WO2017223268A1 - COMPOSITIONS AND METHODS OF RESENSITIZING CELLS TO BROMODOMAIN AND EXTRATERMINAL DOMAIN PROTEIN INHIBITORS (BETi) - Google Patents

Abstract

The present invention provides compositions and methods to increase anti-tumor sensitivity of a cell or tumor to bromodomain and extraterminal domain protein inhibitors (BETi). In one aspect, a composition comprises a BETi and a TGFβ pathway inhibitor, TRIM33 or a fragment thereof, or a nucleic acid encoding TRIM33. In another aspect, a method is described for increasing anti-tumor sensitivity to BETi. Methods for a tumor with BETi are also described.

Description

COMPOSITIONS AND METHODS OF RE SENSITIZING CELLS TO BROMODOMAIN AND EXTRATERMINAL DOMAIN PROTEIN

INHIBITORS (BETi) CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/353,424, filed June 22, 2016, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

Epigenetic regulation of transcription is central to control of cell fate and proliferation. Enzymatic addition or removal of a variety of specific post-translational modifications of histones support the recruitment of epigenetic "readers," proteins that selectively bind to modified sites and recruit transcriptional activators or repressors. Alterations in this complex epigenetic code contribute to development of a range of diseases, including cancer. Consequently pharmacological modulation of enzymes that generate or remove epigenetic modifications and their readers offer new therapeutic opportunities for cancer treatment.

The bromodomain and extraterminal domain (BET) proteins are one class of epigenetic readers involved in transcriptional control. The small family of BET proteins (BRD2, BRD3, BRD4 and BRDT) are characterized by tandem

bromodomains, that bind acetylated lysine residues in histones and other proteins, and a C-terminal extraterminal domain responsible for interactions with chromatin regulators. BET proteins, in particular BRD4, have been implicated as general regulators of transcription through recruitment of the elongation factor, P-TEFb, to gene promoters and through interaction with the mediator complex. In addition, high-level recruitment of BRD4 to enhancer regions has been implicated in gene-specific transcriptional activation. Evidence from a variety of approaches, including genetic screens in cultured cells, targeted gene deletion in mice, and analysis of human tumors, have implicated BET proteins, in particular BRD2 and BRD4, in a range of cancers (Baratta et al., 2015, Proc Natl Acad Sci USA 112(l):232-237; Zuber et al., 2011, Nature 478(7370):524-528).

Recent advances allowing small molecule inhibition of BET proteins generated considerable interest in the therapeutic potential of BET inhibitors (BETi). BETi are small molecules that interact with the acetylated lysine binding pocket of the BET family bromodomains, interfering with BET protein binding to chromatin and consequently modulate transcription. BETi were initially shown to be effective in a mouse xenograft model of midline carcinoma, a rare cancer driven by a chromosomal translocation producing a BRD4-NUT fusion protein. BETi have subsequently proven to be effective in multiple models of hematologic malignancies and solid tumors that are not characterized by mutated oncogenic BET protein activation. One key mechanism by which BETi suppress growth and survival of at least some types of cancer cells is by preferentially repressing transcription of the proto-oncogene, MYC, which is often under the control of BRD4 (Dawson et al., 2011, Nature 478(7370):529- 533; Delmore et al., 2011, Cell 146(6):904-917; Mertz et al., 2011, Proc Natl Acad Sci USA 108(40): 16669-16674). Thus, BETi may provide a new mechanism to target MYC and other oncogenic transcription factors, which lack obvious binding pockets for small molecules and are thus typically considered to be "undruggable".

The excitement surrounding the potential of targeting BET proteins in cancer has fueled the development of a variety of BETi, some of which are currently undergoing clinical trials (Filippakopoulos and Knapp, 2014, Nat Rev Drug Discov 13(5):337-356). However, lessons from other targeted cancer therapies suggest that acquired resistance will limit long-term responsiveness to BETi treatment. Acquired resistance to kinase inhibitors, for example, is often accompanied by outgrowth of clones harboring mutations to the kinase itself that disrupt inhibitor binding. However, in many cases resistance to targeted therapy occurs independently of mutations to the drug target, being driven by re-activation of signaling pathways suppressed by the drug or activation of bypass pathways that facilitate cell growth and survival despite inhibition of the target. A recent study showed that acquired resistance to JQ1 in cultured pancreatic cancer cells was associated with BRD4-independent MYC expression (Kumar et al., 2015, Sci Rep 5:9489). However, specific molecular lesions leading to BETi resistance have not been identified. Identifying such lesions may suggest specific therapeutic strategies for re-sensitizing cells to BETi. Therefore a need exists for methods that identify lesions for acquired resistance and for the development of therapeutics to resensitize tumor cells to BETi. SUMMARY OF THE INVENTION

The present invention provides a method of increasing anti-tumor sensitivity to a bromodomain and extraterminal domain protein inhibitor (BETi) in a cell in a subject, the method comprising administering a composition comprising a TGFP pathway inhibitor and a BETi to a subject in need thereof, wherein the TGFP pathway inhibitor sensitizes the cell to the BETi.

The present invention further provides a method of treating a tumor comprising administering to a subject in need thereof a composition comprising a TGFP pathway inhibitor and a BETi, wherein the TGFP pathway inhibitor sensitizes the tumor to the BETi.

The present invention further provides a method of increasing anti-tumor sensitivity to a BETi of a cell in a subject, the method comprising administering a composition comprising a BETi and one selected from the group consisting of TRTM33 or a fragment thereof, and a nucleic acid encoding TREVI33 to a subject in need thereof, wherein the TRF 33 sensitizes the cell to the BETi.

The present invention further provides a method of treating a tumor comprising administering to a subject in need thereof a composition comprising a BETi and at least one selected from the group consisting of TRF 33 or a fragment thereof, and a nucleic acid encoding TRF 33, wherein TRF 33 sensitizes the tumor to the BETi.

In certain embodiments, the TGFP pathway inhibitor is selected from the group consisting of a small molecule inhibitor, an inhibitory nucleic acid, neutralizing antibody, and an antagonist. In other embodiments, the small molecule inhibitor is selected from the group consisting of SB431542, A83-01, RepSox, SB208, SB505124, LY364947, LY2157299, R268712, D4476, SB525334, GW788388, TEW-7197, and any combination thereof. In other embodiments, the antagonist is selected from the group consisting of HtrAl, decorin, biglycan, fibromodulin, lumican, endoglin, somatostatin, follistatin, RAP-1332, pirfenidone (5-methyl-l-phenyl-2(lH)-pyridone), soluble ectodomains of TGFp receptor type II (RII) or betaglycan (BG), and any combination thereof. In yet other embodiments, the neutralizing antibody specifically binds one selected from the group consisting of TGF and a TGF receptor.

In certain embodiments, the nucleic acid encoding TREVI33 is an expression vector comprising a TRIM33 gene. In other embodiments, the vector is a viral vector. In certain embodiments, the BETi is selected from the group consisting of JQ1, GS-626510, GS-5829, BMS 986158, RVX2135, CPI203, CPI-0610, ABBV-075, BAY1238097, INCB054329, FT-1101, PFI-1, 1-BET151, ZEN-3365, 1-BET762, OTX015, TEN-010, and any combination thereof.

In certain embodiments, the subject has a cancer. In other embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. In yet other embodiments, the method treats a tumor wherein the tumor is a cancer selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer.

In certain embodiments, the cell in the subject is BETi resistant. In other embodiments, the tumor is BETi resistant.

The invention further provides a composition comprising a TGFP pathway inhibitor and a bromodomain and extraterminal domain protein inhibitor (BETi), wherein the TGFP pathway inhibitor sensitizes cells to the BETi.

In certain embodiments, the the TGFP pathway inhibitor is selected from the group consisting of a small molecule inhibitor, an inhibitory nucleic acid, a neutralizing antibody, and an antagonist. In other embodiments, the small molecule inhibitor is selected from the group consisting of SB431542, A83-01, RepSox, SB208, SB505124, LY364947, LY2157299, R268712, D4476, SB525334, GW788388, TEW-7197, and any combination thereof. In yet other embodiments, the neutralizing antibody specifically binds one selected from the group consisting of TGF and a TGF receptor. In yet other embodiments, the antagonist is selected from the group consisting of HtrAl, decorin, biglycan, fibromodulin, lumican, endoglin, somatostatin, follistatin, RAP-1332, pirfenidone (5-methyl-l-phenyl-2(lH)-pyridone), soluble ectodomains of TGFP receptor type II (RII) or betaglycan (BG), a neutralizing antibody against TGFP or a TGFP receptor, and any combination thereof.

The invention also provides a composition comprising a BETi and at least one selected from the group consisting of TREVI33 or a fragment thereof, and a nucleic acid encoding TREVI33, wherein the TGF pathway inhibitor sensitizes cells to the BETi. In certain embodiments, the nucleic acid encoding TRIM33 is a vector comprising a TRIM33 gene. In other embodiments, the vector is a viral vector.

In certain embodiments, the BETi is selected from the group consisting of JQl, GS-626510, GS-5829, BMS 986158, RVX2135, CPI203, CPI-0610, ABBV-075, BAY1238097, INCB054329, FT-1101, PFI-1, 1-BET151, ZEN-3365, 1-BET762, OTX015, TEN-010, and any combination thereof.

In certain embodiments, the compositions of the invention can be formulated in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition can be used in the

manufacture of a medicament for the treatment of a tumor or cancer in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments, which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figures 1 A-1H are images showing that the shRNA screening revealed TREVI33 as a regulator of BETi resistance in cancer cells. Figure 1A shows the structures of the two different BETi used in the study, JQl and GS-626510. Figure IB shows the ^D values of GS-626510 for 40 bromodomains (Table 3) determined with a

BROMOscan™ (DiscoveRx). The dendrogram image was generated using

TREEspot™ Software Tool (DiscoveRx Corporation). Figure 1C shows the dose- dependent inhibition of RKO cell proliferation by JQl and GS-626510 in a 5-day assay. Relative viable cell number was determined by CellTiter Glo assay. Figure ID shows that both GS-626510 and JQl down-regulate MYC protein levels. RKO cells were treated with increasing concentrations of BETi for 3 h and MYC levels in whole cell lysates were assessed by immunoblotting. Actin was used as a loading control. Figure IE shows gene expression changes induced by JQl and GS-626510 in shCTRL cells were highly correlated. The log2 [fold change (BETi/DMSO)] of all gene expression following 3 h treatment with 1 μΜ JQl or 0.3 μΜ GS-626510 was fitted to a line. Two replicate experiments result in an R2 of 0.93 and 0.92 respectively. Figure IF shows the scheme of the shRNA screening procedure. Cells infected by the pooled shRNA library were propagated through 8 doublings in presence of either DMSO vehicle control or different concentrations of JQl or GS-626510. Genomic DNA was extracted from the TO (reference) and T4 conditions for determination of proviral shRNA abundance. Figure 1G shows the top 10 enriched target genes revealed by RIGER analysis in each condition. TRJJVI33 was among the top 3 ranked genes in all four BETi conditions but not in the DMSO condition. Figure 1H shows multiple individual TRJJVI33 shRNAs were enriched in BETi-treated, but not in DMSO control treated conditions. Log₂ fold change (T4/T0) of each shRNA is plotted from the most depleted to the most enriched. Each red line represents a single shRNA targeting TRJJVI33.

Figures 2A-2J are images showing loss of TRJJVI33 conferred resistance to BETi. Figure 2A shows (top) a schematic of TRIM33 domain organization and positions of two pairs of RT-PCR primers, (middle) TRIM33 mRNA levels determined by RT-PCR in shCTRL cell line and cell lines expressing four different TRJJVI33- targeting shRNAs, and (bottom) TRJJVI33 protein levels in these cell lines. Figure 2B shows shCTRL or shTRJJVI33 cells were seeded in a 6-well plate (3 x 10⁵ cells per well) in the presence of DMSO, 100 nM JQl or 50 nM GS-626510 and cumulative cell numbers were assessed every 3 days for up to 15 days. Figure 2C shows the growth inhibition assay. shCTRL and shTRJJVI33 cells were cultured with different

concentrations of JQl or GS-626510 for 120 h and relative cell numbers were determined using CellTiter Glo. Figure 2D shows the IC₅₀ values (mean ± SEM) calculated from 5 independently performed growth inhibition assays using shCTRL and shTRJJVI33 cells. P values are based on paired t-test. Figure 2E is a set of graphs showing the effect of TRJJVI33 depletion on JQl or GS-626510 sensitivity in a panel of cancer cell lines; Figure 2E shows the IC50 values for each cell line expressing either shCTRL or shTRJJVI33 derived from 3 independent growth inhibition assays and the mean ± SEM of the fold change in IC50 (shTRFM33/shCTRL) calculated (*P < 0.05, paired t-test). Figure 2F shows 2 x 10⁴ shCTRL or shTRJJVI33 cells plated in 6-well plates, treated with DMSO, 100 nM JQl, or 50 nM GS-626510 for two weeks and then stained with crystal violet. The crystal violet staining was quantified at 590 nm absorbance. Figure 2G shows the cell proliferation assay of cell lines expressing two independent shRNAs (B5 and A 12) targeting TRJJVI33. Cells were cultured in 100 nM JQl, or 50 nM GS-626510 for two weeks and then stained with crystal violet. Figure 2H shows the shCTRL or shTRJJVI33 cells transduced with either an empty vector control or TRIM33-expressing lentivirus and cell growth was assessed as in Figure 2F. Figure 21 shows TRHVI33 expression levels in cells from Figure 2H assessed by immunoblotting. Figure 2J shows the crystal violet quantification measured at 590 nm absorbance corresponding to Figure 2H.

Figures 3 A-3E are graphs showing RT-PCR quantification of mRNA levels of

15 selected genes whose expression was changed with shTREVI33 or BETi treatment (left graphs; error bars represent SD (n =3)), and normalized RNAseq reads of the 15 genes above from two replicate experiments (right graphs; error bars represent the SD between the two replicates).

Figures 3F-3J are images showing RNAseq analysis of vehicle or BETi -treated shCTRL or shTRFM33 cells. Waterfall plots show gene expression changes induced by 3 h treatment of shCTRL RKO cells with 1 μΜ JQl (Figure 3F) or 0.3 μΜ GS-626510 (Figure 3G). MYC is down-regulated by both JQl and GS-626510. Figure 3H shows the top 10 sequence motifs enriched in promoter regions of genes down-regulated >2- fold by JQl and GS-626510 in shCTRL cells were determined by Gene Set Enrichment Analysis (Broad Institute). Figure 31 shows the gene expression changes induced by shTRJJVI33 in RKO cells. Figure 3 J shows an immunoblot showing that BRD4 protein level is not changed by TRJJVI33 knockdown.

Figures 4A-4F are a panel of images showing TRIM33 modulated MYC sensitivity to BETi. Figure 4 A shows the normalized RNAseq reads of MYC mRNA from two replicate experiments before and after JQl or GS626510 treatment. Figure 4B shows the RT-PCR quantification of MFC mRNA in shCTRL, shTRJJVI33 and shTRJJVI33 rescued (shTRJJVI33^RES) cells, either untreated or treated with BETi for 3 h. Figure 4C shows cells treated similarly as in panel Figure 4B \ analyzed for MYC protein. Figure 4D shows the MYC protein levels in control or MYC over-expressing cells before and after BETi treatment for 3 h. Figure 4E shows crystal violet staining of control or MYC over-expressing cells growing with DMSO, JQl or GS-626510 for two weeks. Figure 4F shows the cumulative cell growth of control or MYC-overexpressing cells over 15 days.

Figures 4G and 4H show the ChIP at MYC gene promoter region. Line threshold indicates IgG control level. Figure 4G shows the TRJJVI33 ChIP using 4 different primer pairs (#2, #14, #15 and #16) in the MYC promoter region. Figure 4H shows the BRD4 ChIP using the same set of primers as in Figure 4G. Figure 5 A shows the shCTRL or shTREVB 3 -transduced RKO and SK-CO-1 cells treated with 1 μΜ of JQ1 or 0.3 μΜ of GS626510 for 24 hours. MYC levels were determined by immunoblotting and ERKl/2 was used as a loading control.

Figures 5B-5E show the gene set enrichment analysis (GSEA) and down- regulation of TGFP and MYC signatures by JQ1 was significantly decreased in shTREVI33 in comparison to shCTRL cells ( ES: normalized enrichment score).

Figures 5F-5V are a set of images showing inhibition of TGFP signaling potentiated the anti-proliferative effects of BETi. Figure 5F shows the TGFpi ligand stimulated phosphorylation of SMAD2 potentiated in shTRFM33 cells. shCTRL or shTREVI33 RKO cells were treated with increasing doses of TGFpi for 25 min (left panel) or with 2 ng/ml TGFpi for various times (right panel), cells were lysed and immunoblotted for phospho-SMAD2 (pSMAD2), total SMAD2 and TREVI33. Figure 5G shows the shCTRL or shTREVI33 cells untreated or treated with 100 pM of TGFpi for 25 min and SMALM was immunoprecipitated. Co-precipitating pSMAD2 was assessed by immunoblotting. Figure 5H shows the shCTRL or shTRFM33 cells infected with lentivirus encoding shCTRL or one of two hairpins targeting SMALM (shSMALM- 3 or shSMAD4-4). Cells were untreated or treated with 100 pM of TGFp 1 for 25 min SMALM, pSMAD2 and total SMAD2 levels were assessed by immunoblotting. Figure 51 shows the TGFp receptor II (ΤβϋΠ) mRNA from RNAseq in shCTRL and shTRFM33 cells. Figures 5J and 5K show the ChIP at TGFBR2 (TpRII) gene promoter region. Line threshold indicates IgG control level. Figure 5 J shows the TRFM33 ChIP using two different pair of primers (#4 and #3) amplifying TGFBR2 gene promoter region. Figure 5K shows the BRD4 ChIP using the same set of primers as in Figure 5 J amplifying TGFBR2 gene promoter region. Figures 5L-5N show the inhibition of TGFP pathway by silencing TpRII increases the magnitude of cell growth inhibition by BETi. Figure 5L shows the RT-PCR quantification of Τβ ΙΙ mRNA levels in shCTRL and shTRFM33 cells expressing control (shCTRL) or two different TpRII-targeting shRNAs (shTpRII-3 and shTpRII-4). Figure 5M shows cells from Figure 5L stimulated with 100 pM of TGFpi for 25 min and pSMAD2 levels assessed by immunoblotting. Figure 5N shows shCTRL cells (left) or shTRFM33 cells (right) expressing control and TpRII-targeting shRNAs cultured for 2 weeks with DMSO or different concentrations of BETi (as indicated) and then stained with crystal violet. Figure 50 shows the crystal violet quantification measured at 590 nm absorbance corresponding to Figure 5N. Figures 5P and 5Q show the TpRI inhibitor LY2157299 potentiated BETi-mediated inhibition of cell proliferation. Figure 5P shows the shTREVI33 cells pre-treated with increasing doses of LY2157299 and then exposed to 100 pM TGFpi for 25 min.

Immunoblotting shows dose-dependent inhibition of pSMAD2 by LY2157299. Figure 5Q shows the shCTRL and two shTREVI33 KD cell lines cultured in the presence of JQ 1 or GS-626510, with or without LY2157299 for 2 weeks and stained with crystal violet. Figure 5R shows the crystal violet quantification measured at 590 nm absorbance corresponding to Figure 5Q. Figure 5S shows the shCTRL or shTREVI33 cells treated with 1 μΜ JQ1 or 0.3 μΜ GS-626510 with or without 5 μΜ LY2157299 overnight and MYC protein levels assessed by immunoblotting. Figures 5T, 5U and 5 V show that the over-expression of TpRII was not sufficient to induce resistance to BETi. Figure 5T shows the pLentiCMV-EV or pLentiCMV-TpRII transduced stable cell lines treated with increasing doses of TGFpi for 25 min and pSMAD2 levels assessed by immunoblotting. Figure 5U shows the cells from Figure 10D cultured in the presence of DMSO, 100 nM JQ1 or 50 nM GS-626510 for 2 weeks and stained with crystal violet. Figure 5 V shows the pLentiCMV-EV or pLentiCMV-TpRII stable cell lines treated with 1 μΜ JQ1 or 0.3 μΜ GS-626510 for overnight either in the presence or in the absence of 100 pM TGFpi . MYC levels were determined by immunoblotting and actin was used as a loading control. DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term "about" is meant to encompass variations of ±20% or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%) of the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The term "antagonist" as used herein refers to a compound or molecule that inhibits or decreases a biological response. An antagonist has affinity for a target, such as a receptor, and binding to the target disrupts or prevents the interaction between the target and its cognate binding partner to inhibit or decrease activity of the target. An antagonist may also block the action of a stimulating ligand by binding to an allosteric site that may "lock" the target in an inactive state or prevent the dimerization or oligomerizarion of the target protein

As used herein, "bromodomain and extraterminal domain" or "BET" proteins refer to epigenetic readers involved in transcriptional control. The small family of BET proteins are characterized by tandem bromodomains, that bind acetylated lysine residues in histones and other proteins, and a C-terminal extraterminal domain responsible for interactions with chromatin regulators. Examples of BET proteins include, but are not limited to, BRD2, BRD3, BRD4 and BRDT.

As used herein, "bromodomain and extraterminal domain inhibitor" or "BETi" refers to a small molecule that interacts with the acetylated lysine binding pocket of the BET family bromodomains and displaces the BET proteins from binding to chromatin. BETi demonstrate anti-tumor activity in a range of malignancies. Some BETi exert antiproliferative effects that disrupt oncogenic pathways. Examples of BETi include, but are not limited to, JQ1, GS-626510, GS-5829, BMS 986158, RVX2135, CPI203, CPI-0610, ABBV-075, BAY1238097, INCB054329, FT-1101, PFI-1, 1-BET151, ZEN- 3365, 1-BET762, OTX015, and TEN-010. Additional examples of BETi include compounds disclosed in U.S. Patent Nos. 9,255,089 and 9,108,953, and U.S. Patent Publication No. 2014/0336190, all of which are hereby incorporated herein in their entireties.

The term "BETi resistant" as used herein refers to a loss or decrease in anti- tumor efficacy or response of a cellular target to a BETi.

The term "BETi sensitivity" as used herein refers to anti-tumor activity of a BETi in a cellular target. "Increasing anti-tumor sensitivity" as used herein refers to increasing an anti-tumor response in a cellular target to the BETi by increasing a basal level of the anti-tumor response or resensitizing the cellular target to the BETi. The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, myeloma and the like.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open- ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By "effective amount" is meant the amount required to reduce or improve at least one symptom of a disease relative to an untreated patient. The effective amount of an active compound(s) used for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject.

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

By "fragment" is meant a portion of a polynucleotide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acids. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or 2500 (and any integer value in between) nucleotides. The fragment, as applied to a nucleic acid molecule, refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid molecule may be at least about 15 nucleotides in length; for example, at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides, at least about 1000 nucleotides to about 1500 nucleotides; or about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between).

As used herein, the term "functional fragment" refers to a truncated peptide or polypeptide of the parent that retains at least one biological, physiological, and/or pharmacological property of the parent. As used herein, the term "inhibit" is meant to refer to a decrease in a biological state. For example, the term "inhibit" may be construed to refer to the ability to negatively regulate expression, stability or activity of an expression product, wherein such inhibition may affect expression of a gene, protein mRNA, stability of a protein mRNA, translation of a protein mRNA, stability of a protein, a protein post- translational modifications, and/or a protein activity.

As used herein, the term "inhibitory nucleic acid" refers to small RNAs that inhibit gene expression. Examples of inhibitory nucleic acids include, but are not limited to, microRNAs (miRNA) and siRNA.

"Instructional material," as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that may be used to communicate the usefulness of the compounds of the invention. In some instances, the instructional material may be part of a kit useful for effecting alleviating or treating the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit may, for example, be affixed to a container that contains the compounds of the invention or be shipped together with a container that contains the compounds. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. For example, the instructional material is for use of a kit; instructions for use of the compound; or instructions for use of a formulation of the compound.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By "microRNA" or "miRNA" or "miR" is meant a small non-coding RNA, which functions in transcriptional and/or post-transcriptional regulation of gene expression.

As used herein, "neutralizing antibody" refers to an antibody that binds an antigen and prevents the biological effects of the antigen. In one embodiment, the neutralizing antibody binds TGFP or another effector in the TGFP signaling pathway.

"Pharmaceutically acceptable" refers to those properties and/or substances that are acceptable to the patient from a pharmacological/toxicological point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding composition, formulation, stability, patient acceptance and bioavailability. "Pharmaceutically acceptable carrier" refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.

As used herein, the term "pharmaceutical composition" or "pharmaceuticaly acceptable composition" refers to a mixture of at least one compound or molecule useful within the invention with a pharmaceutically acceptable carrier. The

pharmaceutical composition facilitates administration of the compound or molecule to a patient. Multiple techniques of administering a compound or molecule exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound or molecule useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;

powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound or molecule useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which may be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides may be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences that are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means. The following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine. The term "RNA" as used herein is defined as ribonucleic acid. The term "recombinant DNA" as used herein is defined as DNA produced by joining pieces of DNA from different sources.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

As used herein, the terms "prevent," "preventing," "prevention," and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

By "reduces" or "decreases" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By "reference" is meant a standard or control. A "reference " is a defined standard or control used as a basis for comparison.

As used herein, "sample" or "biological sample" refers to anything, which may contain the cells of interest (e.g., cancer or tumor cells thereof) for which the screening method or treatment is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. In one embodiment, a biological sample is a tissue sample including pulmonary arterial endothelial cells. Such a sample may include diverse cells, proteins, and genetic material. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s). Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like. As used herein, the term "sensitivity" as used herein refers to the ability of a target to respond in a qualitative fashion to a pharmacologic action of a compound or agent.

The term "small molecule inhibitor" as used herein refers to a compound or agent that inhibits a target. In one embodiment, the small molecule inhibitor inhibits a target in the TGFP signaling pathway. Examples of the small molecule inhibitor include, but are not limited to, SB431542, A83-01, RepSox, SB208, SB505124, LY364947, LY2157299 R268712, D4476, SB525334, GW788388 and TEW-7197.

By "small interfering RNA" or "siRNA" is meant a short RNA molecule that may be double stranded, which interferes with the expression of a specific gene that includes a nucleotide sequence complementary to the RNA molecule.

By the term " specifically binds," as used herein with respect to an antibody, is meant an antibody or antibody fragment which recognizes and binds with a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.

A "subject" or "patient," as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80%> or 85%>, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

As used herein, the term "TGFP pathway inhibitor" refers to an agent that inhibits the TGFP signaling pathway. Examples of a TGFP pathway inhibitor include, but are not limted to, a small molecule, an inhibitory nucleic acid, neutralizing antibody, and an antagonist.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or improving a disorder and/or symptom associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely ameliorated or eliminated.

As used herein, "tripartitie motif-containing 33" or "TRHVI33" refer to a transcriptional corepressor protein and tumor suppressor with three zinc binding domains, a RING, a B-box type 1, a B-box type 2 and a coiled-coil region. TREVI33 is also known as PTC7, RFG7, TFIG, TIFIG, FLJ32925, TIFGAMMA, ECTODERMIN or TIF 1 GAMMA. Three alternatively spliced transcript variants for this gene have been described; however, the full-length nature of one variant has not been determined. TRFM33 regulates TGF -beta/BMP signaling cascade and promotes physiological differentiation of hematopoietic stem cells by associating with SMAD2 and SMAD3. TRFM33 also acts as an E3 ubiquitin-protein ligase to promote SMAD4 ubiquitination, nuclear exclusion and degradation via the ubiquitin proteasome pathway. An exemplary embodiment includes TRFM33 nucleic acid sequence comprising GenBank Accession No. NM_015906 or NM_033020 for human TRFM33 or NM_001079830 or NM 053170 for mouse TRFM33. In another exemplary embodiment includes TRFM33 polypeptide sequence comprising GenBank Accession No. NP 056990.3 or

NP_148980.2 human TRFM33 or NP_001073299.1 or NP_444400.2 for mouse TRFM33.

A "vector" is a composition of matter that comprises an isolated nucleic acid and that may be used to deliver the isolated nucleic acid to the interior of a cell.

Numerous vectors are known in the art including, but not limited to, linear

polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Compositions

It has been discovered that acquired resistance to bromodomain and

extraterminal domain protein inhibitors (BETi) limits long-term responsiveness to BETi treatment. It has also been discovered that loss of TRTM33 or over-activation of a TGFP signaling pathway decreases sensitivity to BET inhibitors. As described herein, increased expression of TREVI33 or inhibition of a TGFP signaling pathway increases anti-tumor sensitivity. The invention therefore includes compositions and methods to increase anti-tumor sensitivity in a cell or tumor. BETi are small molecules that interact with BET family bromodomains and displace the BET proteins, such as BRD2, BRD3, BRD4 and BRDT, from binding to chromatin. BETi demonstrate anti-tumor activity, such as antiproliferative effects, to disrupt oncogenic pathways. Examples of BETi include, but are not limited to, JQl, GS-626510, GS-5829, BMS 986158, RVX2135, CPI203, CPI-0610, ABBV-075, BAY1238097, INCB054329, FT-1101, PFI-1, 1-BET151, ZEN-3365, 1-BET762, OTX015, and TEN-010. In one embodiment, the BETi is selected from the group consisting of JQl, GS-626510, GS-5829, BMS 986158, RVX2135, CPI203, CPI-0610, ABBV-075, BAY1238097, INCB054329, FT-1101, PFI-1, 1-BET151, ZEN-3365, 1- BET762, OTX015, TEN-010, and any combination thereof.

In one aspect, the present invention includes a composition comprising a TGFP pathway inhibitor and a bromodomain and extraterminal domain protein inhibitor (BETi), wherein the TGFP pathway inhibitor sensitizes cells to the BETi.

The TGFP pathway inhibitor inhibits at least one TGFP signaling pathway, such as TGFP receptor signaling. In one embodiment, the TGFP pathway inhibitor is selected from the group consisting of a small molecule inhibitor, an inhibitory nucleic acid, a neutralizing antibody, and an antagonist.

In some embodiments, the TGFP pathway inhibitor is a small molecule inhibitor. The small molecule inhibitor is a compound or agent that inhibits TGFP signaling pathway, such as inhibiting TGFP receptor signaling. In one embodiment, the small molecule inhibitor is selected from the group consisting of SB431542, A83-01, RepSox, SB208, SB505124, LY364947, LY2157299 R268712, D4476, SB525334, GW788388, TEW-7197, and any combination thereof.

In some embodiments, the TGFP pathway inhibitor is an inhibitory nucleic acid. The inhibitory nucleic acid includes small RNAs that inhibit gene expression. In some embodiments, the inhibitory nucleic acid inhibits expression of at least one gene in a TGFP signaling pathway, such as TGFP or a TGFP receptor. In one embodiment, the inhibitory nucleic acid is selected from the group consisting of a miRNA and a siRNA. In another embodiment, the siRNA inhibits expression of TGFP or a TGFP receptor.

In some embodiments, the TGFP pathway inhibitor is a neutralizing antibody.

The neutralizing antibody specifically binds TGFP or another effector in the TGFP signaling pathway. In some embodiments, the neutralizing antibody specifically binds TGFP or a TGFP receptor. In one embodiment, the neutralizing antibody specifically binds one selected from the group consisting of TGFP and a TGFP receptor. In another embodiment, the neutralizing antibody is selected from the group consisting of ab66043, ab61213, ab31013, 341-BR, 241-R2, AF-241-NA, AF1003, 1600-R2, AF532, MAB1835, MAB240, MAB2411, MAB532, 3C11, V, D-12, 2E5, 500-M66, TB21, H- 100, T-19, V-22, G-16, R-20, RM— 10-3A11, C-4, D-2, E-6, 1-20, L-21, S-20, T-20, H- 567, C-16, A-4, C-20, H-280, F-20, ABF17, H-112, 3711, 56E4, LY3022859, and LY238770.

In some embodiments, the TGFP pathway inhibitor is a TGFP pathway antagonist. The TGFP pathway antagonist has affinity for a TGFP pathway target, such as a TGFp receptor, and binding to the TGFp pathway target disrupts or prevents the interaction between the TGFP pathway target and its cognate binding partner to inhibit or decrease activity of the TGFp pathway target. In one embodiment, the antagonist includes agents that bind TGFP and prevent TGFP from binding to a TGFP receptor, such as blocking (neutralizing) antibodies specific for a TGFP (NAbs) or TGFP receptor (Types I, II or III) such as those described by Dasch et al. (J. Immunol. (1989) 142: 1536) and Lucas et al. (J. Immunol. (1990) 145: 1415), soluble TGFp receptors, protease inhibitors that inactivate a protease responsible for activating a precursor TGFP into mature TGFP, and combinations thereof. Examples of such antagonists include monoclonal and polyclonal antibodies directed against one or more isoforms of TGFp (U.S. Pat. No. 5,571,714 and PCT patent application WO 97/13844), TGFp receptors, fragments thereof, derivatives thereof and antibodies directed against TGFp receptors (U.S. Pat. Nos. 5,693,607, 6,008,011, 6,001,969 and 6,010,872 and PCT patent applications WO 92/00330, WO 93/09228, WO 95/10610 and WO 98/48024); latency associated peptide (WO 91/08291), large latent TGFp (WO 94/09812), fetuin (U.S. Pat. No. 5,821,227), decorin and other proteoglycans such as biglycan, fibromodulin, lumican and endoglin (U.S. Pat. Nos. 5,583,103, 5,654,270, 5,705,609, 5,726, 149, 5,824,655, 5,830,847, 6,015,693 and PCT patent applications WO

91/04748, WO 91/10727, WO 93/09800 and WO 94/10187). Further examples of such antagonists include follistatin, somatostatin (PCT patent application WO 98/08529), mannose-6-phosphate or mannose-1 -phosphate (U.S. Pat. No. 5,520,926), prolactin (PCT patent application WO 97/40848), insulin-like growth factor II (PCT patent application WO 98/17304), IP-10 (PCT patent application WO97/00691), arg-gly-asp containing peptides (U.S. Pat. No. 5,958,411 and PCT patent application WO 93/10808 and), extracts of plants, fungi and bacteria (European patent application 813875, Japanese patent application 8119984 and U.S. Pat. No. 5,693,610), antisense oligonucleotides (U.S. Pat. Nos. 5,683,988, 5,772,995, 5,821,234 and 5,869,462 and PCT patent application WO 94/25588), and a host of other proteins involved in TGFP signaling, including SMADs and MADs (European patent application EP 874046, PCT patent applications WO 97/31020, WO 97/38729, WO 98/03663, WO 98/07735, WO 98/07849, WO 98/45467, WO 98/53068, WO 98/5,5512, WO 98/56913, WO

98/53830, and WO 99/50296, and U.S. Pat. Nos. 5,834,248, 5,807,708 and 5,948,639) and Ski and Sno (G. Vogel, Science, 286:665 (1999) and Stroschein et al., Science, 286:771-74 (1999)) and fragments and derivatives of any of the above molecules that retain the ability to inhibit the activity of TGFp. In one embodiment, the antagonist is selected from the group consisting of HtrAl, decorin, biglycan, fibromodulin, lumican, endoglin, somatostatin, follistatin, RAP-1332, pirfenidone (5-methyl-l-phenyl-2(lH)- pyridone), soluble ectodomains of TGFp receptor type II (RII) or betaglycan (BG), and any combination thereof.

In another aspect, the present invention includes a composition comprising a BETi and at least one selected from the group consisting of TRFM33 or a fragment thereof, and a nucleic acid encoding TRFM33, wherein the TGFP pathway inhibitor sensitizes cells to the BETi.

In some embodiments, the composition of the present invention comprises TRFM33. TRFM33 includes a polypeptide that is recombinantly or synthetically produced. In one embodiment, TRFM33 comprises an isolated TRFM33 polypeptide. In another embodiment, TRFM33 comprises a polypeptide having a GenBank

Accession No selected from the group consisting of NP_056990.3, NP_148980.2, NP_001073299.1 and NP_444400.2. In some embodiments, TRFM33 includes at least one post-translational modification.

In some embodiments, the composition of the present invention comprises a functional fragment of TRFM33. The functional fragment of TRFM33 includes fragments that retain at least one biological, physiological, and/or pharmacological property of TRFM33. In one embodiment, the functional fragment of TRFM33 comprises about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or any percent therebetween of TRFM33.

In some embodiment, the composition of the present invention comprises a nucleic acid encoding TRFM33. In one embodiment, the nucleic acid encoding TRFM33 comprises a nucleic acid sequence having a GenBank Accession No selected from the group consisting M_015906, M_033020, NM_001079830, and

M_053170.

In another embodiment, the nucleic acid encoding TRXM33 is an expression vector comprising a TRIM33 gene. In another embodiment, the the vector is a viral vector, such as an adenoviral vector, an adeno-associated virus vector, a retroviral vector, a lentiviral vector, and the like.

Methods

The present invention also includes a method for increasing expression of TRIM33 or inhibiting a TGFP signaling pathway to increase anti -turn or sensitivity. As described herein, loss of TREVI33 or over-activation of a TGFP signaling pathway decreases sensitivity to BET inhibitors. Administering a composition that includes a TREVI33 or a TGFP pathway inhibitor with a BETi to a subject in need thereof increases anti -tumor sensitivity in a cell or tumor in the subject.

In one aspect, the invention includes a method of increasing anti -tumor sensitivity to a bromodomain and extraterminal domain protein inhibitor (BETi) in a cell in a subject. The method comprising administering a composition comprising a TGFP pathway inhibitor and a BETi to a subject in need thereof, wherein the TGFP pathway inhibitor sensitizes the cell to the BETi.

In another aspect, the invention includes a method of treating a tumor comprising administering to a subject in need thereof a composition comprising a TGFP pathway inhibitor and a bromodomain and extraterminal domain protein inhibitor (BETi), wherein the TGFP pathway inhibitor sensitizes the tumor to the BETi.

In yet another aspect, the invention includes a method of increasing anti-tumor sensitivity of a cell in a subject to a bromodomain and extraterminal domain protein inhibitor (BETi). The method comprising administering a composition comprising a BETi and one selected from the group consisting of TREVI33 or a fragment thereof, and a nucleic acid encoding TREVI33 to a subject in need thereof, wherein the TREVI33 sensitizes the cell to the BETi.

In still another aspect, the invention includes a method of treating a tumor comprising administering to a subject in need thereof a composition comprising a BETi and at least one selected from the group consisting of TREVI33 or a fragment thereof, and a nucleic acid encoding TREVI33, wherein TREVI33 sensitizes the tumor to the BETi. In one embodiment, the tumor is a cancer selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. In another embodiment, the subject has a cancer, such as a cancer selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer.

The methods and compositions of the present invention are useful to increase an anti-tumor response in a cellular target to a BETi by increasing a basal level of the anti- tumor response or resensitizing the cellular target to the BETi. In another embodiment, the cell or tumor is BETi resistant. In yet another embodiment, cell or tumor has acquired decreased BETi sensitivity. In still another embodiment, administration of the composition to the subject increases an anti-tumor response to the BETi. Nucleic Acids

The present invention includes, in some embodiments, a composition comprising a TGFP pathway inhibitory nucleic acid, and in some embodiments, a composition comprising a nucleic acid encoding TREVI33. Such nucleic acids are introduced into a cell for the benefit of a subject.

Methods of introducing nucleic acids into a cell include physical, biological and chemical methods. Physical methods for introducing a polynucleotide, such as DNA like cDNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. DNA, like cDNA, can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)),

(ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany). DNA can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl

phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or

chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also

encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

Pharmaceutical Compositions

The invention also encompasses the use of a pharmaceutical composition of the invention to practice the methods of the invention. In one aspect, the invention includes a pharmaceutical composition comprising the composition as described herein and a pharmaceutically acceptable carrier. In another aspect, the composition described herein is used in the manufacture of a medicament for the treatment of a tumor or cancer in a subject in need thereof. In yet another aspect, the invention includes a pharmaceutical composition comprising the composition as described herein in combination with another therapeutic agent used in the treatment of a tumor or cancer. Such pharmaceutical compositions may be provided in a form suitable for

administration to a subject, and may comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The composition described herein may comprise a physiologically acceptable salt, such as a compound contemplated within the invention in combination with a physiologically acceptable cation or anion, as is well known in the art.

Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi- dose unit.

In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction:

Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out embodiments of the present invention, and are not to be construed as limiting in any way.

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. As acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Described herein, through pooled shRNA screening of colorectal cancer cells, tripartite motif-containing protein 33 (TRTM33) was identified as a factor promoting sensitivity to BETi. Loss of TRTM33 was

demonstrated to reprogram cancer cells to a more resistant state through at least two mechanisms. TREVI33 silencing attenuates downregulation of MYC in response to BETi. Moreover, loss of TREVI33 enhances TGFP receptor expression and signaling, and blocking TGFp receptor activity potentiates the anti-proliferative effect of BETi. Described herein is a mechanism for BETi resistance and combining inhibition of TGFP signaling with BET bromodomain inhibition may offer new therapeutic benefits.

The Materials and Methods used in the performance of the experiments disclosed herein are now described.

Cell lines, antibodies and drugs. Cell lines 293 T, RKO, HCT 15, HCT 116,

LoVo, SW620, SW837, SK-CO-1, SW480, SW1463, MDA-MB-231, MDA-MB-415, MDA-MB-468, ZR-75-1, LNCap and PC-3 were obtained from ATCC and maintained as suggested. Antibodies were purchased from Cell Signaling Technology and Abeam: TRFM33 (#13387), SMAD2 (#5339), pSMAD2 (#3108), SMAD4 (#9515), BRD4 (#13440), actin (#3700) and MYC (ab32072). Recombinant human TGFpl was from Cell Signaling Technology (#8915LC). (+)-JQl (11187) was purchased from Cayman Chemical and LY2157299 (S2230) was purchased from Selleck Chemical. GS-626510 was synthesized at Gilead Sciences.

Stable knockdown and expression cell lines. Lentiviral expression vectors for shRNAs in the pLKO.1 puro vector (Sigma) were used to stably knockdown TRFM33, TpRII or SMAD4. For stable knockdown of two genes, the shTRFM33-B5 sequence was cloned into pLKO. l blast (Addgene #26655) to silence TRFM33 expression. The shRNA target sequences used are listed in Table 1. For expression of TGF RII and TRFM33, cDNAs from Addgene #19147 and Addgene #15734 respectively, were cloned into pLentiCMV-hygro(DEST) (Addgene #17454)) through Gateway cloning (Invitrogen). Seven silent mutations were made to TRIM33 cDNA to render resistance to shTREVI33-B5. MYC lentiviral expression vector is from Addgene (#46970).

Table 1 : shRNA target sequences.

shRNA Target sequence shCTRL C AAC AAGATGAAGAGC AC C AA SEQ ID NO: l shTRIM33-B5 GTACTAGTTGTGAAGACAATG SEQ ID NO:2 shTRIM33-A12 GCTCCTGGTTATACTCCTAAT SEQ ID NO: 3 shT RII-3 GCTCCCTAAACACTACCAAAT SEQ ID N0:4 shT RII-4 AATGACGAGAACATAACACTC SEQ ID NO: 5 shSMAD4-3 CAGATTGTCTTGCAACTTCAG SEQ ID NO:6 shSMAD4-4 TACCATACAGAGAACATTGGA SEQ ID NO:7

Cell lysis for immunoblotting and immunoprecipitation. For immunoblotting, cells in 6-well plates were quickly rinsed twice with PBS and directly lysed in 150 μΙ_, SDS lysis buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol). The lysate was then transferred to 1.5 mL Eppendorf tubes and heated for 10 min at 95-100 °C with intermittent vortexing. After spinning to remove any undissolved material and measuring the protein concentration using BCA assay, 20-40 μg total lysate was fractionated by SDS-PAGE and transferred to nitrocellulose membrane for

immunoblotting. For immunoprecipitation, cells were rinsed quickly with ice-cold PBS and lysed in buffer (50 mM HEPES pH7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 25 mM NaF, 1 mM Na₃V0₄, 1 mM PMSF and Roche Complete Protease Inhibitor Cocktail) on ice for 15 min. Scraped cell lysate was centrifuged at 13,200 rpm for 10 min at 4 °C and 1 mg of supernatant was incubated with 1-5 μg primary antibody overnight at 4 °C. 25 μΙ_, of protein A sepharose 4B (Invitrogen) was added to the tube for another 2 h, and the precipitate was washed 3 times and then eluted in 60 μΙ_, of Laemmli sample buffer. 20 μΙ_, of the elution was used for immunoblotting.

Quantitative RT-PCR analysis. Total RNA was extracted using an R EASY® mini kit (Source) with on-column DNA digestion. 1 μg of total RNA was used for cDNA synthesis with the ISCRIPT™ cDNA synthesis kit (Bio-Rad) as per

manufacturer's suggestion. Real-time PCR was performed on a Bio-Rad CFX

CONNECT™ Real-Time System and relative mRNA level was calculated in CFX Manager software using the 2^A (-AACt) method. GAPDH mRNA was used as internal control. PCR primer sequences are listed in Table 2.

Table 2: PCR primer sequences (5 '-3')

Gene

Forward primer Reverse primer

GAPDH GAAGGTGAAGGTCGGAGTCA TTGAGGTCAATGAAGGGGTC

SEQ ID NO:8 SEQ ID NO:9

TRIM33 GGAGTGCTTGCATGTTGAG CCAATTCACTTTCTAGATGCAGG

SEQ ID NO: 10 SEQ ID NO: 11

TRIM33 TTACAGCAATAGAGCTAATCCC ACAACGTTTGCCTGTATGG

SEQ ID NO: 12 SEQ ID NO: 13

MYC GGCTCCTGGCAAAAGGTCA CTGCGTAGTTGTGCTGATGT

SEQ ID NO: 14 SEQ ID NO: 15

TGFBR2 GTAGCTCTGATGAGTGCAATGAC CAGATATGGCAACTCCCAGTG

SEQ ID NO: 16 SEQ ID NO: 17

HBE1 ATGGTGCATTTTACTGCTGAGG GGGAGACGACAGGTTTCCAAA

SEQ ID NO: 18 SEQ ID NO: 19

PDE4B AACGCTGGAGGAATTAGACTGG GCTCCCGGTTCAGCATTCT

SEQ ID NO:20 SEQ ID NO:21

ZNF474 ATATCGGAAAGCCAGCTTAGC GACCCAAATTCTCGGCCAC

SEQ ID NO:22 SEQ ID NO:23

RGS4 ACATCGGCTAGGTTTCCTGC GTTGTGGGAAGAATTGTGTTCAC

SEQ ID NO:24 SEQ ID NO:25

BCL2A1 TACAGGCTGGCTCAGGACTAT CGCAACATTTTGTAGCACTCTG

SEQ ID NO:26 SEQ ID NO:27

MAP1LC3C CCCAAGCGTCAGACCCTTC GGGGAACTTTGCCCGGATT

SEQ ID NO:28 SEQ ID NO:29

FBX08 AGCAAGGCTACCTCACCAGA TCCTTCCTGTTCTTTCGATTTCC

SEQ ID NO:30 SEQ ID NO:31

CHCHD3 GAGGCGGACGAGAATGAGAAC ACCAGAATACCGCTGAGACTTC

SEQ ID NO:32 SEQ ID NO:33

DUSP10 ATCGGCTACGTCATCAACGTC TCATCCGAGTGTGCTTCATCA

SEQ ID NO:34 SEQ ID NO:35

ITGA6 ATGCACGCGGATCGAGTTT TTCCTGCTTCGTATTAACATGCT

SEQ ID NO:36 SEQ ID NO:37

MYCT1 CAATCGGGCTGGTACTTGGAG CGTGGGTGTAAGAAGACCTAGA

SEQ ID NO:38 SEQ ID NO:39

TRIML2 GCCACCGAGCTAGAGGAGAT CTTGAGCAATGCCAAGGTGC

SEQ ID NO:40 SEQ ID NO:41

MARCH4 CTGTAAGGAGAAGACCGAGGA ATCCACTTGATGAGGCAAGGC

SEQ ID NO:42 SEQ ID NO:43

ADRA2C GCCTCAACGACGAGACCTG CCCAGCCCGTTTTCGGTAG

SEQ ID NO:44 SEQ ID NO:45

ERRFIl CTGGAGCAGTCGCAGTGAG GCCATTCATCGGAGCAGATTTG

SEQ ID NO:46 SEQ ID NO:47 Cumulative cell growth assay. RKO cells (3 x 10⁵) transduced with the indicated virus were plated in a single well of a 6-well plate at day 0 in the presence or absence of inhibitors. Three days later cells were detached, counted, and 3 x 10⁵ cells were transferred to a new well. The process was repeated until day 15. The cumulative cell number was then calculated from fold changes and the individual cell counts at each passage.

Crystal violet cell proliferation assay. Cells (5-20 x 10³) were plated in each well of a 6-well plate with 3 mL of media with or without inhibitors and cultured for 14 days undisturbed. Medium was aspirated, and cells were stained with crystal violet staining solution (0.05% w/v crystal violet, 1% formaldehyde, 1% methanol in PBS) for 30 minutes and washed with water several times. Stained plates were then air-dried and imaged with CHEMIDOC® using Image Lab software (Bio-Rad). To quantify the crystal violet staining, 1 mL of 10% acetic acid was added to each well to solubilize the stain for 20 min and the stain was diluted 1 :4 in water and absorbance was measured at 590 nm.

Growth inhibition assay and IC₅₀ value determination. Cells (1000 per well) were plated in 96-well plates in duplicate with 1 :3 serial dilutions of BETi ranging from 0.169 nM to 10 μΜ or 0.1% DMSO vehicle and cultured for 120 h. The end point relative viable cell number was determined using CELLTITER GLO^® by quickly decanting the media, adding 100 μΕ of 1 :2 CellTiter Glo reagent diluted in PBS to the well and incubating for 10 min. The luminescence of each well was read with a

TEC AN Infinite® MIOOOPro plate reader. IC₅₀ values were calculated with

GRAPHPAD PRISM 6® by fitting the data to the "3 -parameter log (inhibitor) vs response" equation. At least three independent growth inhibition assays were performed for each pair of cell lines to derive mean IC₅₀ values.

shRNA screening. The Mission human shRNA library (Luo et al., 2008, Proc Natl Acad Sci USA 105(51):20380-20385) generated in pLKO lentiviral delivery vectors by The RNAi Consortium (TRC) was obtained as arrayed bacterial stocks (Sigma). All shRNAs targeting 517 genes annotated as protein kinases (5634 shRNAs in total) and 85 non-targeting control shRNA vectors were picked from the library, cultured on LB-agar plates and plasmid DNA was prepared from a mixture of these cultures using GeneElute HP Endotoxin Free Plasmid Maxi-prep kit (Sigma). Lentiviral particles were generated by co-transfecting 293T cells with the pLKO plasmid mixture, pCMV dR8.91 packaging vector, and pCMV-VSV-G envelope vector in a 10: 10: 1 ratio. Viral supernatant was collected 48 and 72 h after transfection and stored at -80°C. RKO cells were transduced by incubating for 24 h with the pool of shRNA-expressing viruses diluted to give a MOI (multiplicity of infection) of -0.3 to ensure that most of the cells received a single viral integration. Care was taken to ensure that the initial number of infected cells exceeded 6 x 10⁶ resulting in at least 1000-fold coverage of the -6000 unique shRNAs in the pool, and 1000-fold coverage was strictly maintained at all steps of the screening protocol. Infected cells were selected with 1 μg/mL

puromycin for 2 days, and 6 x 10⁶ cells were removed for genomic DNA extraction to serve as the TO reference sample. Remaining cells were split into 5 parallel 15 cm plates each with 6 x 10⁶ cells to be treated with 0.1 % DMSO vehicle, 0.1 μΜ JQ1, 0.3 μΜ JQ1, 0.05 μΜ GS-626510 or 0.1 μΜ GS-626510. When plates approached confluence, 6 x 10⁶ cells were re-seeded into fresh plates until T4. Genomic DNA from TO and the five T4 samples was extracted and shRNA integrants were PCR amplified with barcoded primers. All the samples were sequenced on an Illumina HiSeq instrument and the relative abundance of each shRNA from T4 was compared to those of TO. To minimize error due to stochastic effects, hairpins with fewer than 50 raw reads in TO were not considered. Each sample in the sequencing library preparation was normalized to a total read depth of 1 x 10⁶ to correct for variation in read depth across samples. RIGER algorithm in the GENE-E java package from the Broad Institute was used to rank each gene by their enrichment. The log fold change metric and second-best hairpin method was used to score the genes so that at least two hairpins against each gene were enriched in each condition.

RNAseq data analysis and gene set enrichment analysis. RKO cells expressing shCTRL or shTREVI33 were treated with 0.1% DMSO vehicle, 1 μΜ JQ1 or 0.3 μΜ GS-626510 for 3 h, and mRNA was extracted using RNEASY® mini kit with on column DNase I digestion option (Qiagen) and submitted to the Yale Center for Genome Analysis for RNAseq analysis. Low quality reads and bases were trimmed, and filtered reads were then mapped to the human reference genome (hgl9) using Tophat™ v2.0.13. Only reads that mapped to a single unique location within the genome were reported. Tophat alignments from duplicate RNAseq experiments were then processed through DESeq to produce one differential expression data set. When a single RNAseq data set was analyzed, differential expression was calculated as fold change in the normalized raw counts of each transcript. For gene set enrichment analysis, the R software package edgeR was used to normalize the gene level read counts across samples. Genes with less than one shortread count per million (CPM) in at least one sample were filtered out to remove genes with low levels of expression. Generalized linear regression in edgeR was then used to estimate log₂ fold changes and p values. To identify the genes that respond differently to BETi in the shTRXM33 cells relative to the shCTRL cells, the following contrast was specified in the edgeR analysis: (BETi in shTRIM33 - DMSO in shTRIM33) - (BETi in shCTRL - DMSO in shCTRL). Multiple testing was controlled by using false discovery rate (FDR). Next, the estimated p values of all the genes were converted using the zScores function in the R package gCMAP to Z scores to generate the ranked list of genes. The ranked list of genes was then analyzed with GSEA Preranked included in the Broad GSEA java tool for Gene set enrichment analysis against MSigDB, C2 (curated gene sets), C6

(oncogenic signatures), and C7 (immunologic signatures) collections (total 5285 gene sets). The Results of the experiments disclosed herein are now described.

Example 1: Pooled shRNA library screening identified TRIM33 as a negative regulator of BETi resistance

To identify genes whose loss confers resistance to the anti-proliferative effects of BET bromodomain inhibitors, a pooled shRNA screen was performed in a BETi- sensitive colorectal cancer cell line (RKO). Screening was carried out in the presence of one of two structurally unrelated inhibitors: the widely used compound JQl and a novel BETi GS-626510 (Figure 1A). GS-626510 binds with high affinity and specificity to BET family bromodomains (Figure IB, Table 3). Both JQl and GS-626510 potently inhibited growth of RKO cells with IC₅₀ values of 81 nM and 33 nM respectively (Figure 1C). As anticipated for BRD4 inhibition, both compounds strongly decreased MYC levels in RKO cells (Figure ID). RNAseq analysis showed a strong correlation between genes up- and down-regulated following 3 h treatment of RKO cells with 1 μΜ of JQl or 0.3 μΜ of GS-626510 (Figure IE), suggesting that growth suppression by these compounds is attributable to a common mechanism of action. Table 3 : Kd values of bromodomains to BETi GS-626510

GS-626510 GCN5L2 KAT2A > 10,000

GS-626510 PBRM1 (2) PBRM1 > 10,000

GS-626510 PBRM1 (5) PBRM1 > 10,000

GS-626510 PCAF KAT2B > 10,000

GS-626510 SMARCA2 SMARCA2 > 10,000

GS-626510 SMARCA4 SMARCA4 > 10,000

GS-626510 TAF1 (2) TAF1 = 8,000

GS-626510 TAF1L (2) TAF1L > 10,000

GS-626510 TREVI24 (Bromo.) TRIM24 = 4,100

GS-626510 TRIM24 (PHD, TRIM24 6,900

Bromo.)

GS-626510 TRIM33 (PHD, TRIM33 > 10,000

Bromo.)

GS-626510 WDR9 (2) BRWD1 > 10,000

A custom lentiviral shRNA library was generated containing 5634 shRNA constructs targeting 517 genes annotated as protein kinases and 85 non-targeting control shRNAs. RKO cells were infected with the pooled shRNA virus, and following puromycin selection for infected cells, 6 x 10⁶ cells were removed for genomic DNA extraction to serve as a reference (TO) population. The remaining cells were placed into each of 5 different inhibitor conditions: DMSO vehicle control and low and high doses of either JQ1 or GS-626510 (Figure IF). Cells were allowed to proliferate and were passaged when they approached confluence. This treatment was maintained until cells reached passage 4 (T4). Genomic DNA was extracted and the relative abundance of each shRNA in each treatment condition at T4, and in the reference TO condition, was assessed by PCR amplifying the integrated shRNA followed by next generation sequencing (Figure IF). This allowed calculation of the relative enrichment or depletion of each individual shRNA at T4 compared with TO.

As the library contained multiple shRNAs targeting each gene, RIGER analysis was used to identify and rank genes preferentially targeted by hairpins enriched upon drug treatment but not in the DMSO-treated control cells. These genes presumably encode proteins that promote susceptibility to BETi. Silencing expression of these genes thus causes drug resistance, resulting in cells harboring their respective hairpins being enriched at the end of the screen. Strikingly, TRIM33 was the top ranked enriched target gene in all four BETi- treated conditions, but was not enriched in the absence of inhibitor (Figure 1G).

Tracking individual shRNAs revealed clear enrichment of most shRNAs targeting TRJJVI33 at T4 in the presence of JQl or GS-626510 (Figure 1H). By contrast, TRJJVI33 hairpins appear to be preferentially depleted in the DMSO vehicle control sample.

An independent replicate of this screen, carried out to passage 5 (T5) produced very similar results with TREVI33 ranked in the top 3 of all four drug conditions (Figure 1G). Notably, TRJJVI24, the most closely related TRJJVI33 family member, was also highly enriched in all four inhibitor treated conditions but not in the DMSO control (Figure 1G), supporting the potential functional relevance of TRJJVI33 to modulate BETi sensitivity.

Thus, data from two independent screens, each performed with two doses of two chemically unrelated BET bromodomain inhibitors, indicated that TRJJVI33 knockdown conferred a selective growth advantage in BETi-treated RKO cells.

TRJJVI33 and TRJJVI24 were included in the shRNA library on the basis of early reports identifying TRJJVI24 and TREVI28 as protein kinases, but the absence of a recognizable kinase catalytic domain and lack of subsequent verification suggests that these proteins are unlikely to have such activity. Example 2: BETi resistance in shTRIM33 cells was due to the specific loss of TRIM33 protein

To verify the screening data suggesting that TRJJVI33 promotes sensitivity to BETi in cancer cells, a stable TRJJVI33 knockdown was established in RKO cells by lentiviral transduction and evaluated their sensitivity to JQl or GS-626510. Among four individual shRNAs tested, shTRJJVI33-B5 (hereafter referred to as shTRJJVI33 unless otherwise noted) was chosen to silence expression of TRJJVI33 as it produced the most efficient TRJJVI33 knockdown at the protein level (Figure 2A).

Comparison of cell proliferation of shCTRL and shTRJJVI33 cells in 15-day cultures confirmed that knocking down TRJJVI33 conferred a growth advantage in the presence of BETi (Figure 2B). Notably, consistent with the screening data, shTRJJVI33 cells cultured in the absence of inhibitor exhibited a growth disadvantage (Figure 2B), suggesting that the effect of TRJJVI33 on growth in the presence of BETi was not due to a basal increase in cell proliferation. These studies were extended to compare the potency of JQl and GS-626510 in shCTRL and shTRJJVI33 cells. Cells were incubated with varying concentrations of JQl or GS-626510 for 5 days and the relative cell number was determined. TRJJVI33 knockdown produced a rightward shift in the growth inhibition curves for both JQl and GS-626510 (Figure 2C). Multiple replicates revealed that the IC₅₀ value of JQl and GS-626510 was increased by approximately 3-fold in shTRJJVI33 cells, suggesting the shTRJJVI33 cells were more resistant to BETi (Figure 2D). This effect was not limited to RKO cells as similar experiments performed in a panel of colorectal, breast and prostate cancer cell lines revealed that TRJJVI33 knockdown also decreased sensitivity to JQl and GS-626510 in a subset of the cell lines tested (Figures 2E and Table 4).

Finally, in prolonged culture, TRJJVI33 knockdown facilitated outgrowth of BETi-treated RKO cells (Figure 2F). Similar effects were observed with a different shRNA targeting TRJJVI33 (A 12) (Figures 2 A and 2G), suggesting that the results were not due to off target effects.

To further confirm that BETi resistance caused by TRJJVI33 -directed shRNA was due to the loss of TRJJVI33 protein and not due to off target silencing of other genes, rescue RKO cell lines were generated re-expressing a knockdown-resistant TRJJVI33 cDNA (Figures 2H and 21). shTRJJVI33 cells re-expressing TRJJVI33 (pLenti- TRJJVI33), but not those infected with an empty vector (pLenti-EV), became more sensitive to both JQl and GS-626510 in long-term culture assays (Figures 2H, bottom panel, and 2J). Furthermore, in these experiments, overexpression of TRIM33 in shCTRL cells increased sensitivity to both compounds (Figures 2H, top panel, and 2J). Together, these data support the idea that TRJJVI33 promotes sensitivity to BET bromodomain inhibition.

Table 4

SKCOl 0.495±0.050 1.404±0.231 0.259±0.035 0.649±0.120

SW480 0.172±0.060 0.376±0.147 0.161±0.066 0.285±0.142

SW1463 0.603±0.151 0.794±0.437 2.107±1.739 0.974±0.434

RKO 0.065±0.005 0.191±0.027 0.026±0.005 0.071±0.013

MDA-MB-231 0.114±0.019 0.267±0.025 0.085±0.022 0.216±0.024

MDA-MB-415 0.334±0.012 0.401±0.044 0.375±0.093 0.378±0.091

ZR75-1 0.215±0.100 0.210±0.099 0.145±0.069 0.150±0.072

MDA-MB-468 0.166±0.006 0.133±0.021 0.122±0.023 0.087±0.011

LNCap 0.033±0.006 0.049±0.019 0.011±0.001 0.031±0.012

PC3 0.12±0.008 O. l l liO.011 0.05±0.011 0.055±0.013

Example 3: TRIM33 knockdown maintains MYC expression following BETi

Given the established role of both TRIM33 and BET proteins as transcriptional regulators, it was hypothesized that shTREVB 3 -mediated BETi resistance could be due to deregulated gene transcription. RNAseq was used to investigate changes in gene expression resulting from treatment with BETi and with loss of TREVI33. RNAseq was performed in shCTRL and shTRIM33 cells after 3h treatment with JQ1 (1 μΜ), GS- 626510 (0.3 μΜ) or vehicle control (DMSO). Results from two independent replicate experiments were analyzed by DESeq. Results consistent with RNAseq data were obtained by measuring mRNA levels for 15 genes by qRT-PCR (Figures 3A-3E).

Similar to previous reports, 3 -hour BETi treatment had a broad impact on gene expression: among the 11,277 genes reliably detected by RNAseq, approximately 1200 genes changed by greater than 2-fold (Figure 3F-3G). Consistent with prior studies in other cell types, BETi treatment of RKO cells strongly reduced levels of MFC (5 to 6- fold). Furthermore, gene set enrichment analysis (GSEA) of transcripts down-regulated by both inhibitors revealed significant enrichment for genes having target motifs for MYC or the MYC co-activator MAZ in their promoter regions (20% of downregulated genes, Figure 3H).

In contrast to BET bromodomain inhibition, TRDVI33 KD influenced the expression of a relatively small fraction of genes (Figure 31). Following TRPM33 knockdown, 272 transcripts were up-regulated by at least 2-fold, while only 84 were down-regulated by at least 2-fold, arguing that TRDVI33 works preferentially as a transcriptional repressor rather than an activator. Notably, loss of TRPM33 had no effect on expression of BET genes (BRD2, BRD3 and BRD4) themselves and did not affect BRD4 protein levels (Figure 3 J).

Repression of MYC is believed to be a major mechanism by which BETi suppress growth of some cell types (10, 12). It was therefore examined whether there was a potential role for MYC in mediating the effect of TRFM33 knockdown.

Consistent with the RNAseq data (Figure 4 A), 3hr treatment with either JQl or GS- 626510 strongly suppressed MYC mRNA levels as measured by qRT-PCR (Figure 4B). Furthermore, presumably due to the short (20-30 min) half-life of MYC protein, MYC protein levels were also strongly suppressed (Figure 4C).

While basal levels of MFC mRNA and protein were modestly increased in shTREVI33 cells, their downregulation by BETi was substantially attenuated (Figure 4B-4C). Furthermore, rescue of TRFM33 protein expression in shTRFM33 cells partially restored MYC sensitivity to JQl and GS-626510 (Figure 4B-4C). These results suggest that TRFM33 was required for the ability of BET inhibitors to maximally down-regulate MYC.

To determine whether stabilization of MYC may play a role in conferring resistance to BETi, MYC was stably over-expressed in RKO cells. Ectopically expressed MYC was resistant to BETi-mediated down regulation (Figure 4D). While RKO cells overexpressing MYC proliferated at the same rate as control cells, possibly reflecting the high basal levels of MYC expression in this cell line, MYC over- expressing cells had a growth advantage in long-term culture in the presence of JQl or GS-626510 (Figures 4E-4F). Thus, protection of MYC levels from downregulation is likely to contribute to BETi resistance in shTRFM33 RKO cells.

Consistent with a role for TRFM33 in regulation of MYC expression, chromatin immunoprecipitation (ChIP) revealed that TRFM33 associated with the MYC promoter in BETi-treated RKO cells (Figure 4G). Notably, BRD4 ChIP showed that BRD4 associated with similar sites in the MYC promoter and that BRD4 was displaced following BETi treatment (Figure 4H). These data suggest that BETi may suppress MYC expression by displacing BRD4 from the MYC promoter to allow recruitment of the transcriptional repressor TREVI33 at that site. In the absence of TRFM33, this negative regulation would be lost, rendering cells less sensitive to BETi

Example 4: TRIM33 knockdown potentiates TGFp signaling and inhibition of TGFp pathway increased BETi sensitivity While the efficacy of BETi has been linked to down-regulation of MYC expression in hematopoietic cancers and a subset of solid tumors, in other tumor cells BETi-mediated growth suppression is independent of MYC. Notably, in contrast to what was observed in RKO cells, MYC levels in another colorectal cancer cell line, SK-CO-1, were much less sensitive to either BETi treatment or TRFM33 knockdown (Figure 5A). Nonetheless, in this cell line TRFM33 knockdown conferred resistance to BETi (Figure 2E and Table 4). This observation suggests that other pathways in addition to MYC signaling can contribute to shTRFM33 cell resistance to BETi.

Gene set enrichment analysis (GSEA) of the RNAseq data revealed that the two signatures most differentially regulated by BETi-treatment in shCTRL vs. shTRFM33 RKO cells corresponded to genes targeted by TGFp signaling (Figures 5B-5E).

Modulation of TGFP target genes in the context of BET inhibition was of interest because TRFM33 has been implicated as a regulator of TGFp signaling. Furthermore, as TGFp signaling can promote resistance to other targeted therapies, it was investigated how the pathway was altered in shTRFM33 RKO cells.

Canonical TGFP signaling involves TGFP ligand-induced formation of heterotetramers containing dimers of the TGFP receptor I (TpRI) and TGFP receptor II (TpRII) serine-threonine kinases. Receptor clustering promotes TpRII phosphorylation of TpRI, leading to recruitment and phosphorylation of regulatory SMADs (SMAD2/3) by TpRI. Phosphorylated SMAD2/3 then binds to SMAD4 to form a complex that enters the nucleus to drive transcription of target genes. Stimulation of control and shTRFM33 cells with recombinant TGFpi ligand revealed that phosphorylation of SMAD2 was dramatically potentiated in the absence of TRFM33 (Figure 5F). Thus, under conditions where control cells exhibited barely detectable responses to TGFpi, SMAD2 was robustly phosphorylated in shTRFM33 cells. These changes were not due to differences in the expression level of SMAD2, which appeared uniform in control and shTRFM33 cells (Figure 5F).

TGFpl-induced phosphorylated SMAD2 (pSMAD2) seen in shTRFM33 cells co-immunoprecipitated with SMAD4, suggesting that the pSMAD2 enters functional complexes with SMAD4 (Figure 5G). Previous reports have suggested that TRFM33 antagonizes TGFP signaling by negatively regulating SMAD4 through either mono- ubiquitinating SMAD4 or competing with SMAD4 for phosphorylated SMAD2/3. However, knockdown of SMAD4 in shTRFM33 cells had no impact on the TGFpi- mediated induction of pSMAD2 (Figure 5H). These results suggest that loss of TREVI33 in RKO cells potentiates TGFp signaling upstream of SMAD4, at the level of SMAD2 phosphorylation.

The RNAseq data showed that the TpRII mRNA was upregulated ~2 fold in shTRIM33 cells (Figure 51). Furthermore, ChIP experiments revealed that TREVI33 association with the TPRII promoter was increased by BETi, while BRD4 association was decreased (Figures 5J-5K), similar to the manner that MYC is regulated by TREVI33 and BRD4.

To investigate whether TpRII up-regulation could underlie the potentiation of TGFp signaling that accompanies loss of TREVI33, two different shRNAs were used to knockdown TpRII and assess SMAD2 phosphorylation. Both shRNAs efficiently reduced TpRII mRNA levels (Figure 5L) and in shTREVI33 cells they dramatically reduced TGFP 1 -induced pSMAD2 levels (Figure 5M). Notably, when the sensitivity of these cells to JQ1 or GS-626510 growth inhibition was assessed, the loss of TpRII re- sensitized the shTREVI33 cells to the BET bromodomain inhibitors (Figures 5N, right panel, and 10B). TpRII knockdown also increased sensitivity of control cells to BETi (Figures 5G, left panel, and 50).

These data suggest that a combination of TGFp pathway inhibitors and BET bromodomain inhibitors may provide a more potent inhibition of cell growth and may provide a means to overcome resistance to BET bromodomain inhibitors. To test this possibility directly, the small molecule TpRI inhibitor, LY2157299 (galunisertib), was used.

Treatment with LY2157299 at a dose that can substantially block TGFpi- stimulated pSMAD2 (Figure 5P) greatly increased the anti -proliferative effect of JQ1 or GS-626510 in shTRFM33 cells, yet alone had no effect on cell growth (Figures 5Q and 5R). As with silencing of TpRII expression, chemical inhibition of TpRI also sensitized shCTRL cells to BETi. Interestingly, sensitization of shTREVI33 cells to BETi by treatment with LY2157299 was not accompanied by down regulation of MYC (Figure 5S). Thus, results with both TpRII knockdown and small molecule inhibitors of TpRI strongly suggest that TRFM33 promotes sensitivity to BETi at least in part through attenuation of TGFP signaling.

In order to determine whether enhanced TGFP signaling is sufficient to induce resistance to BETi, the consequences of over-expressing TpRII were examined. Robust TGFP 1 -induced SMAD2 phosphorylation was detected in TpRII-overexpressing cells, but not in the empty vector control cells (Figure 5T). However, this was insufficient to confer resistance to either JQ1 or GS-626510 (Figure 5U).

ΤβΜΙ over-expression also failed to protect MYC levels from downregulation by BETi treatment, even in the presence of exogenously added TGFpi (Figure 5V). Taken together, these results suggest that TRFM33 knockdown confers resistance to BETi through combined independent effects on MYC transcription and TGFP signaling.

The recent discovery of small molecule BET bromodomain inhibitors and the demonstration of their potent anti-proliferative activity in hematological and solid tumors highlights the potential of BETi as anti-cancer agents. Acquisition of drug resistance is a recurring limitation to targeted anti-cancer therapies. Described herein, pooled shRNA screening was used to identify genes, whose silencing protects RKO colon cancer cells from two chemically distinct BETi: the originally characterized BET inhibitor, JQ1, and a newly developed inhibitor GS-626510.

The top hit from the screen was TRFM33, with its close family member,

TRFM24, also being identified. These data suggest that loss of TRFM33 confers resistance to BETi, and this was confirmed in both short and long-term growth assays. Mechanistically, loss of TRFM33 reduces BETi-mediated down-regulation of MYC and sensitizes cells to TGFP signaling. Notably, inhibition of TGFP signaling re-sensitizes TRFM33 knockdown cells to BETi, suggesting that combining TGFP inhibitors with BETi may have therapeutic benefit.

Multiple studies have pointed to the oncogenic transcription factor MYC as a target of BETi in both hematopoietic and solid tumor cell lines. As shown previously for JQ1 treatment, both BETi employed in the study strongly decreased MYC mRNA and protein levels in RKO colorectal cancer cells, and potently inhibited cell growth. Previously it was shown that ectopic expression of MYC partly protected a multiple myeloma cell line from the growth inhibitory effects of JQ1, affirming MYC suppression to be a major mechanism underlying growth suppression by BETi.

By contrast, it was reported that in lung adenocarcinoma cell lines, JQ1 suppressed growth by downregulating the transcription factor FOSL1 rather than MYC, suggesting that alternative mechanisms may underlie the activity of BETi in solid tumors.

It was observed that MYC overexpression in RKO cells attenuated the efficacy of BETi. In addition, RNAseq analysis showed no reduction in FOSL1 transcript level upon BETi treatment of RKO cells. These observations support a central role for MYC as a key transcriptional target for BET bromodomains in colorectal cancer.

To identify genes whose loss conferred resistance to BETi, a pooled shRNA screen was performed with a library targeting genes annotated as protein kinases. It was found that loss of TRTM33 conferred resistance to either JQ1 or GS-626510 treatment, indicating that TRIM33 is required, in at least some cell types, for cells to be fully sensitive to BETi. In such cells, TREVI33 appears to promote downregulation of MYC by BETi.

Classically TREVI33, TREVI24 and TREVI28 act as potent transcriptional co- repressors when recruited to the promoters of target genes, and consistent with this mechanism, it was found that TREVI33 associates with the MYC promoter. Notably this association is enhanced by BETi, possibly due to direct competition between BRD4 and TREVI33 for binding at these sites.

Transcriptional modulation of MYC by TREVI33 could involve its E3 ligase activity, for example by triggering ubiquitin-mediated degradation of factors co- associated with promoter or enhancer regions. Attempts were made to test this model using TREVI33 mutants with impaired E3 ligase activity. Mutant TREVI33, while unable to restore JQ1 sensitivity in shTRFM33 cells, was also very poorly expressed, making it unclear whether its ligase activity was essential.

While this study was underway, several other groups reported alternative mechanisms of BETi resistance in other cancer lines. While the details of the specific adaptive pathways vary across cell types, a common feature of BETi resistance appears to be reactivation of BRD4-dependent target genes. Most of these reported models of resistance involve the emergence of mechanisms to drive MYC expression in the presence of BETi.

For example, up-regulation of the transcription factor GLI2 contributes to acquired BETi-resistance in pancreatic cancer cells (Kumar K, et al. (2015). Sci Rep 5:9489) by driving MYC expression, and in models of acute myeloid leukemia (AML), increased WNT signaling apparently bypasses BET bromodomain-mediated transcription to maintain MYC expression through utilization of a cryptic enhancer region.

The data described herein show that loss of TRFM33 partially protects MYC levels after BETi treatment, but loss of TRFM33 was not found to affect β-catenin levels or localization in RKO cells. Furthermore, as judged by RNAseq analysis, TRIM33 knockdown did not induce GLI2 in RKO cells.

Thus, while TRIM33 knockdown apparently confers BETi-resistance at least in part by preventing MYC downregulation, the pathways involved are distinct from those previously characterized. In cell lines where BETi function independently of MYC, reported mechanisms of resistance likewise appear to involve maintaining expression of BRD4-target genes. For example, triple negative breast cancer cells can acquire BETi- resistance through BRD4 hyperphosphorylation, which drives expression of target genes through interactions with the mediator complex in a manner independent of the acetylated lysine binding pocket of its bromodomains. As with each of these described mechanisms of resistance, sparing of critical target genes appears to be an important component of BETi resistance caused by loss of TRFM33.

While multiple studies have addressed adaptive responses to BETi and mechanisms of acquired resistance, much less is understood about factors controlling intrinsic susceptibility of tumors to BETi. Mutations in PIK3CA appear to confer intrinsic resistance to BETi in breast cancer cell lines, yet the molecular basis for this phenomenon is currently unknown. Across a panel of cell lines tested, no correlation between the level of TRIM33 protein expression and sensitivity to BETi was found, suggesting that TRFM33 status is not predictive of intrinsic resistance.

A short isoform of BRD4 was recently shown to be an inhibitor of DNA damage response signaling by influencing chromatin structure independently of its role as a transcriptional activator. Resistance to BETi could theoretically arise by reduction of DNA damage signaling, bypassing growth arrest. However, it was found that TRFM33 knockdown did not alter DNA damage signaling as assessed by γΗ2ΑΧ staining, suggesting that an alternative resistance pathway must be involved.

Consistent with prior reports implicating TRFM33 in TGFP signaling, loss of TRFM33 sensitized cells to TGFp. However, in contrast to previous studies suggesting that TRFM33 acts as an E3 ubiquitin ligase for SMAD4, loss of TRFM33 strongly enhanced SMAD2 phosphorylation independently of SMAD4 and was associated with increased expression of TpRII. TRFM33 may therefore act as a direct modulator of TpRII gene transcription.

Importantly, downregulation of TGFP signaling, either by silencing TpRII expression or with a small molecule inhibitor of TpRI, sensitized TRFM33 knockdown cells to BETi. Notably, while overexpressing TpRII was sufficient to sensitize cells to TGFpi, it did not prevent BETi-mediated suppression of MYC levels or cell growth. Thus, while promoting TGFP signaling cannot explain all of the effects of TREVI33 knockdown on BETi sensitivity, inhibition of TGFP signaling was sufficient to sensitize cells to BETi.

How increased TGFP signaling contributes to BETi resistance is unclear, but it is noteworthy that in non-small cell lung cancer cell lines, knockdown of mediator complex component MED12 confers resistance to multiple kinase inhibitors through a transcription-independent mechanism that results in stabilization of TpRII. Likewise, knockdown of the transcription factor, SOX10, in melanoma cell lines induces BRAF inhibitor resistance by induction of TpRII and TGFP signaling, ultimately resulting in increased receptor tyrosine kinase expression. In both of these contexts, TGFP-induced resistance to targeted therapies is associated with enhanced signaling through the ERK MAP kinase pathway.

Notably, in addition to up-regulated Wnt signaling, BETi-resistance in AML was also associated with up-regulated TGFP-dependent gene expression. These observations are consistent with the finding that potentiated TGFP signaling contributes to shTRFM33 -mediated BETi resistance and suggests that TGFP inhibitors may be valuable in combination with BETi in a range of malignancies. The ability of TGFP inhibitors to potentiate the effect of BETi and to function in the setting of TRFM33 loss provides a potential clinical strategy to overcome or delay acquired resistance.

Other Embodiments

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A method of increasing anti -tumor sensitivity to a bromodomain and

extraterminal domain protein inhibitor (BETi) in a cell in a subject, the method comprising administering a composition comprising a TGFP pathway inhibitor and a BETi to a subject in need thereof, wherein the TGFP pathway inhibitor sensitizes the cell to the BETi.

2. A method of treating a tumor comprising administering to a subject in need thereof a composition comprising a TGFP pathway inhibitor and a

bromodomain and extraterminal domain protein inhibitor (BETi), wherein the TGFP pathway inhibitor sensitizes the tumor to the BETi.

3. A method of increasing anti -tumor sensitivity to a bromodomain and

extraterminal domain protein inhibitor (BETi) of a cell in a subject, the method comprising administering a composition comprising a BETi and one selected from the group consisting of TRTM33 or a fragment thereof, and a nucleic acid encoding TRTM33 to a subject in need thereof, wherein the TRTM33 sensitizes the cell to the BETi.

4. A method of treating a tumor comprising administering to a subject in need thereof a composition comprising a bromodomain and extraterminal domain protein inhibitor (BETi) and at least one selected from the group consisting of TRTM33 or a fragment thereof, and a nucleic acid encoding TRTM33, wherein TRTM33 sensitizes the tumor to the BETi.

5. The method of any one of claims 1 or 2, wherein the TGFP pathway inhibitor is selected from the group consisting of a small molecule inhibitor, an inhibitory nucleic acid, neutralizing antibody, and an antagonist.

6. The method of claim 5, wherein the small molecule inhibitor is selected from the group consisting of SB431542, A83-01, RepSox, SB208, SB505124, LY364947, LY2157299, R268712, D4476, SB525334, GW788388, TEW-7197, and any combination thereof.

7. The method of claim 5, wherein the antagonist is selected from the group consisting of HtrAl, decorin, biglycan, fibromodulin, lumican, endoglin, somatostatin, follistatin, RAP-1332, pirfenidone (5-methyl-l-phenyl-2(lH)- pyridone), soluble ectodomains of TGFp receptor type II (RII) or betaglycan (BG), and any combination thereof.

8. The method of claim 5, wherein the neutralizing antibody specifically binds one selected from the group consisting of TGF and a TGF receptor.

9. The method of any one of claims 3 or 4, wherein the nucleic acid encoding TRIM33 is an expression vector comprising a TRIM33 gene.

10. The method of claim 9, wherein the vector is a viral vector.

11. The method of any one of claims 1-4, wherein the BETi is selected from the group consisting of JQ1, GS-626510, GS-5829, BMS 986158, RVX2135, CPI203, CPI-0610, ABBV-075, BAY1238097, INCB054329, FT-1101, PFI-1, I-BET151, ZEN-3365, 1-BET762, OTX015, TEN-010, and any combination thereof.

12. The method of any one of claims 2 or 4, wherein the tumor is a cancer selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer.

13. The method of any one of claims 1-4, wherein the subject has a cancer.

14. The method of claim 13, wherein the cancer is selected from the group

consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer.

15. The method of any one of claims 1 or 3, wherein the cell is BETi resistant.

16. The method of any one of claims 2 or 4, wherein the tumor is BETi resistant.

17. A composition comprising a TGFP pathway inhibitor and a bromodomain and extraterminal domain protein inhibitor (BETi), wherein the TGFP pathway inhibitor sensitizes cells to the BETi.

18. A composition comprising a BETi and at least one selected from the group

consisting of TRIM33 or a fragment thereof, and a nucleic acid encoding TRIM33, wherein the TGFP pathway inhibitor sensitizes cells to the BETi.

19. The composition of claim 17, wherein the TGFP pathway inhibitor is selected from the group consisting of a small molecule inhibitor, an inhibitory nucleic acid, a neutralizing antibody, and an antagonist.

20. The composition of claim 19, wherein the small molecule inhibitor is selected from the group consisting of SB431542, A83-01, RepSox, SB208, SB505124, LY364947, LY2157299, R268712, D4476, SB525334, GW788388, TEW-7197, and any combination thereof.

21. The composition of claim 19, wherein the neutralizing antibody specifically binds one selected from the group consisting of TGF and a TGF receptor.

22. The composition of claim 19, wherein the antagonist is selected from the group consisting of HtrAl, decorin, biglycan, fibromodulin, lumican, endoglin, somatostatin, follistatin, RAP-1332, pirfenidone (5-methyl-l-phenyl-2(lH)- pyridone), soluble ectodomains of TGFp receptor type II (RII) or betaglycan (BG), a neutralizing antibody against TGF or a TGF receptor, and any combination thereof.

23. The composition of claim 18, wherein the nucleic acid encoding TREVI33 is a vector comprising a TRIM33 gene.

24. The composition of claim 23, wherein the vector is a viral vector.

25. The composition of any one of claims 17 or 18, wherein the BETi is selected from the group consisting of JQ1, GS-626510, GS-5829, BMS 986158,

RVX2135, CPI203, CPI-0610, ABBV-075, BAY1238097, INCB054329, FT- 1101, PFI-1, 1-BET151, ZEN-3365, 1-BET762, OTX015, TEN-010, and any combination thereof. A pharmaceutical composition comprising the composition of any one of claims 17 or 18 and a pharmaceutically acceptable carrier.

Use of the composition of claim 26 in the manufacture of a medicament for the treatment of a tumor or a cancer in a subject.