS ERK l ie, XMD 8-92, DEL-22379, or any combination thereof.

INCORPORATION BY REFERENCE

[0006] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Figure 1 A illustrates FACS analysis of SW620 cells sorted based on Annexin V-FITC (apoptotic marker, x-axis) and propidium iodide (DNA content and cell viability marker, y-axis) after the indicated treatment (control, Cobimetinib, TX803, and TX803 and Cobimetinib(Cobi)) with and without TNFa (T);

Figure IB illustrates a graph of percentage of apoptosis of SW620 cell line treated with

Cobimetinib (1 μΜ), TX803 (5 μΜ), and a combination of Cobimetinib (1 μΜ) and TX803 (5 μΜ) with and without TNFa (25 ng/mL); Figure 2A illustrates FACS analysis of HCT116 cells sorted based on Annexin V-FITC

(apoptotic marker, x-axis) and propidium iodide (DNA content and cell viability marker, y-axis) after the indicated treatment (control, Cobimetinib, TX803, and TX803 and Cobimetinib(Cobi)) with and without T Fa (T);

Figure 2B illustrates a graph of percentage of apoptosis of HCT116 cell line treated with Cobimetinib (1 μΜ), TX803 (5 μΜ), and a combination of Cobimetinib (Cobi) (1 μΜ) and TX803 (5 μΜ) with and without TNFa (25 ng/mL);

Figure 3 illustrates a graph of percentage of survival in SW620 cells treated with Cobimetinib (Cobi) or a combination of Cobimetinib (Cobi) and TX803 for the indicated concentrations; Figure 4 illustrates a graph of percentage of survival in SW620 cells treated with Cobimetinib (Cobi) or a combination of Cobimetinib (Cobi) and TX803 with and without TNFa;

Figure 5 illustrates a graph of percentage of survival in HCT116 cells treated with Cobimetinib (Cobi) or a combination of Cobimetinib (Cobi) and TX803 for the indicated concentrations; Figure 6 illustrates a graph of percentage of survival in HCT116 cells treated with Cobimetinib (Cobi) or a combination of Cobimetinib (Cobi) and TX803 with and without TNFa;

Figure 7A illustrates SW620 mice after treatment with a negative control, positive control, or TX803 respectively and the extracted tumors from each;

Figure 7B illustrates the average weight of tumors extracted from SW620 mice as depicted in Figure 7A;

Figure 8 illustrates Western blots indicating presence of full-length (FL) or truncated (t) RXRa in the indicated tissue types from colon cancer patients or rectal cancer patients;

Figure 9A illustrates Western blots of TX803 treatment (0, 2.5, 5, 10, 20, and 40 uM) and measuring apoptosis in SW620 cells by detecting PARP and cleaved PARP. β-actin is used as a loading control;

Figure 9B illustrates Western blots of TX803 treatment (0, 1.25, 2.5, 5, 10, and 20 uM) and measuring apoptosis in HCT116 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 9C illustrates Western blots of TX803 treatment (0, 2.5, 5, 10, and 20 uM) and measuring apoptosis in H292 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 9D illustrates Western blots of TX803 treatment (0, 2.5, 5, 10, and 20 uM) and measuring apoptosis in MCF-7 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control; Figure 9E illustrates Western blots of TX803 treatment (0, 2.5, 5, 10, and 20 uM) and measuring apoptosis in MDA-MB-231 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 10A illustrates Western blots of control (C), TNFa (T), TX803, and TX803 and TNFa (T) treatment measuring apoptosis in SW620 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 10B illustrates Western blots of control (C), TNFa (T), TX803, and TX803 and TNFa (T) treatment measuring apoptosis in HCT116 cells by detecting PARP and cleaved PARP;

Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure IOC illustrates Western blots of control (C), TNFa (T), TX803, and TX803 and TNFa (T) treatment measuring apoptosis in HT-29 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 10D illustrates Western blots of control (C), TNFa (T), TX803, and TX803 and TNFa (T) treatment measuring apoptosis in HepG2 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 10E illustrates Western blots of control (C), TNFa (T), TX803, and TX803 and TNFa (T) treatment measuring apoptosis in H292 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 10F illustrates Western blots of control (C), TNFa (T), TX803, and TX803 and TNFa (T) treatment measuring apoptosis in A549 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 10G illustrates Western blots of control (C), TNFa (T), TX803, and TX803 and TNFa (T) treatment measuring apoptosis in MCF-7 cells by detecting PARP and cleaved PARP. β-actin is used as a loading control;

Figure 10H illustrates Western blots of control (C), TNFa (T), TX803, and TX803 and TNFa (T) treatment measuring apoptosis in MDA-MB-231 cells by detecting PARP and cleaved PARP. Truncated (t) RXRa and RXRa are also detected, β-actin is used as a loading control;

Figure 11 A is a plot of body weight of mice (n=8, per group) treated with either vehicle

(control), TX803 (30 mg/kg daily for two weeks), Cobimetinib (10 mg/kg daily for two weeks), or Cobimetinib and TX803 (TX803 15 mg/kg plus Cobimetinib 5 mg/kg daily for two weeks). Body weight in grams (g) is shown on a Y-axis;

Figure 1 IB is an image of tumors from mice (n=8, per group) treated with vehicle (control), TX803 (30 mg/kg daily for two weeks), Cobimetinib (10 mg/kg daily for two weeks), or

Cobimetinib and TX803 (TX803 15 mg/kg plus Cobimetinib 5 mg/kg daily for two weeks); Figure 11C is a graph and statistical analysis of body weight from mice (n=8, per group) treated with vehicle (control), TX803 (30 mg/kg daily for two weeks), Cobimetinib (10 mg/kg daily for two weeks), or Cobimetinib and TX803 (TX803 15 mg/kg plus Cobimetinib 5 mg/kg daily for two weeks). Body weight in grams (g) is shown on a Y-axis;

Figure 1 ID is a graph and statistical analysis of tumor weight from mice (n=8, per group) treated with vehicle (control), TX803 (30 mg/kg daily for two weeks), Cobimetinib (10 mg/kg daily for two weeks), or Cobimetinib and TX803 (TX803 15 mg/kg plus Cobimetinib 5 mg/kg daily for two weeks). Tumor weight in grams (g) is shown on a Y-axis;

Figure 12A is a Western blot analysis of control, TX803, Cobimetinib, and Cobimetinib and TX803 detecting RXR-A197, p-ERK, and PARP. β-actin is used as a loading control;

Figure 12B is an image of an immunohistochemical analysis of control, TX803, Cobimetinib, and Cobimetinib and TX803 on p-ERK and Ki-67 expression from tissue from mice (n=3); Figure 13 is a plot of tumor volume in a PDX colon cancer mouse model in mice treated with control, TX803, and Cobimetinib (Cobi) and TX803. Tumor volume (mm ³ ) is plotted on a Y- axis versus days on a X-axis; and

Figure 14 is a plot of body weight in a PDX colon cancer mouse model in mice treated with control, TX803, and Cobimetinib (Cobi) and TX803. Body weight (g) is plotted on a Y-axis versus days on a X-axis.

DETAILED DESCRIPTION OF THE INVENTION

[0008] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "the cell" includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary between 1% and 15% of the stated number or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, "consist of or "consist essentially of the described features. [0009] As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.

[0010] "Pharmaceutically acceptable salt" includes both acid and base addition salts. A pharmaceutically acceptable salt of TX803 described herein is intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts of TX803 are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0011] "Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, 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 that are formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. and include, 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. Exemplary salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinate suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates,

dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, and the like. Also contemplated are salts of amino acids, such as arginates, gluconates, and galacturonates (see, for example, Berge S.M. et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66: 1 -19 (1997)). Acid addition salts of basic compounds are, in some embodiments, prepared by contacting the free base forms with a sufficient amount of the desired acid to produce the salt according to methods and techniques with which a skilled artisan is familiar.

[0012] "Pharmaceutically acceptable base addition salt" refers to those salts that retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Pharmaceutically acceptable base addition salts are, in some embodiments, formed 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, for example, isopropylamine, trimethylamine,

diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine,

2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, Ν,Ν-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine,

methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. See Berge et al., supra.

[0013] As used herein, "treatment" or "treating" or "palliating" or "ameliorating" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the 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, notwithstanding that the patient is still afflicted with the underlying disorder. For prophylactic benefit, the compositions are, in some embodiments, administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease has not been made. Retinoid X Receptors

[0014] Retinoid X Receptors (RXRs) are type II nuclear receptors that mediate biological effects of retinoids. RXRs are activated by retinoic acid and subsequently activate specific target gene expression. There are three RXRs, RXRa, -β, and -γ. RXRs mediate retinoic acid-mediated gene activation through homodimerization, or heterodimerization with subfamily 1 nuclear receptors including CAR, FXR, LXR, PPAR, PXR, TR, and VDR.

[0015] Retinoid X receptor a (RXRa), also known as R2B1 (nuclear receptor subfamily 2, group B, member 1), is a ligand-dependent transcription factor that regulates a wide range of biological functions, including cell differentiation, growth, and apoptosis (Germain et al., 2006; Szanto et al., 2004). RXRa resides in the cytoplasm at certain stages during development (Dufour and Kim, 1999; Fukunaka et al., 2001) and migrates from the nucleus to the cytoplasm in response to differentiation, apoptosis, and inflammation (Cao et al., 2004; Casas et al., 2003; Zimmerman et al., 2006).

[0016] RXRa exhibits a modular organization structurally consisting of three main functional domains: an N-terminal region, a DNA-binding domain and a ligand-binding domain (LBD). The LBD possesses a ligand-binding pocket (LBP) for the binding of small molecule ligands, a transactivation function domain termed AF-2 composed of Helix 12 (HI2) of the LBD, a coregulator binding surface, and a dimerization surface (Germain et al., 2006; Szanto et al., 2004). The ligand-dependent transcription regulation is predominately mediated through H12 that is highly mobile.

[0017] Upon agonist binding, RXRa dissociates from the corepressor and recruits a coactivator protein, which subsequently promotes transcription of downstream target genes. Agonist ligand binds to the LBP and helps the H12 to adopt the active conformation that forms a surface to facilitate the binding of coactivators and subsequent transactivation.

[0018] In contrast, in the absence of an agonist ligand or in the presence of an antagonist ligand, the H12 adopt an inactive conformation that favors the binding of corepressors to inhibit target gene transcription. Such inactivation of RXRa can induce histone deacetylation, chromatin condensation, and transcriptional suppression.

[0019] Due to its role in many regulatory processes, RXRa is an attractive molecular target for drug development. Natural RXRa ligand 9-cis-retinoic acid (9-cis-RA) and synthetic ligands have been effective in preventing tumorigenesis in animals and RXRa has been a drug target for therapeutic applications, especially in the treatment of cancer (Bushue and Wan, 2010; Yen and Lamph, 2006; Altucci and Gronemeyer, 2001; Dawson and Zhang, 2002). 9-cis-retinoic acid (9- cis-RA), several polyunsaturated fatty acids, and the NSAID Etodolac (Kolluri et al., 2005) can bind to RXRa to regulate different biological functions. Targretin, a synthetic RXR ligand, is currently used for treating cutaneous T-cell lymphoma (Dawson and Zhang, 2002),

demonstrating the suitability of targeting RXRa for cancer therapy. Consistently, the oncogenic potential of RXRa has been demonstrated. Genetic disruption of RXRa enhances tumorigenesis (Huang et al., 2002; Li et al., 2001), and RXR binding to PML/RAR is essential for the development of acute promeylocytic leukemia (Zeisig et al., 2007; Zhu et al., 2007). However, inhibitors designed to target full-length RXRa can be toxic to other non-cancerous cells.

[0020] In many cancer types, including colorectal, gastric, and breast cancer, RXRa is cut at the N-terminus. The resulting truncated RXRa (tRXRa) migrates from the nucleus and recruits PI3K to the cell membrane through its interaction with p85a. This recruiting of PI3K leads to its activation, by receptors such as TNFR1, and initiation of the PI3K/AKT survival pathway. Therefore, tRXRa, unlike full-length RXRa, activates cell survival pathways in cancer cells (Zhou et al., 2010, Cancer Cell 17:560-573).

[0021] tRXRa is often produced in tumor tissues but not in normal tissues. For example RXRa is cleaved in tumor but not in premalignant or normal tissues from patients with prostate or thyroid cancer (Takiyama et al., 2004; Zhong et al., 2003). Thus, agents targeting tRXRa- mediated pathway can be effective and tumor specific.

[0022] AKT has been shown to be overexpressed in many cancers, including colon, pancreatic, ovarian, and some breast cancers (Roy et al., 2002, Carcinogenesis 23 :201-205; Asano et al., 2004, Oncogene 23 :8571-8580). Phosphorylated, and thereby activated, AKT delivers survival signal (Datta et al., 1997, Cell 91 :231-241). Conversely, inhibition of AKT signaling can lead to induction of apoptosis in some cancers (Yuan et al., 2000, Oncogene 19:2324-2330; Page et al., Int J Oncol 17:23-28).

tRXRa Antagonists

[0023] Disclosed herein are inhibitors or antagonists of tRXRa activity. In some embodiments, the tRXRa antagonist is TX803, or a pharmaceutically acceptable salt thereof. TX803 lacks COX-1 and COX-2 inhibitory activities (IC ₅₀ >1000 μΜ for both), and thereby mitigates the potential for gastrointestinal and cardiotoxicities which are associated with NSAIDs. The synthesis and characterization of TX803 is provided in WO 2011/140525 or US 9,611,235. TX803 is also known as (Z)-2-(5-fluoro-l-(4-isopropylbenzylidene)-2-methyl-lH-inden -3- yl)acetic acid and has the chemical structure shown below.

[0024] Disclosed herein are methods for using antagonists of tRXRa, such as TX803 or a pharmaceutically acceptable salt thereof, to inhibit tRXRa-mediated PI3K/AKT activation. For example, binding of TX803 to tRXRa dissociates it from PI3K. Dissociation of PI3K from tRXRa turns off the erroneous PI3K/AKT mediated cell survival pathway.

[0025] Additionally disclosed herein are methods for using antagonists of tRXRa, such as TX803 or a pharmaceutically acceptable salt thereof, in combination with other therapeutic agents. For example, disclosed herein are methods of synergistically inhibiting tRXRa- dependent AKT activation with TX803 and T Fa.

[0026] Furthermore, TX803 -tRXRa binding converts the cytokine TNFa from a cancer cell survival factor to a cancer cell killer via apoptosis. [0027] TNFa plays important roles in diverse cellular events such as cell survival and death. However, it often fails to induce apoptosis in cancer cells due to its simultaneous activation of the NF-κΒ and/or the PI3K/AKT pathway (Aggarwal, 2003; Balkwill, 2009).

[0028] TNFa is elevated in tumor environments, including following immuno-therapy.

Retinoids in combination with cytokines, such as TNFa and TNF-related apoptosis inducing ligand (TRAIL), can synergistically induce differentiation or apoptosis of human transformed cells (Altucci et al., 2005) whereas combination of retinoids and TNFa can overcome retinoic acid resistance in some cancers (Witcher et al., 2004). Previous studies with Sulindac and TNFa show these two agents synergistically inhibit AKT activation in cancer cells, which implies that TNFa, and possibly other cytokines, can prime cancer cells for their responsiveness to RXRa ligands by converting AKT activation from a RXRa-independent to a RXRa-dependent manner (Zhou et al., 2010, Cancer Cell 17:560-573).

[0029] In addition to the PI3K/AKT pathway, the MAPK/ERK pathway is also commonly up- regulated in cancer cells. The MAPK pathway is initiated by a ligand binding to a receptor, such as EGFR, and culminates with transcription activation or repression of target genes. In many cancers, the MAPK pathway is stuck in the on position. Inhibitors of MAPK pathway components, such as MEK inhibitors, can reverse this mis-activation. MEK inhibitors include, for example, trametinib (GSK1120212), cobimetinib, XL518, binimetinib, selumetinib, PD- 325901, CI-1040, PD035901, and TAK-733 (Wang et al, 2007, Biochem Biophys Acta

1773(8): 1248-55; Yeh et al., 2009, Mol Cancer Ther, 8(4):834-43). Additional MEK inhibitors include MEK162, AZD6244, R05126766, and GDC-0623.

[0030] In the MAPK/ERK pathway, MEK is activated by a RAF protein kinase, such as BRAF, CRAF, or ARAF. BRAF inhibitors include vemurafenib, dabrafenib, encorafenib (formerly LGX818), PLX-4720. Other RAF inhibitors include sorafenib, TAK-632, MLN2480, SB90885, XL281, and RAF265.

[0031] The downstream target of MEK kinases is ERK (ERK1 and/or ERK2), also called mitogen-activated protein kinase (MAPK). Inhibitors of ERK include SCH772984, VTXl le, BIX02189, ERK5-IN-1, FR180204, Pluripotin, TCS ERK l ie, XMD 8-92, and DEL-22379.

[0032] Disclosed herein are tRXRa antagonists. In some aspects, the tRXRa antagonist is TX803 or a pharmaceutically acceptable salt thereof. In some cases, the tRXRa antagonist lacks COX-1 and/or COX-2 inhibitory activities.

[0033] Disclosed herein are methods of using tRXRa antagonists, such as TX803 or a pharmaceutically acceptable salt thereof, to block TNFa-induced AKT-mediated survival function. Since TNFa can induce both apoptosis and cell survival, by blocking the cell survival pathway, T Fa function in cancerous cells shifts from initiating cell survival to initiating cell death.

[0034] Disclosed herein are methods of the use of tRXRa antagonists, such as TX803 or a pharmaceutically acceptable salt thereof, which results in a two-fold therapeutic effect:

dissociation of PI3K from tRXRa leads to 1) inactivation of AKT -mediated cell survival pathways and 2) removes the blockage of T Fa-induced apoptosis pathways. Therefore, tRXRa antagonists, as disclosed herein, turn off cell survival pathways and turn on apoptosis pathways in targeted cancerous cells.

[0035] Disclosed herein are methods for altering the PI3K and MAPK pathways in cancerous cells, comprising co-administering tRXRa antagonists and MEK inhibitors. In some aspects, TNFa is also co-administered.

[0036] In some embodiments, methods disclosed herein comprise administration of tRXRa antagonists. In some cases, the antagonist comprises TX803 or a pharmaceutically acceptable salt thereof.

[0037] In some embodiments, methods disclosed herein comprise administration of tRXRa antagonists in combination with other agents. In some examples, other agents comprise TNFa. In some cases, TNFa is endogenously overexpressed in cells in which the treatment is being administered. In some cases, TNFa is co-administered with a tRXRa antagonist, such as TX803 or a pharmaceutically acceptable salt thereof.

[0038] In some embodiments, methods disclosed herein comprise administration of tRXRa antagonists in combination with other agents. In some examples, the tRXRa antagonist comprises TX803 or a pharmaceutically acceptable salt thereof. In some embodiments, the coadministered agent comprises a MEK inhibitor. In some embodiments, the MEK inhibitor comprises cobimetinib. In some embodiments, the MEK inhibitor comprises trametinib. In some embodiments, the MEK inhibitor is selected from trametinib, cobimetinib, XL518, binimetinib, selumetinib, PD-325901, CI-1040, PD035901, TAK-733, MEK162, AZD6244, R05126766, and GDC-0623, or any combination thereof. In some cases, TNFa is endogenously overexpressed in cells in which the treatment is being administered. In some cases, TNFa is co-administered with the tRXRa antagonist and MEK inhibitor.

[0039] In some embodiments, the co-administered agent comprises a RAF inhibitor. In some embodiments, the RAF inhibitor is selected from vemurafenib, dabrafenib, encorafenib

(formerly LGX818), PLX-4720, sorafenib, TAK-632, MLN2480, SB90885, XL281, RAF265, or any combination thereof.

[0040] In some embodiments, the co-administered agent comprises an Erk 1/2 inhibitor. In some embodiments, the Erk 1/2 inhibitor is selected from SCH772984, VTXl le, BIX02189, ERK5-IN-1, FR180204, Pluripotin, TCS ERK l ie, XMD 8-92, DEL-22379, or any combination thereof.

Pharmaceutical Compositions

[0041] In certain embodiments, the TX803, or a pharmaceutically acceptable salt thereof is administered as a pure chemical. In other embodiments, the TX803, or a pharmaceutically acceptable salt thereof, is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Remington: The Science and Practice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005)).

[0042] Provided herein is a pharmaceutical composition comprising the TX803, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers. The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject or patient) of the composition.

[0043] One embodiment provides a pharmaceutical composition comprising the TX803, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0044] In certain embodiments, the TX803, or a pharmaceutically acceptable salt thereof, is substantially pure, in that it contains less than about 5%, or less than about 1%, or less than about 0.1%, of other organic small molecules, such as unreacted intermediates or synthesis byproducts that are created, for example, in one or more of the steps of a synthesis method.

[0045] Suitable oral dosage forms include, for example, tablets, pills, sachets, or capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. In some embodiments, suitable nontoxic solid carriers are used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. (See, e.g., Remington: The Science and Practice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005)).

[0046] The dose of the composition comprising TX803, or a pharmaceutically acceptable salt thereof, differ, depending upon the patient's condition, that is, stage of the disease, general health status, age, and other factors.

[0047] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the

composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome), or a lessening of symptom severity. Optimal doses are generally determined using experimental models and/or clinical trials. The optimal dose depends upon the body mass, weight, or blood volume of the patient.

[0048] Oral doses typically range from about 1.0 mg to about 1000 mg, one to four times, or more, per day.

Methods of Treatment

[0049] Colorectal cancer (CRC) is one of the most common cancers worldwide (Jemal et al., 2010 Cancer J. Clin. 60:277-300). With the emergence of two anti-epidermal growth factor receptor (EGFR)-targeted antibodies, cetuximab (Erbitux) and panitumumab (Vectibix), the treatment of metastatic CRC has entered into the era of personalized treatment. However, EGFR, the target of these drugs, which is overexpressed in approximately 80% of colorectal

carcinomas, failed to predict a therapeutic response when used clinically (Chung et al., 2005, J. Clin. Oncol. 23 : 1803-1810; Sartore-Bianchi et al., 2007, J. Clin. Oncol. 25:3238-3245). It was later found that the KRAS gene is a strong negative predictive biomarker to indicate whether a CRC patient will respond to anti-EGFR treatment. As the target treatment may also be toxic and expensive, KRAS mutation status detection has become a common diagnostic tool. Furthermore, new and effective therapeutics are needed for the treatment of patients which harbor such KRAS mutation.

[0050] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a composition comprising (Z)-2-(5-fluoro-l-(4- isopropylbenzylidene)-2-methyl-lH-inden-3-yl)acetic acid, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising cobimetinib, or a pharmaceutically acceptable salt thereof. Another embodiment provides the method wherein the cobimetinib is administered as the fumarate salt. One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a composition comprising (Z)-2- (5-fluoro-l-(4-isopropylbenzylidene)-2-methyl-lH-inden-3-yl) acetic acid, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising trametinib, or a pharmaceutically acceptable salt thereof. Another embodiment provides the method wherein the composition compri sing (Z)-2-(5 -fluoro- 1 -(4-i sopropylbenzylidene)-2-methyl - 1 H-inden-3 - yl)acetic acid, or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition comprising cobimetinib or trametinib, or pharmaceutically acceptable salts thereof, are provided in separate dosage forms. Another embodiment provides the method wherein the composition comprising (Z)-2-(5-fluoro-l-(4-isopropylbenzylidene)-2-methyl-lH-inden -3-yl)acetic acid, or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition comprising cobimetinib or trametinib, or pharmaceutically acceptable salts thereof, are provided in the same dosage form. Another embodiment provides the method wherein the composition comprising (Z)-2-(5-fluoro-l-(4-isopropylbenzylidene)-2-methyl-lH-inden -3-yl)acetic acid, or a

pharmaceutically acceptable salt thereof, and the pharmaceutical composition comprising cobimetinib or trametinib, or pharmaceutically acceptable salts thereof, are provided in fixed dose combination form. Another embodiment provides the method wherein the composition comprising (Z)-2-(5-fluoro-l-(4-isopropylbenzylidene)-2-methyl-lH-inden -3-yl)acetic acid, or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition comprising cobimetinib or trametinib, or pharmaceutically acceptable salts thereof, are administered together or separately. Another embodiment provides the method wherein the cancer is selected from colon cancer, rectal cancer, gastric cancer, or breast cancer. Another embodiment provides the method wherein the cancer is selected from colon cancer or breast cancer. Another embodiment provides the method wherein the cancer is selected from gastric cacncer. Another embodiment provides the method wherein the cancer is colon cancer. Another embodiment provides the method wherein the cancer is breast cancer. Another embodiment provides the method the breast cancer does not express the genes for estrogen receptor (ER), progesterone receptor (PR) or Her2/neu. Another embodiment provides the method wherein the breast cancer does not express the genes for estrogen receptor (ER), or progesterone receptor (PR). Another embodiment provides the method wherein the cancer is characterized by a KRAS mutant.

Another embodiment provides the method wherein the cancer is further characterized by expression of tRXRa.

[0051] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a composition comprising (Z)-2-(5-fluoro-l-(4- isopropylbenzylidene)-2-methyl-lH-inden-3-yl)acetic acid, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising a MEK inhibitor. Another embodiment provides the method wherein the MEK inhibitor is selected from trametinib, cobimetinib, XL518, binimetinib, selumetinib, PD-325901, CI-1040, PD035901, TAK-733, MEK162, AZD6244, R05126766, and GDC-0623, or any combination thereof. Another embodiment provides the method wherein the cancer is selected from colon cancer, rectal cancer, gastric cancer, or breast cancer.

[0052] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a composition comprising (Z)-2-(5-fluoro-l-(4- isopropylbenzylidene)-2-methyl-lH-inden-3-yl)acetic acid, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising a RAF inhibitor. Another embodiment provides the method wherein the RAF inhibitor is selected from vemurafenib, dabrafenib, encorafenib (formerly LGX818), PLX-4720, sorafenib, TAK-632, MLN2480, SB90885, XL281, RAF265, or any combination thereof. Another embodiment provides the method wherein the cancer is selected from colon cancer, rectal cancer, gastric cancer, or breast cancer.

[0053] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a composition comprising (Z)-2-(5-fluoro-l-(4- isopropylbenzylidene)-2-methyl-lH-inden-3-yl)acetic acid, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising a Erk 1/2 inhibitor. Another embodiment provides the method wherein the Erk 1/2 inhibitor is selected from SCH772984, VTXl le, BLX02189, ERK5-IN-1, FR180204, Pluripotin, TCS ERK l ie, XMD 8-92, DEL-22379, or any combination thereof. Another embodiment provides the method wherein the cancer is selected from colon cancer, rectal cancer, gastric cancer, or breast cancer.

EXAMPLES

Example 1 : Increasing apoptosis in SW620 colorectal cells

[0054] Colorectal cells of the SW620 cell line were plated from 1000-10000 cells per well in a 96-well plate and incubated for 24 hours. The tRXRa antagonist TX803, the MEK inhibitor Cobimetinib, and/or T Fa were added in the amounts and combinations indicated in Table 1. Where indicated, Cobimetinib was added at 0 hours to a final concentration of 1 uM, TX803 was added at 2 hours to a final concentration of either 3 μΜ or 5 μΜ, and TNFa was added at 3 hours to a final concentration of 25 ng/mL. Following the indicated treatment cycle, the cells were incubated at 37 degrees Celsius for 24 hours in a C0 ₂ incubator.

Table 1. 3 μΜ TX803 treatment

Table 2. 5 μΜ TX803 treatment

[0055] IX annexin-binding buffer was prepared by diluting lmL of 5X annexin binding -buffer with 4 mL of deionized water. Propidium Iodide (PI) was prepared as a 100 μg/mL working solution by diluting 5 μΙ_, of the 1 mg/mL PI stock solution in 45 IX annexin-binding buffer.

[0056] Following incubation, cells were incubated for 24 hours at 37 °C in C0 ₂ incubator. Cells were then harvested and washed in cold phosphate-buffered saline (PBS). After washing, cells were centrifuged, the supernatant discarded, and the cells were then re-suspended in 100 μΐ _^ IX annexin-binding buffer.

[0057] 5 μΙ_ Alexa Fluor® 488 annexin V (Component A) and 1 μΐ. 100 μg/mL PI working solution was added to each 100 μΐ _^ of cell suspension. Cells were then incubated at room temperature for 15 minutes. After the incubation period, 400 μΐ _^ IX annexin-binding buffer was added, cells were mixed gently, and then kept on ice until ready for use.

[0058] Stained cells were analyzed by flow cytometry, measuring the fluorescence emission at 530 nm and 575 nm (or equivalent) using 488 nm excitation. The counting cell number was 10000 every time. Annexin binds and detects the apoptosis marker phosphatidyl serine located on the cell surface of apoptotic cells. Propidium iodide is a DNA intercalating agent and is used as a marker for DNA content, cell cycle stage, and cell viability.

[0059] As seen in Figure 1 A, apoptosis of SW620 cell line treated with the Cobimetinib (1 μΜ) and TX803 (5 μΜ) and TNFa (25 ng/mL) was measured by flow cytometry. The combination of TNFa with either TX803 or Cobimetinib results in increased induction of apoptosis compared to either TNFa, TX803, or Cobimetinib alone.

[0060] As seen in Figure IB, the combination of TX803, Cobimetibib, and TNFa had a drastic effect on apoptosis induction, resulting in 74% total apoptotic cells (early and late stage) compared to TNFa and Cobimetinib (9.5% apoptotic cells) or TNFa and TX803 (22.5% apoptotic cells). The significant increase in apoptotic cells in the three agent treatment is greater than that of the two two-agent combinations added together. This suggests the effect is not simply additive, but instead has a true synergistic effect. [0061] As seen in Figure 2A and Figure 2B, apoptosis of HCT116 cell line treated with the Cobimetinib (1 μΜ) and TX803 (3 μΜ) and T Fa (25 ng/mL) was measured by flow cytometry. The combination of TNFa with either Cobimetinib, TX803, or Cobimetinib and TX803 results in increased induction of apoptosis compared to either TNFa, TX803,

Cobimetinib, or Cobimetinib and TX803 alone.

[0062] Cell survival was also measured using an MTT assay. Briefly, cells were seeded at a density of 8,000 -10,000 cells per well in 96-well plates in RPMI 1640 or DMEM containing 10% FBS. Cells were replenished with fresh complete medium containing either compound or 0.1% DMSO. After 48 hours of incubation, 15 μΐ of 5 mg/ml MTT (Thiazolyl Blue Tetrazolium Bromide) was added to each well. One set of wells included MTT but no cells and served as a control. Plates were incubated for 4 hours at 37 °C in a culture hood. Media was then removed and 150 μΐ of MTT solvent (DMSO) was added. Plates were covered with foil and agitated on an orbital shaker for 10 minutes. Absorbance was read at 490 nm with a reference filter of 490 nm.

[0063] Using the MTT assay, survival of SW620 cells was assessed. As seen in Figure 3, percentage of survival was measured in SW620 cells cultured in FBS free medium and treated with Cobimetinib (1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) for 2 hours followed by Cobimetibib and TX803 for 46 hours. As seen in Figure 4, percentage of survival was measured in SW620 cells cultured in FBS free medium and treated with Cobimetinib (1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) for 2 hours followed by Cobimetinib and TX803 for 1 hour, and followed by Cobimetinib, TX803, and TNFa (25 ng/mL) for 45 hours.

[0064] Using the MTT assay, survival of HCT116 cells was assessed. As seen in Figure 5, percentage of survival was measured in HCT116 cells cultured in FBS free medium and treated with Cobimetinib (1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) for 2 hours followed by Cobimetinib and TX803 for 46 hours. As seen in Figure 6, percentage of survival was measured in HCT116 cells cultured in FBS free medium and treated with Cobimetinib (1 nM, 10 nM, 100 nM, 1000 nM, 10000 nM) for 2 hours followed by Cobimetinib and TX803 for 1 hour and followed by Cobimetinib, TX803, and TNFa (25 ng/mL) for 45 hours.

Example 2: TX803 efficacy in colorectal cancer model (SW620)

[0065] SW620 colorectal cancer model mice were treated with the truncated RXRa antagonist TX803, a positive control molecule, or negative control. The positive control mice and TX803 treated mice were injected with 60 mg/kg of the respective therapeutic intraperitoneally (IP), every other day (Q2D). At the end of the study, the tumor size and weight were measured (Figures 7A and 7B). Mice treated with TX803 had reduced tumor size compared to both the positive and negative control mice. Example 3: Treatment of colorectal patients with TX803

[0066] Truncated-RXRa is elevated in tumor tissues from colorectal cancer patients (Figure 8). Elevated truncated -RXRa levels indicates misregulation of cell survival pathways, such as the PI3K/AKT pathway. Such patients can be treated with TX803 in combination with TNFa in order to turn off the cell survival pathway and turn on apoptosis, as was described and shown in vitro in Example 1.

[0067] The colorectal cancer patients described above can also be treated with a combination of TX803, TNFa, and the MEK inhibitor Cobimetinib. In this scenario, in addition to turning off the P13K/AKT cell survival pathway and turning on the TNFa-mediated apoptosis pathway, the MEK inhibitor will turn off the MAPK pathway, which is often also misregulated in colon cancer cells. This combination therapy can lead to a synergistic effect of apoptosis induction greater than any of the three therapeutic agents individually, as was demonstrated in vitro in Example 1.

Example 4: Growth inhibition and induction of apoptosis of TX803 in cancer cell lines

[0068] Proliferation and apoptosis were assessed in different cancer cell lines.

[0069] Cells were seeded at a density of 8,000 cells per well in 96-well plates in RPMI 1640 or DMEM containing 10% FBS. Cells were replenished with fresh complete medium containing either compounds or 0.1% DMSO. After 48 hours of incubation, 15 μΐ of 5 mg/ml MTT

(Thiazolyl Blue Tetrazolium Bromide) was added to each well. One set of wells included MTT but no cells and served as a control. Plates were then incubated for 4 hours at 37°C in a culture hood. Media was removed and 150 μΐ MTT solvent (DMSO) was added. Plates were covered with foil and agitated on an orbital shaker for 10 minutes. Absorbance was read absorbance at 490 nm with a reference filter of 490 nm.

[0070] Growth inhibition was measured in different cell lines and can be seen in Table 3 and Table 4. Referring to Table 3, cancer cells were treated with TX803 for 48 hours in the presence of TNFa (25 ng/mL). Referring to Table 4, cancer cells were cultured in medium with 0% FBS and treated with TX803 for 48 hours in the presence of TNFa (25ng/ml).

Table 3. Growth inhibition in SW620, HCT116, HT29, MDA-MB-231, and HepG2 cell lines

Table 4. Growth inhibition in MCF-7, H292, and A549 cell lines

[0071] Apoptosis was measured by Western blotting. Cell lysates were prepared by cell incubation in radio immunoprecipitation assay buffer (RTPA: 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, 1% Sodium deoxycholic acid, 0.1% SDS, 2 mmol/L phenylmethylsulfonyl fluoride (PMSF), 30 mmol/L Na ₂ HP0 ₄ , 50 mmol/L NaF, and 1 mmol/L Na ₃ V0 ₄ ). Cell lysates were then boiled in SDS sample loading buffer, resolved by 10% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked in 5% milk in TBST (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20) for 1 hour at room temperature. Membranes were washed twice with TBST and incubated with primary antibody for 1 hour. Following incubation, the membranes were washed twice with TBST and probed with horseradish peroxide-linked anti-immunoglobulin for 1 hour at room temperature.

Membranes were then washed three times with TBST and immunoreactive products were visualized using enhanced chemiluminescence reagents and autoradiography.

[0072] As seen in Figure 9A, the apoptotic effects of TX803 were assayed in SW620 cells. SW620 cells were cultured in medium with 0% FBS and treated with TX803 (0, 2.5, 5, 10, 20, and 40 uM) for 6 hours followed by analysis by Western blotting.

[0073] As seen in Figure 9B, the apoptotic effects of TX803 were assayed in HCT116 cells. HCT116 cells were cultured in medium with 0% FBS and treated with TX803 (0, 1.25, 2.5, 5, 10, and 20 uM) for 6 hours followed by analysis by Western blotting.

[0074] As seen in Figure 9C, the apoptotic effects of TX803 were assayed in H292 cells. H292 cells were cultured in medium with 0% FBS and treated with TX803 (0, 2.5, 5, 10, and 20 uM) for 6 hours followed by analysis by Western blotting.

[0075] As seen in Figure 9D, the apoptotic effects of TX803 were assayed MCF-7 cells. MCF- 7 cells were cultured in medium with 0% FBS and treated with TX803 (0, 2.5, 5, 10, and 20 uM) for 8 hours followed by analysis by Western blotting.

[0076] As seen in Figure 9E, the apoptotic effects of TX803 were assayed in MDA-MB-231 cells. MDA-MB-231 cells were cultured in medium with 0% FBS and treated with TX803 (0, 2.5, 5, 10, and 20 uM) for 6 hours 30 minutes followed by analysis by Western blotting. [0077] As seen in Figure 10A apoptotic effects of TX803 and T Fa combination were measured in SW620 cells. SW620 cells were cultured in medium with 0% FBS and treated with TX803 (10 uM) for 1 hour followed by TNFa (25 ng/ml, T: TNFa) for 8 hours. Apoptosis was analyzed by Western blotting.

[0078] As seen in Figure 10B apoptotic effects of TX803 and TNFa combination were measured in HCT116 cells. HCT116 cells were cultured in medium with 0% FBS and treated with TX803 (10 uM) for 1 hour followed by TNFa (25 ng/ml, T: TNFa) for 6 hours. Apoptosis was analyzed by Western blotting.

[0079] As seen in Figure IOC apoptotic effects of TX803 and TNFa combination were measured in HT-29 cells. HT-29 cells were cultured in medium with 0% FBS and treated with TX803 (10 uM) for 1 hour followed by TNFa (25 ng/ml, T: TNFa) for 6 hours. Apoptosis was analyzed by Western blotting.

[0080] As seen in Figure 10D apoptotic effects of TX803 and TNFa combination were measured in HepG2 cells. HepG2 cells were cultured in medium with 0% FBS and treated with TX803 (10 uM) for 1 hour followed by TNFa (25 ng/ml, T: TNFa) for 6 hours. Apoptosis was analyzed by Western blotting.

[0081] As seen in Figure 10E apoptotic effects of TX803 and TNFa combination were measured in H292 cells. H292 cells were cultured in medium with 0% FBS and treated with TX803 (10 uM) for 1 hour followed by TNFa (25 ng/ml, T: TNFa) for 6 hours. Apoptosis was analyzed by Western blotting.

[0082] As seen in Figure 10F apoptotic effects of TX803 and TNFa combination were measured in A549 cells. A549 cells were cultured in medium with 0% FBS and treated with TX803 (10 uM) for 1 hour followed by TNFa (25 ng/ml, T: TNFa) for 8 hours. Apoptosis was analyzed by Western blotting.

[0083] As seen in Figure 10G apoptotic effects of TX803 and TNFa combination were measured in MCF-7 cells. MCF-7 cells were cultured in medium with 0% FBS and treated with TX803 (10 uM) for 1 hour followed by TNFa (25 ng/ml, T: TNFa) for 8 hours. Apoptosis was analyzed by Western blotting.

[0084] As seen in Figure 10H apoptotic effects of TX803 and TNFa combination were measured in MDA-MB-231 cells. MDA-MB-231 cells were cultured in medium with 0% FBS and treated with TX803 (10 uM) for 1 1 hour followed by TNFa (25 ng/ml, T: TNFa) for 6 hours and 30 minutes. Apoptosis was analyzed by Western blotting.

[0085] Growth inhibition assay of TX803 in different cancer cell lines demonstrated that TX803 was sensitive toward colon cancer cells. TX803 induced apoptosis in most cancer cell lines. The degree of apoptosis was enhanced by TX803 and TNFa combination. Example 5: TX803 and Cobimetinib treatment in mouse tumor model

[0086] MMTV-PyMT Mouse Model

[0087] A MMTV-PyMT mouse breast cancer model was used to test the effects of TX803 and Cobimetinib treatment. MMTV-PyMT mice of 12 weeks of age were randomly divided into four groups (n=8, per group) and treated with daily oral doses of either TX803 (30 mg/kg), Cobimetinib (10 mg/kg), and combination of TX803 (15 mg/kg) and Cobimetinib (5 mg/kg), or vehicle for two weeks.

[0088] Dosing Solutions

[0089] TX803 was dissolved in DMSO and diluted with normal saline containing 5.0% (WIN) Tween-80 to a final concentration 3 mg/mL. Cobimetinib was dissolved in DMSO and diluted with normal saline containing 5.0% (V/V) Tween-80 to a final concentration 1 mg/mL. Normal saline with DMSO and 5.0% Tween-80 was employed as the vehicle control.

[0090] Western blot analysis

[0091] For Western blot assays, tissues from MMTV-PyMT mice of 14 weeks of age were harvested by grinding the tissue with a tissue disruptor on ice and lysing with buffer. Tissue extract was centrifuged at 12000 rpm for 10 minutes. The supernatant was added at an equal volume of 2X SDS. Samples were then boiled, resolved by SDS-PAGE, and transferred to a PVDF membrane. The PVDF membrane was blocked in milk, washed with TBST, and incubated with primary antibody. Following incubation with primary antibody, the PVDF membrane was washed and incubated with secondary antibody. Proteins were detected with ECLA and ECLB.

[0092] Hematoxylin-eosin Staining

[0093] Tumor tissues were fixed with 10% buffered formalin in PBS and embedded in paraffin. Tumor sections (4 μπι) were stained with hematoxylin and eosin (H&E).

[0094] Histopathological Analysis

[0095] Small pieces of breast tumor were removed, rinsed with saline, fixed with 10% formalin, and embedded in paraffin. Tissue sections were cut at a thickness of 4 μπι and stained with hematoxylin and eosin (H&E). Primary antibodies targeting Ki-67 (Abeam) and p-ERK (Cell Signaling Technologies) were used to examine cell proliferation and ERK phosphorylation. Stained sections were analyzed with an image analysis system (Olympus, Tokyo, Japan).

[0096] Statistical analyses

[0097] ANOVA with Tukey's post-test (One-way ANOVA) was used for comparisons between groups. Two-way ANOVA was used for comparisons of magnitude of changes between different groups and to compare values among different experimental groups. For experiments with only two groups, Student's t-test was used. P<0.05 was considered statistically significant (*), P<0.01 as highly significant (**), P<0.001 as extremely significant (***), and ns as not significant.

[0098] Results

[0099] As seen in Figures 11 A-l ID, TX803 alone or in combination with Cobimetinib had little effect on the body weight as compared to vehicle control. The data was consistent with a lack of toxicity of the drug at this dose. Administration of Cobimetinib at 10 mg/kg to mice for 14 days resulted in a 23.33% decrease in body weight. In assessing tumor growth, administration of TX830 alone at 30 mg/kg resulted in a significant inhibition (-48%) of tumor growth.

Administration of Cobimetinib at 10 mg/ml resulted in an inhibition of tumor growth (-39%). The combination treatment of both TX830 and Cobimetinib resulted in a significant inhibition of tumor growth (-62%), even though both drugs were administered at lower dosage (15 mg/kg and 5 mg/kg, respectively).

[00100] As seen in Figure 12A, tumor tissues from mice treated with TX803 alone showed reduced expression of RXRa, suggesting that the drug targets RXRa in animals as analyzed by Western blot. ERK was strongly activated as indicated by elevated ERK phosphorylation. The activation of MEK/ERK pathway by TX803 was completed blocked when mice were coadministered with the MEK inhibitor Cobimetinib. In contrast, administration of mice with the MEK inhibitor Cobimetinib had no effect on RXRa expression, while it suppressed ERK activation. As seen in Figure 12B, the immunohistochemical staining showed development of mammary ducts in animals treated with combination of TX803 and Cobimetinib and in TX803 alone group, although to a lesser degree.

[00101] Results demonstrated that TX803 and Cobimetinib combination therapy achieved better efficacy on tumor growth inhibition, while on a dose reduction treatment regimen for the MEK inhibitor to alleviate toxicity. Synergistic inhibition by TX803 and Cobimetinib combination on tumor growth suggests that ERK activation by TX803 serves as an escape mechanism by which tumor cells develop resistance to TX803 treatment.

Example 6: TX803 effects in colon cancer patient-derived mouse model

[00102] PDX colon cancer model

[00103] A PDX colon cancer model was used to assay the effects of TX803. Nude mice of 6 weeks of age were implanted with tumor tissues from a patient (COCA45) who had a KRAS gene mutation Q61K and positive in tRXRa. The mice were allowed to grow to approximately 200 mm ³ . Mice were then randomly divided into three groups (n=5, per group) and treated with daily oral doses of either TX803 (30 mg/kg), combination of TX803 (30 mg/kg) and

Cobimetinib (5 mg/kg), or vehicle for four weeks. Tumor volumes were measured two times per week using caliper, and body weights were measured three times per week. [00104] Dosing Solutions

[00105] TX803 was formulated in 0.5% Carboxymethyl cellulose Sodium (CMC-Na) with 0.2% Tween 80 at 3 mg/mL as a uniform suspension. Cobimetinib was formulated in 0.5% CMC-Na with 0.2%) Tween 80 at 0.5 mg/mL as a uniform suspension.

[00106] Statistical analyses

[00107] Student's t-test was performed between treatment and vehicle control groups. P<0.05 was considered statistically significant (*), P<0.01 as highly significant (**), P<0.001 as extremely significant (***), and ns as not significant.

[00108] Results

[00109] As seen in Figure 13, TX803 was effective against this KRAS mutated tumor model following once-a-day oral treatment for 4 weeks. Further, TX803 mono-therapy was as effective as a combination therapy with a MEK inhibitor Cobimetinib (Figure 13). As seen in Figure 14, all treatments were well tolerated and there was no difference in body weight changes between the vehicle control and either treatment group.

Example 7: Preparation of pharmaceutical dosage forms - oral tablet

[00110] A tablet is prepared by mixing 48%> by weight of TX803, or a pharmaceutically acceptable salt thereof, 45% by weight of microcrystalline cellulose, 5% by weight of low- substituted hydroxypropyl cellulose, and 2% by weight of magnesium stearate. Tablets are prepared by direct compression. The total weight of the compressed tablets is maintained at 250- 500 mg.