WO2022229846A1

WO2022229846A1 - Treatment of cancer using a transforming growth factor beta receptor type 1 inhibitor

Info

Publication number: WO2022229846A1
Application number: PCT/IB2022/053870
Authority: WO
Inventors: Keith Anthony CHING; Maruja Elma Lira; Mack Harrison MABRY; Xinmeng MU; Robert Andrew ROLLINS; David Arlen SCHAER
Original assignee: Pfizer Inc.
Priority date: 2021-04-29
Filing date: 2022-04-26
Publication date: 2022-11-03

Abstract

This invention relates to methods for the treatment and selection of subjects having cancer who may benefit from administration of a TGFβr1 inhibitor.

Description

TREATMENT OF CANCER USING A TRANSFORMING GROWTH FACTOR BETA RECEPTOR TYPE 1 INHIBITOR FIELD OF THE INVENTION The invention relates to methods for the treatment and selection of subjects having cancer who may benefit from administration of a TGFβr1 inhibitor. BACKGROUND TGFβ signalling is an emerging pathway in cancer progression and has a role in modulating immune response, and in many other cancer pathways including metastasis and angiogenesis. Elevated TGFβ expression by tumor and stromal cells in the tumor microenvironment and activation of TGFβ receptor intracellular signalling is observed in many cancers (Massague J., TGFbeta in Cancer, Cell 2008, 134(2):215-30; Neuzillet C, et al., Targeting the TGFβ pathway for cancer therapy, Pharmacol Ther 2015, 147:22- 31). The TGFβ signalling pathway can be activated upon interaction of dimeric TGFβigand with its specific cell-surface transmembrane serine/threonine kinase receptors. The activated TGFβ ligand interacts with TGFβ type II receptors (TGFβr2), which recruit and phosphorylate TGFβ type I receptors (TGFβr1, also known as activin receptor-like kinase (ALK5)) at specific serine and threonine residues (Principe et al., TGF-ß: duality of function between tumor prevention and carcinogenesis, J Natl Cancer Inst 2014, 106(2): djt369). Activation of the TGFβ pathway in cancer cells can induce epithelial-to- mesenchymal transition (EMT) in which epithelial cells lose their apico-basal polarity and cell-cell adhesion, to become highly migratory mesenchymal cells, leading to metastasis.n addition to importance in tumor cell migration and metastasis, EMT has also beeninked to tumor cell evasion of immune surveillance (Akalay I, et al., Epithelial-to- mesenchymal transition and autophagy induction in breast carcinoma promote escaperom T-cell-mediated lysis, Cancer Res 2013, 73(8):2418-27). TGFβ is a potentmmunosuppressive agent on both innate and adaptive immune cells, including dendritic cells, macrophages, natural killer cells, and CD4+ and CD8+ T cells. Conversely, TGFβ has a key role stimulating the differentiation of immune-suppressive regulatory T (Treg) cells and myeloid derived suppressor cells (MDSCs) (Akalay I, et al., 2013). TGFβ pathways have key roles in disease progression and resistance to therapyn a broad spectrum of tumors (Neuzillet C., et. al., 2015; Colak S, et. al., Targeting TGF- ß signaling in cancer, Trends in Cancer 2017, 3(1):56-71). High TGFβ signatures and EMT gene expression are found in a variety of tumors (Mak MP, et al., A Patient-Derived, Pan-Cancer EMT Signature Identifies Global Molecular Alterations and Immune Target Enrichment Following Epithelial-to-Mesenchymal Transition, Clin Cancer Res 2016, 22(3):609-20). TGFβ is an important regulator of the tumor microenvironment by inducing expression of extracellular matrix (ECM) proteins and suppressing expression of chemokines and cytokines required for T cell tumor infiltration, creating a reactive stroma with dense ECM and a T cell excluded infiltrate phenotype, with peritumoral or stromal T cell localization (Hegde PS, et. al., The Where, the When, and the How of Immune Monitoring for Cancer Immunotherapies in the Era of Checkpoint Inhibition, Clin Cancer Res 2016, 22(8):1865-74). The compound, 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N- (1,3-dihydroxypropan-2-yl)nicotinamide (also referred to as “TGFβr1 inhibitor PF- 06952229” “PF-06952229” or “PF-‘2229”), is a potent and selective TGFβr1 (transforming growth factor beta receptor type 1) inhibitor, having the structure:

Formula (I) PF-06952229 and pharmaceutically acceptable salts thereof are disclosed innternational Application No. PCT/US2014/072922, which published as International Publication No. WO 2015/103355 on 9 July 2015, and U.S. Patent No.10,030,004 issued on 10 December 2019. The contents of each of the foregoing references arencorporated herein by reference in their entirety. In normal physiologic conditions, TGFβ proteins are involved in the regulation of critical cellular functions including tissue differentiation, cell proliferation, migration, apoptosis, immune surveillance, and maintaining tissue homeostasis. However, TGFβ is often overexpressed and activated in inflammation, fibrosis and even tumorigenesis. The dual function of tumor suppression and oncogenesis in the tumor microenvironment ishrough activation of cell signaling pathways. Active TGFβ induces a linear signaling pathway through activation of type II and type I receptor kinase. The ultimate response resulting from SMAD-dependent and SMAD-independent responses and ligand-inducedranscription (Derynck R, Zhang YE. Smad-dependent and Smad-independent pathwaysn TGF-beta family signaling. Nature.2003; 425: 577-584). SMAD4 is a common component of TGFβ, activin and BMP signaling (Lagna et al., Nature, 383:832-836, 1996; Zhang et al., Curr. Biol., 7:270-276, 1997; de Winter et al., Oncogene, 14:1891-1900, 1997). SMAD4 phosphorylation has thus far been reported only after activin stimulation of transfected cells (Lagna et al., 1996). After stimulation with TGFβ or activin SMAD4 interacts with SMAD2 or SMAD3, and upon BMP challenge a heteromeric complex of SMAD4 and SMAD1 have been observed (Lagna et. al., 1996). Upon ligand stimulation, SMAD complexes translocate from the cytoplasm to the nucleus (Hoodless et al., 1996; Liu et al., 1996; Baker and Harland, 1996; Macias-Silva et al., 1996), where they, in combination with DNA-binding proteins, may regulate generanscription (Chen et al., Nature 1996, 383:691-696). The cell cycle inhibition or growth inhibitory effect of TGFβ canonical SMAD4 - dependent signaling pathway defines its tumor suppressive role whereas in carcinogenesis overexpression of TGFβ and loss of TGFβ canonical SMAD4 dependent mediated tumor suppressive action leads to tumor progression (Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signaling. Nature. 2003, 425: 577-584). The identification of SMAD4 gene mutations (e.g., a frameshift mutation) predictive of response to treatment with TGFβr1 inhibitors in cancer patients across multiple lines of therapy may be useful. SUMMARY OF THE INVENTION The invention relates to methods for the treatment and selection of subjects having cancer who may benefit from administration of a TGFβr1 inhibitor. In one aspect, the invention provides a method of treating cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a TGFβr1 inhibitor, wherein said cancer comprises a loss or reduction of canonical TGFβ signaling. In some embodiments, the loss or reduction of canonical TGFβ signaling is caused by a mutation, reduced expression or deletion of the SMAD4 gene, or combinations thereof. In some such embodiments, the loss or reduction of canonical TGFβ signaling is caused by a mutation of the SMAD4 gene. In a preferred embodiment, the mutation of the SMAD4 gene is a loss of function mutation, a low expression mutation and/or a loss of copy number mutation. In a preferred embodiment,he mutation of the SMAD4 gene results is a premature stop codon. In some embodiments, the mutation of the SMAD4 gene results in a frame-shift mutation. In a preferred embodiment, the mutation of the SMAD4 gene is c.-430C>T or c.1155delA (deletes A), or combinations thereof. In a preferred embodiment, the mutation of the SMAD4 gene is a loss of function mutation. In a preferred embodiment, the loss of function mutation of the SMAD4 gene is G386fs, -301fs, A199Sfs, A309Sfs*25, A532Pfs*5 or C443*, or combinations thereof. In a preferred embodiment, the loss of function mutation is G386fs. In one embodiment, the mutation of the SMAD4 gene interferes with a SMAD4 protein binding to a SMAD2 or SMAD3 protein. In some embodiments, the loss or reduction of canonical TGFβ signaling is caused by a mutation, reduced expression or deletion of the SMAD2 or SMAD3 gene, or combinations thereof. In some embodiments,he loss or reduction of canonical TGFβ signaling is caused by a mutation of the SMAD2 or SMAD3 gene. In some such embodiments, the mutation of the SMAD2 or SMAD3 gene is a loss of function mutation, a low expression mutation and/or a loss of copy number mutation. In some embodiments, the mutation of the SMAD2 or SMAD3 genenterferes with a SMAD2 or SMAD3 protein binding to a SMAD4 protein. In a preferred embodiment of each of the aspects described herein, the TGFβr1nhibitor is selected from the group consisting of galunisertib, LY2109761, SB525334, SP505124, GW788388, LY364947, RepSox, SD-208, vactosertib, LY3200882 and 4-(2- (5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N-(1,3-dihydroxypropan-2- yl)nicotinamide (PF-06952229), or a pharmaceutically acceptable salt thereof. In a preferred embodiment of each of the foregoing, the TGFβr1 inhibitor is 4-(2-(5-chloro-2-luorophenyl)-5-isopropylpyridin-4-ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof. In further embodiments of each of the foregoing, the method further comprises administering to the subject an amount of one or more anti-cancer agents; wherein the amounts of the TGFβr1 inhibitor and the one or more anti-cancer agents together are effective in treating cancer. In a preferred embodiment, the one or more anti-cancer agents is selected from the group consisting of a further TGFβr1 inhibitor, an anti-tumor agent, an anti-androgen and an anti-angiogenic agent. In one aspect, the invention provides a method of treating cancer in a subject comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; c. selecting the subject for treatment with a TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression level in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product, is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population; and d. administering to the selected subject an effective amount of the TGFβr1 inhibitor, thereby treating cancer in the subject. In another aspect, the invention provides a method of selecting a subject having cancer for treatment with a TGFβr1 inhibitor, comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; and c. selecting the subject for treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression level in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population. In yet another aspect, the invention provides a method of predicting whether a subject having cancer will respond to treatment with a TGFβr1 inhibitor, comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; c. selecting the subject for treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression level in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population; and d. predicting the subject will respond to treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression level in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population. In a preferred embodiment of each of the aspects described herein, the TGFβr1nhibitor is selected from the group consisting of galunisertib, LY2109761, SB525334, SP505124, GW788388, LY364947, RepSox, SD-208, vactosertib, LY3200882 and 4-(2- (5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N-(1,3-dihydroxypropan-2- yl)nicotinamide (PF-06952229), or a pharmaceutically acceptable salt thereof. In a preferred embodiment of each of the foregoing, the TGFβr1 inhibitor is 4-(2- (5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N-(1,3-dihydroxypropan-2- yl)nicotinamide (PF-06952229) having the structure: or a pharmaceutically acc

eptable salt thereof. In some embodiments of each of each of the foregoing, the biological sample comprises cancer tissue, cancer cells or circulating tumor DNA. In some embodiments of the each of the foregoing, the biological sample is a cancer tissue. In some such embodiments, the cancer tissue is obtained from a biopsy. In some such embodiments,he biological sample comprises a bodily fluid. In one such embodiment, the bodily fluids selected from the group consisting of blood, lymph, urine, saliva, fluid from ductalavage, and nipple aspirate. In some embodiments of each of the aspects described herein, the cancer is selected from the group consisting of prostate cancer, breast cancer, ovarian cancer,ung cancer, colon cancer, brain cancer, gastric cancer, liver cancer, thyroid cancer, endometrial cancer, gallbladder cancer, kidney cancer, adrenocortical cancer, sarcoma, skin cancer, head and neck cancer, leukemia, bladder cancer, colorectal cancer, hematopoietic cancer and pancreatic cancer. In a preferred embodiment, the prostate cancer is hormone dependent prostate cancer. In a preferred embodiment, the prostate cancer is hormone dependent prostate cancer. In a preferred embodiment, the cancers a resistant or relapsed cancer. In one embodiment, the cancer is prostate cancer. In another preferred embodiment, the cancer is castration resistant prostate cancer. In yet another preferred embodiment, the prostate cancer is a metastatic castration resistant prostate cancer. In a preferred embodiment, the prostate cancer is hormone dependent prostate cancer. In preferred embodiment, the prostate cancer is advanced prostate cancer. In another preferred embodiment, the prostate cancer is metastatic, non-metastatic, locally advanced, advanced hormone sensitive, advanced castration resistant, or recurrent. In a preferred embodiment of the each of the foregoing, the subject is a human. In another aspect, the invention provides a kit for assaying a biological sample to determine a SMAD4 gene deletion or mutation in the biological sample, comprising a first set of probes for detecting the expression level of the SMAD4 gene in the biological sample. In another aspect, the invention provides a kit for assaying a biological sample to determine a SMAD4 gene deletion or mutation in the biological sample, comprising a first set of probes for detecting the activity level of a SMAD4 gene encoded product in the biological sample. In a preferred embodiment of each of the aspects described herein, the invention provides a kit further comprising a second set of probes for detecting the expression level of a set of normalization genes in the tumor sample. In a preferred embodiment of the each of the foregoing, the subject is a human. Each of the aspects and embodiments of the present invention described herein may be combined with one or more other embodiments of the present invention which is not inconsistent with the embodiment(s) with which it is combined. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1. Shows Prostate Specific Antigen (PSA) levels in the blood over time afternitiating treatment with PF-06952229 for a metastatic castration resistant prostate cancer (mCRPC) patient whose tumor harbors a SMAD4 loss of function mutation. FIG.2. Shows metastatic prostate tumor lesion size measurements over time afternitiating treatment with PF-06952229 for a mCRPC patient whose tumor harbors a SMAD4 loss of function mutation. DETAILED DESCRIPTION OF THE INVENTION The present invention may be understood more readily by reference to theollowing detailed description of the embodiments of the invention and the Examplesncluded herein. It is to be understood that the terminology used herein is for the purpose of describing preferred embodiments only and is not intended to be limiting. It is furthero be understood that unless specifically defined herein, the terminology used herein iso be given its traditional meaning as known in the relevant art. The invention described herein may be suitably practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. Definitions As used herein, the singular form "a," "an," and "the" include plural references unless indicated otherwise. For example, "a" substituent includes one or more substituents. As used herein, the term "about" means having a value falling within an accepted standard of error of the mean, when considered by one of ordinary skill in the art, typically ± 10%. As used herein, the term “loss of function” refers to a reduction or elimination ofhe normal activity of a gene or gene product. Loss of activity can be due to a decreasen transcription and/or processing of the RNA, a decrease in translation, stability,ransport, or activity of the gene product, or any combination thereof. As used herein, “deleterious” is understood as harmful, often in a subtle or unexpected way. For example, a deleterious mutation may not result in any overt phenotypic change (e.g., cell morphology, doubling time, maintenance of cell cycle checkpoints, DNA repair, alterations in motility, etc.) when present alone in the cell, however, a deleterious mutation can increase the susceptibility of the cell to genomicnstability, or result in an overt phenotypic change when present in combination with a mutation in the second copy of the gene in the cell, or with one or more other gene mutations in the cell which, alone, do not result in any overt phenotypic change in the cell. Genomic instability can manifest, for example, as an increased mutation accumulation, aneuploidy, apoptosis, and mitotic catastrophe. As used herein, the term “benign” or “neutral” is understood as a mutation that does not threaten health or life; especially not becoming cancerous or having no significant effect on tissue function. For example, benign mutations do not alter theunction of the gene or gene product e.g., a silent mutation in the coding sequence that does not alter the amino acid sequence of the final protein, mutations of intron or untranslated sequences that do not alter splicing or processing of the mRNA transcript. As used herein, “hypomorphic” is understood as a mutation that reduces, but does not completely eliminate, the function of a gene. As used herein, the terms “deleterious” and “benign” are understood as being thewo ends of a continuum encompassing the natural complexity of human genetics. As used herein, a “transcription factor” is a protein that binds to specific sequences of DNA using DNA binding domains and is part of the system that controls theranscription of genetic information from DNA to RNA. Transcription factors perform this function alone, or by using other proteins in a complex, by increasing (as an activator), or preventing (as a repressor) the presence of RNA polymerase, the enzyme which activates the transcription of genetic informationrom DNA to RNA. Many transcription factors, especially some that are oncogenes orumor-suppressors, help regulate the cell cycle and, as such, determine how large a cell will get and when it can divide into two daughter cells. One example is the Myc oncogene, which has important roles in cell growth and apoptosis. Many transcription factors areumor-suppressor genes or oncogenes, and thus mutations or aberrant regulation of them are associated with cancer. For example, some transcription factors associated with cancer and/or believed to be tumor-suppressor genes include but are not limited to smad2 and smad4. As used herein, the term “apoptosis” refers to an active process of programmed cell death, which occurs normally during growth and development and also under conditions of cellular damage or stress; it is distinguished from necrosis, which is not an active process on the part of the dying cell. Apoptosis is a result of signal transduction cascades activated in the cell, for example by failed cell cycle checkpoints or by alterations in extracellular signaling, e.g., loss of growth factor receptor binding of a ligand such as a growth factor. A “functional assay” is a method to detect the activity of a gene, protein, or cell in response to a stimulus or insult. The specific functional assay performed depends on the specific mutation or mutations incorporated into the genome of the cell. Functional assaysnclude, but are not limited to, kinase assays, transcription assays using, for example, reporter constructs, proliferation assays, apoptosis assays, migration/chemotaxis assays, nutrient sensitivity assay, agent (e.g., drug, chemotherapeutic agent, mutagen) or radiation sensitivity assays, nucleic acid-binding assay or protein-binding assay, all of which are within the ability of those of skill in the art. As used herein, the term “nucleic acid sequence” (or nucleic acid molecule) referso a DNA or RNA molecule in single or double stranded form, particularly a DNA encoding a protein or protein fragment according to the invention. An “isolated nucleic acid sequence” refers to a nucleic acid sequence which is no longer in the natural environmentrom which it was isolated, e.g., the nucleic acid sequence in a bacterial host cell or inhe plant nuclear or plastid genome. As used herein, the term “protein” or “polypeptide” is used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. A “fragment” or “portion” of a SlPP2C1 protein may thus still be referred to as a “protein”. An “isolated protein” is usedo refer to a protein which is no longer in its natural environment, for example in vitro orn a recombinant bacterial or plant host cell. As used herein, the term "gene" refers to a nucleic acid sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by a full-length coding sequence or by any portion ofhe coding sequence. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). A gene may thus comprise several operably linked sequences, such as a promoter, a 5′ leader sequence comprising e.g., sequences involved in translationnitiation, a (protein) coding region (cDNA or genomic DNA) and a 3′ non-translated sequence comprising e.g., transcription termination sites. As used herein, a cDNA is a copy of the mRNA which is translated into the protein after splicing and other post-transcriptional processing events. As used herein, the term “exon” is a nucleic acid sequence that is represented inhe mature form of an RNA molecule after a) portions of a precursor RNA, introns, have been removed by cis-splicing or b) two or more precursor RNA molecules have beenigated by trans-splicing. The mature RNA molecule can be a messenger RNA or aunctional form of a non-coding RNA such as rRNA or tRNA. Depending on the context, exon can refer to the sequence in the DNA or its RNA transcript. As used herein, “UTR,” which stands for “untranslated region,” refers to either ofwo sections on each side of a coding sequence on a strand of mRNA. If it is found onhe 5′ end, it is called the 5′ UTR, or if it is found on the 3′ end, it is called the 3′ UTR. The untranslated regions typically include control regions involved in translation, RNAargeting, and post-transcriptional processing. As used herein, the term “intron,” derived from the term “intragenic region” and also called intervening sequence (IVS), are DNA regions in a gene that are not translatednto proteins. These non-coding sections are present in precursor mRNA (pre-mRNA) and some other RNAs and removed by splicing during the processing to mature RNA. After intron splicing, the mRNA consists only of exons, which are translated into a protein. Mutations present in introns are often silent. However, intronic mutations can result in aberrant or alternative splicing. A gene may contain one or more modifications in either the coding or the untranslated regions which could affect the biological activity or the chemical structure ofhe expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. The gene may constitute an uninterrupted coding sequence, or it may include one or more introns, bound by the appropriate spliceunctions. As used herein, the term "level of expression" or “expression level” refers to theevel of mRNA, as well as pre-mRNA nascent transcript(s), transcript processingntermediates, mature mRNA(s), and degradation products, encoded by a gene in the cell. The phrase "level of expression" also refers to the level of protein or polypeptide in a cell. As used herein, “expression of a gene” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e., which is capable of beingranslated into a biologically active protein or peptide (or active peptide fragment) or whichs active itself (e.g., in posttranscriptional gene silencing or RNAi). The coding sequence may be in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment. In gene silencing approaches, the DNA sequence is preferably present in the form of an antisense DNA or an inverted repeat DNA, comprising a short sequence of the target gene in antisense or in sense and antisense orientation (inverted repeat). “Ectopic expression” refers to expression in a tissue in which the genes normally not expressed. As used herein, the term "differential expression" refers to both quantitative as well as qualitative differences in the genes' expression patterns depending on differential development and/or tumor growth. Differentially expressed genes may represent "marker genes," and/or "target genes". The expression pattern of a differentially expressed gene disclosed herein may be utilized as part of a prognostic or diagnostic cancer evaluation. Alternatively, a differentially expressed gene disclosed herein may be used in methodsor identifying reagents and compounds and uses of these reagents and compounds forhe treatment of breast cancer as well as methods of treatment. As used herein, a “reference gene” or “normalization gene” refers to a gene, expression of which remains consistent in individual cells, even under different conditions, as well as among cells from different samples and origins. As used herein, the term “genome” refers to the total genetic information or hereditary material possessed by an organism (including viruses), i.e., the entire genetic complement of an organism or virus. The genome generally refers to all of the genetic material in an organism's chromosome (s), and in addition, extra chromosomal, geneticnformation that is stably transmitted to daughter cells (e.g., the mitochondrial genome). A genome can comprise RNA or DNA. "Biological activity" or "bioactivity" or "activity" or "biological function," which are used interchangeably, herein mean an effector or antigenic function that is directly orndirectly performed by a polypeptide (whether in its native or denatured conformation), or by any fragment thereof in vivo or in vitro. Biological activities include but are not limitedo binding to polypeptides, binding to other proteins or molecules, enzymatic activity, signal transduction, activity as a DNA binding protein, as a transcription regulator, abilityo bind damaged DNA, etc. A bioactivity can be modulated by directly affecting the subject polypeptide. Alternatively, a bioactivity can be altered by modulating the level of the polypeptide, such as by modulating expression of the corresponding gene. The term "marker," “biological marker” or "biomarker" refers a biological molecule, e.g., a nucleic acid, peptide, hormone, etc., whose presence, concentration or alteredevel of expression in a tissue or cell from its expression level in a control (e.g., normal or healthy tissue or cell) can be detected and correlated with a known condition, such as, cancer or subtype thereof (e.g., a prostate cancer). In some embodiments, a “biomarker” indicates a change in the level of mRNA expression that may correlate with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment. In some embodiments, the biomarkers a nucleic acid, such as mRNA or cDNA. In additional embodiments, a “biomarker” indicates a change in the level of polypeptide or protein expression that may correlate with the risk or progression of a disease, or patient's susceptibility to treatment. In some embodiments, the biomarker can be a polypeptide or protein, or a fragment thereof. The relative level of specific proteins can be determined by methods known in the art. For example, antibody-based methods, such as an immunoblot, enzyme-linked immunosorbent assay (ELISA), or other methods can be used. As used herein, the term "biological sample" or “sample” refers to a material or mixture of materials obtained from a subject (such as a patient), cell line, tissue culture, or other source which may contain cells or cellular products such as extracellular matrix.n certain embodiments, the biological sample comprises cancer tissue, cancer cells or circulating tumor DNA. The sample may be of any biological tissue or bodily fluid. The sample is typically, although not necessarily, in fluid form, containing one or more components of interest. Frequently the sample will be a "clinical sample" which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, blood cells (e.g., white cells), organs, cells, tissue or fine needle biopsy samples, cell-containing bodily fluid, free floating nucleic acids, urine, peritoneal fluid, and pleuralluid, or cells therefrom. Examples of bodily fluid include but are not limited to blood,ymph, urine, saliva, fluid from ductal lavage, and nipple aspirate. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. In one embodiment, the sample is a “tumor sample” or “tumor biological sample,” e.g., includes one or more premalignant or malignant cells. In certain embodiments, the sample, e.g., the tumor sample, is obtained from a solid tumor, a soft tissue tumor or a metastatic lesion. In other embodiments, the sample, e.g., the tumor sample, includesissue or cells from a surgical margin. In another embodiment, the sample, e.g., tumor sample, includes one or more circulating tumor cells (CTC) (e.g., a CTC acquired from a blood sample). In some embodiments, the sample is a “non-tumor sample” or “non-tumor biological sample.” In certain embodiments, the non-tumor sample, is a blood control, a normal adjacent tumor (NAT), or any other non-cancerous sample from the same or a different subject. A "native" or "wild type" nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, a "wild type mRNA"s an mRNA that is naturally occurring in or endogenous to the organism. An “active protein” or “functional protein” is a protein which has protein activity as measurable in vitro, e.g., by an in vitro activity assay, and/or in vivo, e.g., by the phenotype conferred by the protein. A “mutant protein” is herein a protein comprising one or more mutations in the nucleic acid sequence encoding the protein, whereby the mutation results in (the mutant nucleic acid molecule encoding) a “reduced-function” or “loss-of-function” protein, as e.g., measurable by the protein activity in vitro compared to the activity of the wild type protein, e.g., by an activity assay, and/or in vivo, e.g., by the phenotype conferred by the mutant allele. As used herein, “mutation,” “gene comprising a mutation,” “mutant gene” or "mutated gene" as used interchangeably herein refers to alterations in one or more nucleic acids in a genomic sequence, compared to the wild type sequence, including one or more base changes, deletions, and/or insertions of one or more nucleotides, that resultn silent mutations, non-sense mutations, mis-sense mutations, mutations that result in premature stop codons, aberrant splicing, transcription or translation. A mutant gene has undergone a change, such as the loss, gain, or exchange of genetic material, which affects the normal transmission and expression of the gene. A "disrupted gene" as used herein refers to a mutant gene that has a mutation that causes a premature stop codon. The disrupted gene product is truncated relative to a full-length undisrupted gene product. A gene comprising a mutation can have more than one mutation. "Premature stop codon" or "out-of-frame stop codon" as used interchangeably herein refers to nonsense mutation in a sequence of DNA, which results in a stop codon at location not normally found in the wild-type gene. A premature stop codon may cause a protein to be truncated or shorter compared to the full-length version of the protein. Means the mutation inserts a transcription start codon earlier. A "deletion" or “DEL” refers to the absence of one or more amino acid residues in the related protein. As used herein, the term "insertion" or “INS” refers to the addition of one or more amino acids in the related protein. As used herein, the term “frame shift,” “frame-shift” or “Frame_Shift” refers to a sequence change between the translation initiation (start) and termination (stop) codon where, compared to a reference sequence, translation shifts to another reading frame. A “frame-shift mutation” is a mutation a nucleic acid sequence encoding a protein by which the reading frame of the mRNA is changed, resulting in a different amino acid sequence. The resulting protein may have loss of function, a reduced function, a low expression mutation and/or loss of copy number. As used herein, the term “insertion–deletion mutations” or “indels” refers tonsertion and/or deletion of nucleotides into genomic DNA and include events less than 1 kb in length. Where there is a “Frame_Shift” insertion (INS) or a deletion (DEL), the indel causes the change of reading frame, resulting in premature stop codon, change in the amino acids coded by the remaining sequence, or prevents the production of a protein due to induction of nonproductive sequences. As used herein, “fs” means that the type of mutation is a frame-shift mutation. For example, G386fs means frame-shift occurs after position G386. As used herein, mutation are shown as “amino acid” “position” ”new_amino_acid”“fs” “*” “position_termination_site”, e.g., (A309Sfs*25) and A532Pfs*5. As used herein, “fs*” means that a frame-shift results in a stop codon. For example, in A309Sfs*25, “amino_acid” = first amino acid changed = A, “position” = position = 309 “new_amino_acid” = new amino acid = S, “fs” = type of change is a frame shift = fs, “*” = stop codon = *“position_termination_site” = position new termination site = 25. As used herein, mutation are shown as “amino acid” “position” ”new_amino_acid” “fs” “*” “, e.g., “A199Sfs”. As used herein, “C443*” means amino acid C is changed into a stop codon. A minus sign means the mutation occurs upstream of the translational start site. For example, in “-301fs,” the mutation occurs upstream of position 301. As used herein, the term “missense” mutation or “missense variant” refers to a (point) mutation in a nucleic acid sequence encoding a protein, whereby a codon is changed to code for a different amino acid. The resulting protein may have reducedunction or loss of function. The term "locus" is herein defined to be a specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map. Gene mapping is the process of determining the locus for a particular biological trait. The term "polymorphism" is herein defined to be the occurrence of genetic variations that account for alternative DNA sequences and/or alleles among individualsn a population. The term "polymorphic site" is herein defined to be a genetic locus wherein one or more particular sequence variations occur. A polymorphic site can be one or more base pairs. For example, a "single nucleotide polymorphism (SNP)" is a polymorphism that occurs at a single nucleotide. As used herein, a "cluster" of SNPs refers to three or more SNPs that occur within 100 kilobases of each other in a particular polymorphic site, wherein all of the SNPs have a p-value e<"4> (i.e., < 1 x 10<"4>). As used herein, the term “nonsynonymous” refers to mutations that result in changes to the encoded amino acid. As used herein, the term “synonymous” refers to mutations that do not result in changes to the encoded amino acids. As used herein, the term “somatic mutation” or “somatic variation” refers to a mutation in the DNA of somatic cells (i.e., not germ cells), occurring after conception. “Somatic mutagenesis” therefore refers to the process by which somatic mutations occur. As used herein, “variant sequence” or “mutant sequence” means a nucleotide or amino acid sequence that contains one or more differences with respect to a primary sequence. These differences may include alternative residues, modified residues, deletions, insertions, and substitutions. For example, a “mutant polynucleotide,” “mutant nucleic acid,” “variant nucleic acid,” and “nucleic acid with variant nucleotides,” refers to a polynucleotide which has a nucleotide sequence that is different from the nucleotide sequence of the corresponding wild-type polynucleotide. The difference in the nucleotide sequence of the mutant polynucleotide as compared to the wild-type polynucleotide is referred to as the nucleotide “mutation,” “variant nucleotide,” “nucleotide variants” or “variation.” Deletions may be of a single nucleotide base, a portion or a region of the nucleotide sequence of the gene, or of the entire gene sequence. Insertions may be of one or more nucleotide bases. The variants may occur in transcriptional regulatory regions, untranslated regions of mRNA, exons, introns, or exon/intron junctions. The “variant nucleotide” may or may not result in stop codons, frame shifts, deletions of amino acids, altered gene transcript splice forms or altered amino acid sequence. The term “variant nucleotide” also refers to one or more nucleotide(s) substitution, deletion,nsertion, methylation, and/or modification changes. As used herein, “splice-site” mutation is a mutation in a nucleic acid sequence encoding a protein, whereby RNA splicing of the pre-mRNA is changed, resulting in an mRNA having a different nucleotide sequence and a protein having a different amino acid sequence than the wild type. The resulting protein may have reduced function or loss ofunction. As used herein, “a protein altering mutation” refers to a genetic mutation that (a) results in a change in the amino acid sequence of the corresponding protein; or (b) otherwise results in a disruption of the expression, or function of the protein which the gene encodes. Examples of a protein altering genetic mutation includes but is notimited to disruptive in-frame deletion, disruptive in-frame insertion, frame-shift variant,n-frame deletion, in-frame insertion, initiator codon variant, intron variant, missense variant, non-canonical start codon, splice acceptor variant, splice donor variant, splice region variant, start lost, stop gained, stop lost, and stop retained variant. In some embodiments, the insertions can include from 1 to 21 nucleotides, 1 to 12 nucleotides, 1o 6 nucleotides or 1 to 3 nucleotides. In some embodiments, deletions can be of one or more exonic or intronic regions, or about 1 to 21 nucleotides, 1 to 12 nucleotides, 1 to 6 nucleotides or 1 to 3 nucleotides. In some embodiments the mutations are found at thentron exon splice sites, within introns, or within exons. As used herein, the term "single nucleotide variant" or "SNV" refers to a substitution of one nucleotide to a different nucleotide at a position (e.g., site) of a nucleotide sequence, e.g., a sequence read from a sample. A substitution from a first nucleobase X to a second nucleobase Y may be denoted as "X>Y." For example, a cytosine to thymine SNV may be denoted as "C>T." As used herein, “mutation type” refers to the specific nucleotide substitution that comprises the mutation, and is selected from among C>T, C>A, C>G, G>T, G>A, G>C, A>T, A>C, A>G, T>A, T>C and T>G mutations. Thus, for example, a mutation type of C>T refers to a mutation in which the targeted or mutated nucleotide cytosine is replaced with the substituting nucleotide thymine. As used herein, the term “loss of copy number” or “copy number loss” is the deletion of the entire SMAD4 gene through loss of chromosome or deletion of a region of a chromosome containing the SMAD4 gene locus on at least one chromosome allele. A mutation in a regulatory sequence, e.g., in a promoter of a gene, is a change of one or more nucleotides compared to the wild type sequence, e.g., by replacement, deletion or insertion of one or more nucleotides, leading for example to reduced or no mRNA transcript of the gene being made. As used herein, the term “silencing” refers to a down-regulation or completelynhibition of gene expression of the target gene or gene family. As used herein, “point mutation” is the replacement of a single nucleotide, or thensertion or deletion of a single nucleotide. As used herein, the term “non-sense mutation” or “Nonsense_Mutation” is a (point) mutation in a nucleic acid sequence encoding a protein, whereby a codon is changed into a stop (termination) codon. Nonsense mutations, in some aspects, cause and are thusnterchangeably referred to as “premature termination codons” (PTCs). In some aspects, PTCs comprise a triplet nucleotide sequence, for example UGA (e.g., TGA in DNA), UAG (e.g., TAG in DNA), or UAA (e.g., TAA in DNA). For example, mutations (e.g., in DNA, mRNA (RNA), or both) that result in a PTC include, but are not limited to: (1) single base pair substitutions that change a sense codon to an in-frame PTC (e.g., nonsense mutations); (2) insertion or deletion mutations that alter the ribosomal reading frame, causing translating ribosomes to encounter a PTC; (3) an insertion mutation that maintains the proper distal reading frame but introduces an in-frame PTC; and (4) mutations that lead to mRNA splicing defects that cause retention of an intron (or part of an intron) that alters the reading frame, leading translating ribosomes to encounter a PTC. This results in a premature stop codon being present in the mRNA and in aruncated protein. A truncated protein may have reduced function or loss of function. In some aspects, mutations resulting in a PTC have important consequences on gene expression, such as in the context of disease. For example, a PTC will terminate mRNAranslation prior to completion of a full-length polypeptide, leading to production ofruncated proteins that are often nonfunctional and/or unstable and/or have detrimentalunction. In addition, PTC-containing mRNAs are also frequently unstable because the MRNAs are degraded by NMD, resulting in a severe reduction in steady-state mRNAevels. In some examples, the combination of these PTC-induced events reduce the level of functional protein produced to such an extent that a severe disease state results. As used herein, “'Nonsense_Mutation” with a stop codon is referred using “*”. For example, in G358* nonsense mutation,” the “*” means a stop codon, i.e., amino acid G at position 358 is mutated to a stop codon. As used herein, “loss of function mutation” or “inactivating mutation” refers to mutations that result in the gene product having reduced or abolished function (being partially or wholly inactivated). As used herein, “reduced expression mutation” or “low expression mutation” referso lower transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene. As used herein, “loss of copy number mutation” refers to a mutation that an expression of a particular gene is decreased below threshold or reference level. For example, a loss of copy number mutation of SMAD4 may result in consequent downregulation of gene expression and function among the tumors from cancer patients. As used herein, the term "stop codon" intends a three-nucleotide contiguous sequence within messenger RNA that signals a termination of translation. Non-limiting examples include in RNA, UAG, UAA, UGA and in DNA TAG, TAA or TGA. Unless otherwise noted, the term also includes nonsense mutations within DNA or RNA thatntroduce a premature stop codon, causing any resulting protein to be abnormally shortened. tRNA that correspond to the various stop codons are known by specific names: amber (UAG), ochre (UAA), and opal (UGA). As used herein, the term “target gene” in gene silencing approaches is the gene or gene family (or one or more specific alleles of the gene) of which the endogenous gene expression is down-regulated or completely inhibited (silenced) when a chimeric silencing gene (or “chimeric RNAi gene”) is expressed and for example produces a silencing RNAranscript (e.g., a dsRNA or hairpin RNA capable of silencing the endogenous target gene expression). In mutagenesis approaches, a target gene is the endogenous gene whichs to be mutated, leading to a change in (reduction or loss of) gene expression or a changen (reduction or loss of) function of the encoded protein. As used herein, the term “sense” RNA transcript is generally made by operablyinking a promoter to a double stranded DNA molecule wherein the sense strand (coding strand) of the DNA molecule is in 5′ to 3′ orientation, such that upon transcription a sense RNA is transcribed, which has the identical nucleotide sequence to the sense DNA strand (except that T is replaced by U in the RNA). An “antisense” RNA transcript is generally made by operably linking a promoter to the complementary strand (antisense strand) ofhe sense DNA, such that upon transcription an antisense RNA is transcribed. As used herein, the term “transcription regulatory sequence” is herein defined as a nucleic acid sequence that is capable of regulating the rate of transcription of a (coding) sequence operably linked to the transcription regulatory sequence. A transcription regulatory sequence as herein defined will thus comprise all of the sequence elements necessary for initiation of transcription (promoter elements), for maintaining and for regulating transcription, including e.g., attenuators or enhancers. Although mostly the upstream (5′) transcription regulatory sequences of a coding sequence are referred to, regulatory sequences found downstream (3′) of a coding sequence are also encompassed by this definition. As used herein, “library” is a collection of nucleic acid sequences, typically corresponding to a specific gene where the members of the library include one or more mutations in the gene as well as to the corresponding “normal” gene or a known, benign SNP of a “normal” gene. A library can include sequences from multiple genes. The nucleic acid sequence members typically contain a fragment of the sequence of the gene, e.g., intron, exon, and/or transcription or translational regulatory region, for example the 3′-UTR, 5′-UTR, or in a region at the junction of two of the juxtaposed regions, for examplentron/exon junction or 5′-UTR/exon junction. In an embodiment, the library includes a series of mutations, either generated randomly or by design in a gene known to or suspected of being a tumor-suppressor gene or otherwise associated with cancer. Library members also preferably include sequences that facilitate replication of the library, e.g., plasmid sequences for replication in E. coli, chromosomal sequences in mammalian or yeast cells, in viral vectors, e.g., adenoviral vectors, adeno-associated viral vectors. Aibrary can have essentially any number of members greater than 1, for example about 2, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 750, 1000, 1500, 5000, or more. As used herein, “nucleic acid fragment of a gene” is a portion of a gene large enough to include a mutation (e.g., 1 nucleotide) that is flanked by regions that allow for recombination of the fragment into the genome of a cell. Regions that allow for recombination into the genome of mammalian cells are at least about 50, 100, 250, 500, 750, 1000, 1500, or 2000 bp on each side of the mutation. Therefore, the overall length of a gene fragment for recombination should be about 100 to 4000, 250 to 4000, 500 to 2000, or 500 to 3000 bp with the mutation relative to the genomic sequence at least about 50, 100, 250, 500, 750, or 1000 bp of nucleotides from the end of the fragment. The portion of the fragment including the mutation need not necessarily be distinct from thelanking regions to allow recombination. The specific length of the nucleic acid fragment used is not a limitation of the methods of the invention as long as the fragment is of an appropriate size to allow for recombination. The methods herein include recombination into a “functional gene” which is understood as insertion of the nucleic acid fragment into any portion or portions of a gene, e.g., intron, exon, transcriptional/translational control region, 3′UTR, 5′UTR, wherein the “functional gene” encodes a protein that has at least partial function of the wild-type gene,or example at least about 50%, 60%, 70%, 80%, or 90% of the activity of the wild-type gene as determined by standard functional assays for the specific gene type. A “functional gene” can include naturally occurring or artificially inserted mutations or alterations, for example silent mutations that do not alter the coding sequence of the expressed protein, to detect integration of a nucleic acid fragment into the gene, or to determine which strand the gene fragment integrated into. As used herein, “transforming a cell” is understood as contacting a cell with a nucleic acid under conditions to promote uptake of the nucleic acid into the cell. The method of transfection may be selected based on the cell type into which the nucleic acids to be introduced. Commonly used transfection methods include electroporation, calcium phosphate precipitation, cationic lipid reagents (e.g., Lipofectamine®, Oligofectamine^®), dendrimer reagents. The invention is not limited by the method ofransfection. As used herein, a cell transfected with a construct includes cells that containhe construct in the cell, as an episomal plasmid, or integrated into the genome of the cell. It is understood that upon recombination of the fragment into the genome, other sequences included in the originally transfected construct can be lost. Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. The term “cell culture” refers to the culturing of cells derivedrom multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumors, most primary cell cultures have limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing, while generally retaining viability. Primary cells can be obtained from essentially any source, including transgenic animals. An established or immortalized cell line has acquired the ability to proliferatendefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well-established cell lines representative of particular cell types. The methods herein can use essentially any celline, preferably cell lines that are easily transfected by one or more methods. Cell linesor use in the methods of the invention include, but are not limited to cancer cells, near- diploid cancer cell lines having DNA mismatch-repair defects, CHO (Chinese hamster ovary) cells, 293 cells, and cells immortalized with viral transforming proteins or viralnfection. Immortalized cell lines are frequently not precisely diploid and can be aneuploid. As used herein, “selecting” is understood as identifying one or more members of a group. As used herein, the term “detectable label” is understood as a chemical modification, binding agent, or other tag that can be readily observed, preferably in a quantitative manner, such as a fluorescent tag that has a specific wavelength of absorption and emission to allow detection of the compound associated with the detectable label. Detectable labels include vital dyes for use with living cells wherein the dye has little or no effect on cell viability, growth, and function. As used herein, “isolated” or “purified” is understood as being removed from its usual environment or other components with which they are naturally associated. For example, an isolated cell can be removed from an animal and placed in a culture dish or another animal. Isolated is not meant as being removed from all other cells. A group of cells can also be isolated (e.g., bone marrow cells, an organ) from one organism and placed in another organism or in culture. A polypeptide or nucleic acid is isolated when its about 80% free, 85% free, 90% free, 95% free from other cellular material typically associated with the nucleic acid or polypeptide (e.g., material in a cell in which the nucleic acid or peptide is expressed), or substantially free of chemical precursors or other chemicals when chemically synthesized. An “isolated polypeptide” or “isolated polynucleotide” is, therefore, a substantially purified polypeptide or polynucleotide, respectively. Alternately an isolated nucleic acid or polypeptide may be present in a non- native environment for the molecule, e.g., a heterologous cell, tissue, or animal. As used herein, “homogenously purified” polypeptide or nucleic acid is about 90%, 95%, 98%, or 99% pure and removed from biological or synthetic contamination from the synthesis ofhe polypeptide or nucleic acid. A homogeneously purified polypeptide or nucleic acid can be present in a buffer or other carrier such as a pharmaceutically acceptable carrier. The terms “polynucleotides,” “nucleic acid,” “nucleotides” and “oligonucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. As used herein, “nucleic acid sequence fragment” or “gene fragment” and the like are understood as a portion of a gene that includes at least about 8, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 150, 200, 250, 500 contiguous nucleotidesrom a full length naturally occurring gene or genomic sequence. A nucleic acid sequenceragment can include one or more mutations, naturally occurring or artificially (e.g., randomly) generated. As used herein, nucleic acid sequences from “the same gene” and the like are nucleic acid sequences that are fragments of the same genomic sequence and may or may not include mutations or polymorphisms. A nucleic acid sequence from a genencludes fragments of the gene. A fragment from the same gene has at least 80%, 90%, 95%, or more identity with the wild-type sequence of the gene over the length of theragment. Complimentary DNA (cDNA) or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR. The choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences. The term "cell-free DNA" or "cfDNA" refers to nucleic acid fragments that circulaten a subject's body (e.g., bloodstream) and originate from one or more healthy cells and/orrom one or more cancer cells. The term "circulating tumor DNA" or "ctDNA" refers to nucleic acid fragments that originate from tumor cells or other types of cancer cells, which may be released into a subject's bloodstream as a result of biological processes, such as apoptosis or necrosis of dying cells, or may be actively released by viable tumor cells. Techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagents useful in applying suchechniques, such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U. S. Biochemicals, New England Nuclear, and a number of other sources. The recombinant nucleic acid sequences used to produceusion proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See GenBank, National Institutes of Health. As used herein, “expression” refers to the process by which a polynucleotide isranscribed into mRNA, and/or the process by which the transcribed mRNA (also referredo as “transcript”) is subsequently being translated into peptides, polypeptides, or proteins. The transcripts and/or the encoded polypeptides can be assessed as a readoutor expression level. If the polynucleotide is derived from genomic DNA, expression maynclude splicing of the mRNA in a eukaryotic cell. The terms “polypeptide,” “peptide” and “protein” are used interchangeably hereino refer to a polymer of amino acid residues. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation,ipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L opticalsomers, and amino acid analogs and peptidomimetics. A polypeptide "fragment," "portion" or "segment" is a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids. The polypeptides of the present invention, if soluble, may be coupled to a solid- phase support, e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates. Such supports may take the form, for example, of beads, wells, dipsticks, or membranes. "Target region" refers to a region of the nucleic acid which is amplified and/or detected. The term "target sequence" refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions The term “amino acid,” as used herein, encompasses naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics thatunction in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., γ-carboxyglutamate, hydroxyproline, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Exemplary amino acid analogs include but are not limited to homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, as long as they retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. In the context of polypeptides, a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. “Expression level,” “level of expression” and the like refers to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., gene-encoded and/or epigenetic information) is converted into the structures present and operating in the cell. Therefore, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a posttranslational processing of the polypeptide, e.g., by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). "Increased expression," "increased expression level," "increased levels," "elevated expression," "elevated expression levels," or "elevated levels" refers to anncreased expression or increased levels of a biomarker in an individual relative to a reference level or control, such as an individual or individuals who do not have the disease or disorder (e.g., cancer), an internal control e.g., a housekeeping biomarker), or a median expression level of the biomarker in samples from a group/population of patients. "Decreased expression," "decreased expression level," "decreased levels," "reduced expression," "reduced expression levels," or "reduced levels" refers to a decrease expression or decreased levels of a biomarker in an individual relative to a reference level or control, such as an individual or individuals who do not have the disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or a median expression level of the biomarker in samples from a group/population of patients. In some embodiments, reduced expression is little or no expression. An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect in inhibiting the expression of a gene. "The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%dentity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visualnspection. Such sequences are then said to be “substantially identical.” This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least about 20 amino acids or nucleotides in length, or more preferably over a region that is about 25-50, 50-100, 100-200, 20-300, 50-500, 50-1000, 500-1000 amino acids or nucleotides in length. For sequence comparison, typically one sequence acts as a reference sequence,o which test sequences are compared. When using a sequence comparison algorithm,est and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities forhe test sequences relative to the reference sequence, based on the program parameters. Sequence alignments can be used to determine amino acids that are more or less conserved between species. Alteration of non-conserved amino acids is typically has less of an effect on the activity of a polypeptide than mutation of a conserved amino acid. Similarly, more conservative mutations, based on charge and/or size of the amino acid,ypically have less of an effect on the activity of a polypeptide. For example, a conservative mutation would include substitution of one small, neutral, non-polar amino acid such as alanine, glycine, isoleucine, leucine, proline, and valine for another. Similarly, mutation of a glutamic acid to an aspartic acid, or vice versa would also be considered a conservative mutation. Exchange of phenylalanine, tyrosine, andryptophan for each other would be considered a conservative mutation. Protein toleranceo random mutations is understood in the art, for example, see Guo et al. (Proteinolerance to random amino acid change. Proc. Natl. Acad. Sci. USA 101:9205-9210, 2004, incorporated herein by reference). SNPs or other natural variations associated with disease can be used to identify essential amino acids that do or do not tolerate mutation. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitlyndicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Enhanced evolutionary PCR using oligonucleotides with inosine at the 3'-terminus, Nucleic Acid Res. 1991, 19:5081; Ohtsuka et al., An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions, J. Biol. Chem.1985, 260:2605-2608; Rossolini et al., Use of deoxyinosine-containing primers vs degenerate primers for polymerase chain reaction based on ambiguous sequencenformation, Mol. Cell Probes, 1994, 8:91-98). The term nucleic acid is usednterchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, the term “polymerase chain reaction” (PCR) refers to the methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, all of which are herebyncorporated by reference, directed to methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. As used herein, the terms “PCR product” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences. Of the PCR techniques, RT-PCR (Reverse Transcription-PCR), competitive RT- PCR and the like are used for detecting and quantifying a trace amount of mRNA and show their effectiveness. A real-time quantitative detection technique using PCR has been established (TaqMan^® PCR, Real time quantitative PCR, Genome Res., 1996, 6 (10), 986, ABI PRISM™ Sequence Detection System, Applied Biosystems, incorporated herein by reference). The use of PCR and methods to design primers and select polymerases to identify single nucleotide changes are well known in the art. For example, the importance of hybridization of the 3′ ends of primer sequences to allow for extension is well known. Methods to distinguish genomic sequences that have undergone recombination, or not, are well known in the art, including primer design, and methods are further provided herein. As used herein, the term “recombinant DNA molecule” refers to a DNA molecule, which is comprised of segments of DNA joined together by means of molecular biologicalechniques. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5′ of the “downstream” or 3′ elements. This terminology reflects the fact that transcription proceeds in a 5′ to 3′ fashion along the DNA strand. The promoter and enhancer elements which directranscription of a linked gene are generally located 5′ or upstream of the coding region. However, enhancer elements can exert their effect even when located 3′ of the promoter element and the coding region. Transcription termination and polyadenylation signals areocated 3′ or downstream of the coding region. By “detecting” or “detection” and the like is meant the process of performing the steps for determine if an analyte or nucleotide sequence is present, or to determine if a compound, gene, or mutation has an effect, or if a recombination or insertion has taken place, and the like. The amount of analyte present or the effect may be none or belowhe level of detection of the method. As used herein, “correlating a mutation in a gene with a diagnosis or predispositiono a disease or condition” is understood as detecting a mutation determined to be deleterious (or not) present in a sample from a subject and associating the presence ofhe specific mutation with an increased incidence or chance of occurrence (or not) of the development of a specific disease or condition, e.g., a specific type of cancer. By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. Amelioration can require administration of more than one dose. The term "treating", "treat" or "treatment" as used herein embraces both preventative, i.e., prophylactic, and palliative treatment, i.e., relieve, alleviate, or slow the progression of the patient’s disease (or condition) or anyissue damage associated with the disease. As used herein, “susceptible to” or “prone to” or “predisposed to” a specific disease or condition and the like refers to an individual who based on genetic, environmental, health, and/or other risk factors is more likely to develop a disease or condition than the general population. An increase in likelihood of developing a disease may be an increase of about 10%, 20%, 50%, 100%, 150%, 200%, or more. The terms “diagnosing” or “diagnosis” and “prognosticating” or “prognosis," as used herein, are used in the broadest sense, and are commonly used and are well- understood in medical and clinical practice. As used herein, the term “diagnosing” or “diagnosis” refers to a clinical or other assessment of the condition of a subject based on observation, testing, or circumstances for identifying a subject having a disease, disorder, or condition based on the presence of at least one sign or symptom of the disease, disorder, or condition. Typically, diagnosing using the method of the invention includeshe observation of the subject for other signs or symptoms of the disease, disorder, or condition in addition to detection of a loss-of-function mutation in a gene that makes the subject susceptible to a particular disease or condition. As used herein, the term “prognosticating” or “prognosis” refers to the determination of probability, risk or possibility of developing a disease, disorder, or condition, such as cancer, in a subject. As used herein, the term “alteration” refers to a change (increase or decrease) inhe expression level or activity of a gene or polypeptide” as detected by standard art known methods (e.g., ELISA, PCR, mass spectrometry-based methods,mmunohistochemistry, RIA, functional assays). An alteration in activity for example from a gene mutation may only be detectable when present in combination with a second mutation or alteration. As used herein, “obtaining” is understood as purchase, procure, manufacture, or otherwise come into possession of the desired material. As used herein, “providing,” refers to obtaining, by for example, buying or makinghe, e.g., polypeptide, drug, polynucleotide, probe, and the like, including libraries of nucleic acids or cells having a targeted heterozygous for a mutation or deletion in a gene. The material provided may be made by any known or later developed biochemical or other technique, e.g., standard molecular biology techniques. As used herein, the term “differential” or “differentially” generally refers to a statistically significant different level in the specified property or effect. Preferably, the difference is also functionally significant. Thus, “differential binding or hybridization” is a sufficient difference in binding or hybridization to allow discrimination using an appropriate detection technique. Likewise, “differential effect” or “differentially active” in connection with a therapeutic treatment or drug refers to a difference in the level of the effect or activity that is distinguishable using relevant parameters and techniques for measuring the effect or activity being considered. For example, identification of a specificoss of function mutation can provide an indication of a drug that may be most useful forhe treatment of a specific disease (e.g., cells having mutations in a DNA repair gene may be more susceptible to treatment with a drug that induces DNA double strand breaks). Preferably the difference in effect or activity is also sufficient to be clinically significant, such that a corresponding difference in the course of treatment or treatment outcome would be expected, at least on a statistical basis. As used herein, the term “administration” as it applies to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic or diagnostic agent, or composition, to the animal, human, experimental subject, cell, tissue, organ or biological fluid. The term, “administration” may also relate to in vitro and ex vivo treatment, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. Exemplary routes of administration to the human body can be through the mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like. As used herein, the term "co- administration " or “coadministration” refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments,he coadministration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co- administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. As used herein, “small molecule” is meant a compound having a molecular weight of no more than about 1500 daltons, 1000 daltons, 750 daltons, 500 daltons. A small molecule is not a nucleic acid or polypeptide. As used herein, terms, including, but not limited to, “drug,” “agent,” “component,” “composition,” “compound,” “substance,” “targeted agent,” “targeted therapeutic agent,” “therapeutic agent,” “pharmaceutical agent,” and "medicament” may be usednterchangeably to refer to the compounds of the present invention, e.g., a TGFβr1nhibitor. As used herein, the term “inhibits,” or "inhibition" refers to the decrease in active of a target protein product relative to the normal wild type level. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, As used herein, the term “pharmaceutically acceptable salt” refers to those salts which retain the biological effectiveness and properties of the parent compound. The phrase “pharmaceutically acceptable salt(s) ," as used herein, unless otherwisendicated, includes salts of acidic or basic groups which may be present in the compounds of the formulae disclosed herein. For example, the compounds of the invention that are basic in nature may be capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions. Examples of anions suitable for mono- and di- acid addition salts include, but are not limited to, acetate, asparatate, benzenesulfonate, benzoate, besylate, bicarbonate, bisulfate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, decanoate, edetate, edislyate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollate, hexanoate, hexylresorcinate, hydrabamine, hydroxynaphthoate, iodide, isethionate,actate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, octanoate, oleate, pamoate (embonate), pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, succinate, sulfate,annate, tartrate, teoclate, tosylate, triethiodode, and valerate salts. Alternatively, compounds that are acidic in nature may be capable of forming base salts with various pharmacologically acceptable cations which form non-toxic base salts. Such non-toxic base salts include, but are not limited to, those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines. Examples of cations suitable for such salts include alkali metal or alkaline-earth metal salts and other cations, including aluminium, arginine, benzathine, calcium, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenediamine, lysine, magnesium, histidine, lithium, meglumine, potassium, procaine, sodium, triethyamine and zinc. Salts may be prepared by conventional techniques. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Methods for making pharmaceutically acceptable salts are known tohose of skill in the art. The terms “determining," “measuring," “evaluating," “assessing," “assaying," and “analyzing” are used interchangeably herein to refer to any form of measurement andnclude determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something present, as well as determining whether it is present or absent. As used herein, the term “regulate” refers to controlling the activity of a molecule or biological function, such as enhancing or diminishing the activity or function. A “disorder” is any condition that would benefit from treatment with the methods ofhe present invention. This includes chronic and acute disorders or diseases includinghose pathological conditions which predispose the subject to the disorder in question. As used herein, the term “cancer,” “cancerous,” “malignant” refer to or describehe physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, “cancer” refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth. As used herein, “cancer” refers to solid tumors namedor the type of cells that form them, cancer of blood, bone marrow, or the lymphatic system. The term “cancer” includes but is not limited to a primary cancer that originates at a specific site in the body, a metastatic cancer that has spread from the place in which it started to other parts of the body, a recurrence from the original primary cancer after remission, and a second primary cancer that is a new primary cancer in a person with a history of previous cancer of a different type from latter one. A subject may be identified as having de novo metastatic disease or after progression from an earlier-identified cancer. The term, "metastasis," “metastatic cancer” or “metastatic” (also known as “secondary cancer”) as used herein, refers to a type of cancer that originates in one tissueype, but then spreads to one or more tissues outside of the (primary) cancer’s origin. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor,raveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant. The term “refractory” or “resistant” refers to a circumstance where patients, even after intensive treatment, have residual cancer cells (e.g., prostate cancer cells) in their body. The term “relapsed” or “recured” refers to a situation where patients who have had a remission of cancer after therapy have a return of cancer cells (e.g., prostate cancer cells) in their body. A “TGFβ expressing cancer” is one that produces sufficient level of TGFβ at the surface of cells thereof, such that an anti-TGFβ antibody can bind thereto and have aherapeutic effect with respect to the cancer. A cancer “characterized by excessive activation” of a TGFβ receptor (TGFβr) is one in which the extent of TGFβ receptor activation in cancer cells significantly exceedshe level of activation of that receptor in non-cancerous cells of the same tissue type. Such excessive activation may result from overexpression of the TGFβ receptor and/or greater than normal levels of a TGFβ ligand available for activating the TGFβ receptor inhe cancer cells. Such excessive activation may cause and/or be caused by the malignant state of a cancer cell. In some embodiments, the cancer will be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression of a TGFβ receptor is occurring that results in such excessive activation of the TGFβ receptor. Alternatively, or additionally, the cancer may be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression of a TGFβ ligand is occurring in the cancer that attributes to excessive activation of the receptor. In a subset of such cancers, excessive activation of the receptor may resultrom an autocrine-stimulatory pathway. In an autocrine-stimulatory pathway, self-stimulation occurs by virtue of the cancer cell producing both a TGFβ ligand and its cognate TGFβ receptor. For example, the cancer may express or overexpress TGFβ receptor and also express or overexpress a TGFβ ligand (e.g., TGFβ1 ligand). A cancer that “overexpresses” a TGFβ receptor is one that has significantly higherevels of a TGFβ receptor, at the cell surface thereof, compared to a non-cancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. TGFβ receptor overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the TGFβ protein present on the surface of a cell (e.g., via an immunohistochemistry assay;HC). Alternatively, or additionally, one may measure levels of TGFβ encoding nucleic acid in the cell, e.g., via fluorescent in situ hybridization (FISH; see WO 1998/045479 published October, 1998), southern blotting, or polymerase chain reaction (PCR)echniques, such as real-time quantitative PCR (RT-PCR). One may also study TGFβ receptor overexpression by measuring shed antigen (e.g., TGFβ extracellular domain) in a biological fluid such as serum (e.g., U.S. Pat. No.4,933,294 issued Jun.12, 1990; WO 1991/005264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al., ELISA for Quantitation of the Extracellular Domain of p185HER2 in Biological Fluids, J. Immunol. Methods 1990, 132: 73-80). Aside from the above assays, various in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody that is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody. Conversely, a cancer that is “not characterized by overexpression of the TGFβ receptor” is one that, in a diagnostic assay, does not express higher than normal levels of TGFβ receptor compared to a non-cancerous cell of the same tissue type. A cancer that “overexpresses” a TGFβ ligand is one that produces significantly higher levels of that ligand compared to a non-cancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. Overexpression of the TGFβ ligand may be determined diagnostically by evaluating levels of the ligand (or nucleic acid encoding it) in the patient, e.g., in a tumor biopsy or by various diagnostic assays such as the IHC, FISH, southern blotting, PCR, enzyme-linked immunosorbent assay (ELISA) or in vivo assays described above. As used herein, the term “subject” includes organisms which are capable of suffering from a disease of interest that could otherwise benefit from detection of a mutation in a gene that can result in a loss of function of the gene or predispose the subject to a specific disease or condition such that the subject would benefit from screening. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals, e.g., sheep, dog, cow, and primates including non-human primates; e.g., rodents, e.g., mice, and non-mammals, e.g., chickens, amphibians, reptiles, etc. The terms "subject," "individual," or "patient" are used interchangeably herein to refer to any subject for which therapy is being assessed, desired or that is participatingn a clinical trial, epidemiological study or used as a control. The terms "subject," "individual," and "patient" thus encompass individuals having cancer (e.g., prostate cancer), including those who have undergone or are candidates for resection (surgery)o remove cancerous tissue. Accordingly, the term “subject” refers to any animal species of the Mammalia class. Examples of mammals include: humans; non-human primates such as monkeys; laboratory animals such as rats, mice, guinea pigs; domestic animals such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like. In a preferred embodiment, the subject is a human and may be referred to as a patient. In yet another preferred embodiment, the subject is a male having a prostate cancer. Those skilled in the medical art are readily able to identify individual patients who are afflicted with prostate cancer. In a preferred embodiment, the subject may be a human who is at risk, or genetically or otherwise predisposed (e.g., risk factor) to developing cancer who has or has not been diagnosed. As used herein, an “at risk” subject is a subject who is at risk of developing cancer. The subject may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein. An at-risk subject may have one or more so-called risk factors, which are measurable parameters that correlate with development of cancer, which are described herein. A subject having one or more of these risk factors has a higher probability of developing cancer than an individual without these risk factor(s). These risk factors maynclude, for example, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposure. In some embodiments, the subjects at risk for cancer include, for example,hose having relatives who have experienced the disease, and those whose risk is determined by analysis of genetic or biochemical markers. A “control population” refers to a population of individuals who do not have cancer but are otherwise matched to the subject. The skilled person will be able to select an appropriate control population to provide the requisite reference value. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cell; inhibiting metastasis or neoplastic cells; shrinking or decreasing the size of a tumor; remission of the cancer; decreasing symptoms resultingrom the cancer; increasing the quality of life of those suffering from the cancer; decreasing the dose of other medications required to treat the cancer; delaying the progression of the cancer; curing the cancer; overcoming one or more resistance mechanisms of the cancer; and/or prolonging survival of patients the cancer. Positive therapeutic effects in cancer can be measured in a number of ways (see, for example, W. A. Weber, Assessing tumor response to therapy, J. Nucl. Med.50 Suppl.2009, 1:1S- 10S. For example, with respect to tumor growth inhibition (T/C), according to the National Cancer Institute (NCI) standards, a T/C less than or equal to 42% is the minimum level of anti-tumor activity. A T/C <10% is considered a high anti-tumor activity level, with T/C (%) = median tumor volume of the treated / median tumor volume of the control x 100. In some embodiments, the treatment achieved by the present invention is defined by reference to any of the following: complete response (CR), disease free survival (DFS), duration of response (DoR), invasive disease free survival (iDFS), overall response (OR), "overall response rate" (ORR), progression free survival (PFS), overall survival (OS), partial response (PR), progressive disease (PD) and stable disease (SD). As used herein, the term "complete response" or "CR" means the disappearance of all signs of cancer (e.g., disappearance of all target lesions) in response to treatment. This does not always mean the cancer has been cured. As used herein, the term “disease-free survival” (DFS) means the length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer. As used herein, the term “duration of response” (DoR) means the length of timehat a tumor continues to respond to treatment without the cancer growing or spreading. Treatments that demonstrate improved DoR can produce a durable, meaningful delay in disease progression. As used herein, the terms "objective response" and “overall response” refer to a measurable response, including complete response (CR) or partial response (PR). Theerm "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate. As used herein, the term “overall survival” (OS) means the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that patients diagnosed with the disease are still alive. OS is typically measured as the prolongation in life expectancy in patients who receive a certain treatment as comparedo patients in a control group (i.e., taking either another drug or a placebo). As used herein, the term "partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response toreatment. For example, in some embodiments, PR refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD. As used herein, the term "progression free survival" or “PFS” refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not get worse. PFS, also referred to as “Time to Tumor Progression,” may includehe amount of time patients have experienced a CR or PR, as well as the amount of time patients have experienced SD. As used herein, the term "progressive disease" or "PD" refers to a cancer that is growing, spreading or getting worse. In some embodiments, PR refers to at least a 20%ncrease in the SLD of target lesions, taking as reference the smallest SLD recorded since the treatment started, or to the presence of one or more new lesions. As used herein, the term “stable disease” or “SD” refers to a cancer that is neither decreasing nor increasing in extent or severity. As used herein, the term "sustained response" refers to the sustained effect on reducing tumor growth after cessation of a treatment. For example, the tumor size may be the same size or smaller as compared to the size at the beginning of the medicament administration phase. In some embodiments, the sustained response has a duration of at least the same as the treatment duration, at least 1.5x, 2x, 2.5x, or 3x length of thereatment duration, or longer. The anti-cancer effect of the method of treating cancer, including “objective response,” “complete response,” “partial response,” “progressive disease,” “stable disease,” “progression free survival,” and “duration of response,” as used herein, may be defined and assessed by the investigators using RECIST v1.1 (Eisenhauer et al., New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1), Eur J of Cancer 2009, 45(2):228-47). The treatment regimen that is effective to treat a cancer patient may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject. While the aspects of the present invention may not be effective in achieving a positive therapeutic effect in every subject, it should do so in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student’s t-test, the chi2-est the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstrat-testy and the Wilcon on-test. The terms “treatment regimen,” “dosing protocol” and “dosing regimen” may be used interchangeably to refer to the dose and timing of administration of each therapeutic agent administered according to the invention herein. “Ameliorating” means a lessening or improvement of one or more symptoms uponreatment with a combination described herein, as compared to not administering the combination. “Ameliorating” also includes shortening or reduction in duration of a symptom. As used herein, an “effective dosage,” “effective amount” or “therapeutically effective amount” of drug, compound or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired, including biochemical, histological and/or behavioral symptoms, of the disease, its complications andntermediate pathological phenotypes presenting during development of the disease. Forherapeutic use, an effective amount refers to that amount which will relieve to some extent one or more of the symptoms of the disorder being treated. In reference to thereatment of cancer, a therapeutically effective amount refers to that amount which hashe effect of (1) reducing the size of the tumor, (2) inhibiting (that is, slowing to some extent, preferably stopping) tumor metastasis, (3) inhibiting to some extent (that is, slowing to some extent, preferably stopping) tumor growth or tumor invasiveness, (4) relieving to some extent (or, preferably, eliminating) one or more signs or symptoms associated with the cancer, (5) decreasing the dose of other medications required to treathe disease, and/or (6) enhancing the effect of another medication, and/or (7) delayinghe progression of the disease in a patient. An effective dosage can be administered in one or more administrations. For the purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition. As used herein, a “normal cell” is a cell that is derived from tissue that does notnclude any known mutations or disruptions that predispose the cell to a particular disease or disorder. “Abnormal cell growth," as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contactnhibition). Abnormal cell growth may be benign (not cancerous), or malignant (cancerous). This includes the abnormal growth of: (1) tumor cells (tumors) that proliferate, for example, by expressing a mutated SMAD4; (2) benign and malignant cells of other proliferative diseases, for example, in which aberrant SMAD4 inactivation occurs; (3) any tumors that proliferate, for example, by SMAD4 mutations; (4) any tumorshat proliferate, for example, by aberrant SMAD4 inactivation; and (5) benign and malignant cells of other proliferative diseases, for example, in which aberrant SMAD4nactivation occurs. Abnormal cell growth can refer to cell growth in epithelial (e.g., carcinomas, adenocarcinomas): mesenchymal (e.g., sarcomas (e.g., leiomyosarcoma. Ewing's sarcoma)); hematopoetic (e.g., lymphomas, leukemias, myelodysplasias (e.g., pre-malignant)); or other (e.g., melanoma, mesothelioma, and other tumors of unknown origin) cell. As used herein, a “lesion” is a localized change or abnormalities in a tissue or an organ. Tumors are types of lesions. In the terminology of RECIST, “lesion” is generally used instead of “tumor.” “Target lesions” are lesions that have been specifically measured. “Non-target lesions” are lesions whose presences have been noted, but whose measurements have not been taken. At the beginning of a target lesion evaluation, certain lesions are measured in order to provide bases for comparison. Response assessment and evaluation criteria forarget lesions are as follows: for example, Complete Response, or CR – Signifies that allarget lesions have disappeared during the course of treatment. Partial Response, or PR – Signifies that decreases of at least 30% have been noted in the lesion that has theargest diameter, or LD. Stable Disease, or SD – Signifies that there has been no significant decrease or increase in the size of target lesions, based on the smallest sum LD. Progressive Disease, or PD – Signifies that there has been an increase of at least 20% in the sum of the LD of targeted lesions. The criteria for non-target lesions are similar to those of target lesions: for example, Complete Response, or CR – Signifies the disappearance of all non-targetesions. Non-Complete Response or Non-Progressive Disease – Signifies the continued presence of one or more non-target lesions. Progressive Disease, or PB – Signifies that appearance of at least one new lesion, or the increasing size of at least one existing non-arget lesion. Ideally a drug trial will return results like CR or PR. Responses of SD or PD could be used to show that a drug is not an effective treatment for cancer. Although RECIST has its detractors, it continues to be an important set of evaluation rules in the medical and pharmaceutical communities. The World Health Organization has evaluation criteria that differ somewhat. In the WHO system, Partial Response means the largest lesion has decreased in size (diameter) by 50 percent or more, and Progressive Disease means the largest lesion hasncreased in diameter by 35 percent or that new lesions are visible. As used herein, the term “tumor” as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm orissue mass of any size and includes primary tumors and secondary neoplasms. A solidumor is an abnormal growth or mass of tissue that usually does not contain cysts oriquid areas. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National Cancernstitute, Dictionary of Cancer Terms). As used herein, the term “tumor burden” or “tumor load’, refers to the total amount of tumorous material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of tumor(s), throughout the body, including lymph nodes and bone marrow. Tumor burden can be determined by a variety of methods known in the art, such as, e.g., using calipers, or while in the body using imagingechniques, e.g., ultrasound, bone scan, computed tomography (CT), or magnetic resonance imaging (MRI) scans. The term “tumor size” refers to the total size of the tumor which can be measured as the length and width of a tumor. Tumor size may be determined by a variety of methods known in the art, such as, e.g., by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imagingechniques, e.g., bone scan, ultrasound, CR or MRI scans. A "kit" is any manufacture (e.g. a package or container) comprising at least one reagent, e.g., a probe, for specifically detecting or modulating the expression of a marker of the invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. TGFβr1 Inhibitor Transforming growth factor β (also known as TGFβ, TGF-β, TGFB, TGF-B, TGFb, TGF-b, TGFbeta or TGF-beta) is a multifunctional cytokine having three forms designated TGFβ1, TGFβ2 and TGFβ3. TGFβ1 to 3 form a subfamily of highly similar proteins withinhe TGFβ superfamily of cytokines. As used herein, the term “TGFβ” refers to TGFβ from any species and includessoforms, fragments, variants or homologues of a TGFβ from any species. In some embodiments, the TGFβ is a human TGFβ, primate TGFβ, non-human primate TGFβ, rodent TGFβ, murine TGFβ, or mammalian TGFβ. The “TGFβ family” is a class within the TGFβ superfamily and in human containshree members: TGFβ1, TGFβ2, and TGFβ3, which are structurally similar. The three growth factors are known to signal via the same receptors. Human TGFβ1 (NCBI Reference Sequence: NP_000651.3) is a 390 amino acid protein, whilst TGFβ2 (NCBI Reference Sequence: NP_001129071.1) is a 442 amino acid protein, and TGFβ3 (NCBI Reference Sequence: NP_001316868.1) contains 412 amino acids. Each of TGFβ1-3 have a 20-30 amino acid signal peptide at the N-terminus which is necessary for secretion, a pro-region called latency associated peptide (LAP), and a 112-114 amino acid C-terminal region that becomes the mature TGFβ moleculeollowing proteolytic cleavage from the pro-region (Khalil et al., TGF-beta: from latent to active, Microbes Infect. 1999, 1 (15): 1255-63). Mature monomeric TGβ dimerize to produce the biologically active 25 KDa protein. TGFβ1 to 3 can form homodimers withhe same type of TGFβ, or can form heterodimers with another type of TGFβ (e.g., a TGFβ1:TGFβ2 heterodimer). TGFβ comprises nine conserved cysteine residues, eight of which form disulfide bonds to form the cysteine knot structure which is characteristic of the TGFβ superfamily. The remaining cysteine is involved interacts with that of another TGFβ monomer to form the dimer. The surface-exposed region between the fifth and sixth conserved cysteine residues is the region, which is least conserved between TGFβ1o 3 proteins, and is thought to be important for receptor binding and specificity of TGFβ. TGFβ1 was originally defined by its ability to cause the phenotypic transformation of rat fibroblasts. TGFβ1 is a multipotent cytokine with cell- and dose-dependent activities. Although TGFβ1 is a growth inhibitor for most cell types, it can act as a stimulator for some cell types. TGFβ1 has ubiquitous distribution. For reviews on TGFβ1, see Massagué, The transforming growth factor-beta family, J. Ann. Rev. Cell Biol.1990, 6, 597; Letterio et al., Regulation of immune responses by TGF-beta, Ann. Rev. Immunol. 1998, 16, 137. TGFβ1 demonstrates regulatory effects on a wide range of cell types, and modulates embryonic development, bone formation, mammary development, wound healing, haematopoiesis, angiogenesis, cell cycle progression and the production of the extracellular matrix. With respect to the immune system, TGFβ1 inhibits T and B cell proliferation and acts as an anti-inflammatory molecule both in vitro and in vivo. TGFβ1nhibits macrophage maturation and activation, and also inhibits the activity of natural killer cells and lymphokine-activated killer (LAK) cells and blocks cytokine production. In some embodiments, a TGFβ ligand binds to and activates TGFβ receptor. Theerm “TGFβ receptor” refers to a TGFβ receptor from any species and includes isoforms,ragments, variants or homologues of a TGFβ receptor from any species. In some embodiments, the TGFβ receptor is a human TGFβ receptor, primate TGFβ receptor, non-human primate TGFβ receptor, rodent TGFβ receptor, murine TGFβ receptor, or mammalian TGFβ receptor. TGFβ exerts its functional consequences through binding to and activating signaling through TGFβ receptors. The term “TGFβ receptor,” unless otherwisendicated, refers to any receptor that binds at least one TGFβ receptor. TGFβ receptors comprising an extracellular domain having a TGFβ binding region, a single passransmembrane domain and an intracellular domain comprising a serine/threonine kinase domain. There are three main types of TGFβ receptors; TGFβ receptor type 1 or TGFβr1 (NCBI Reference Sequence: NP_004603.1), TGFβ receptor type 2 or TGFβr2 (NCBI Reference Sequence: NP_001020018.1) and TGFβ receptor type 3 or TGFβr3 (NCBI Reference Sequence: NP_003234.2). In some embodiments, the TGFβ receptor is TGFβ receptor 1 (TGFβr1), TGFβ receptor 2 (TGFβr2), or TGFβ receptor 3 (TGFβr3). As used herein, the term “TGFβ1-positive cancer,” “TGFβ1-positive tumor,” “TGFβ-positive cancer” or TGFβ-positive tumor refers to a cancer or tumor with aberrant TGFβ1, and/or other TGFβ isoform expression (overexpression). Many human cancer/tumor types show predominant expression of the TGFβ1 isoform. The term “TGFβ” is sometimes used to refer to the gene as opposed to protein. In some embodiments, such cancer/tumor may show co-dominant expression of another isoform, such as TGFβ3. A number of epithelial cancers (e.g., carcinoma) may co-express TGFβsoforms (e.g., TGFβ1 and TGFβ3). Within the tumor environment of TGFβ1-positiveumors, TGFβ1 may arise from multiple sources, including, for example, cancer cells,umor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and the surrounding extracellular matrix (ECM). In the context of the present disclosure, preclinical cancer or tumor models that recapitulate human conditions are TGFβ1- positive cancer or tumor. The term “TGFβ inhibitor,” “TGFβ receptor inhibitor” or “TGFβr inhibitor” refers to any agent capable of antagonizing biological activities, signaling or function of TGFβ growth factor (e.g., TGFβ1 (TGFβr1), TGFβ2 (TGFβr2), and/or TGFβ3 (TGFβr3)). Theerm is not intended to limit its mechanism of action and includes, for example, neutralizing inhibitors, small molecule inhibitors, receptor antagonists, soluble ligandraps, and activation inhibitors of TGFβ. TGFβr inhibitors also include antibodies that are capable of reducing the availability of latent proTGFβ which can be activated in the niche,or example, by inducing antibody-dependent cell mediated cytotoxicity (ADCC), and/or antibody-dependent cellular phagocytosis (ADPC), as well as antibodies that result innternalization of cell-surface complex comprising latent proTGFβ, thereby removing the precursor from the plasma membrane without depleting the cells themselves.nternalization may be a suitable mechanism of action for LRRC33-containing protein complexes (such as human LRRC33-proTGFβ1) which results in reduced levels of cells expressing LRRC33-containing protein complexes on cell surface. As used herein, a tumor cell that is "responsive" to a TGFβr1 inhibitor, refers to aumor cell in which the growth, division, and/or other metabolic pathways are adversely affected such that the cell is inhibited from growing or dividing and/or undergoes apoptosis or other forms of cell death. In some embodiments of each of the methods, uses and pharmaceutical compositions described herein, the TGFβr1 inhibitor is selected from the group consisting of galunisertib, LY2109761, SB525334, SP505124, GW788388, LY364947, RepSox, SD-208, vactosertib, LY3200882 and 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4- ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229), or a pharmaceutically acceptable salt thereof, or combinations thereof. In a preferred embodiment, the methods, uses and pharmaceutical compositions described herein, the TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-sopropylpyridin-4-ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229), or a pharmaceutically acceptable salt thereof. The compound, 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N- (1,3-dihydroxypropan-2-yl)nicotinamide (“TGFβr1 inhibitor PF-06952229,” “PF- 06952229” or “PF-‘2229”), is a potent and selective TGFβr1 (transforming growth factor beta receptor type 1) inhibitor, having the structure:

PF-06952229 and pharmaceutically acceptable salts thereof and its preparation are disclosed in International Application No. PCT/US2014/072922, which published asnternational Publication No. WO 2015/103355 on 9 July 2015, and U.S. Patent No. 10,030,004 which issued on 10 December 2019. The contents of each of the foregoing references are incorporated herein by reference in their entirety. In a preferred embodiment of each of the foregoing, the TGFβr1 inhibitor is a crystalline form of 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N-(1,3- dihydroxypropan-2-yl)nicotinamide. In some such embodiments, the crystalline form is an anhydrous mono hydrochloride salt. Unless indicated otherwise, all references herein to TGFβr1 inhibitors include references to pharmaceutically acceptable salts, solvates, hydrates and complexeshereof, and to solvates, hydrates and complexes of pharmaceutically acceptable saltshereof, and include amorphous and polymorphic forms, stereoisomers, and isotopicallyabelled versions thereof. SMAD4 As used herein, the tumor suppressor gene, “SMAD

4,” is synonymous with other designations for the same tumor suppressor gene that are known to those of skill in the art, including but not limited to madh4 and dpc4. As is conventional, the products of the expression of this gene is designated herein as SMAD4, which is synonymous with the corresponding other designations for the expression product of this gene that are knowno those of skill in the art, including but not limited to MADH4 and DPC4. SMAD4 is a component of the SMAD pathway that is involved in signalransduction in the TGF-β pathway (Levy, L. and Hill, C. S., Molec. Cell. Biol. 2005, 25:8108-8125; Fukuchi, M. et al., Cancer 2002, 95:737-743). This gene, also known as DPC4 (for “decreased in pancreatic carcinoma”), appears to be a tumor suppressor gene, and a decrease in SMAD4 expression has been observed in a variety of primary carcinomas, including pancreatic carcinomas (Luttges, J. et al., Am. J. Pathol. 2001, 158:1677-1683; Subramanian, G. et al., Cancer Res.2004, 64:5200-5211), esophageal carcinomas (Fukuchi, M. et al., Cancer 2002, 95:737-743, cervical carcinomas (Maliekal, T. T. et al., Oncogene 22:4889-4897 (2003), and other primary human cancers (Iacobuzio-Donahue, C. A. et al., Clin. Canc. Res.2004, 10:1597-1604, as well as in cell line cancer models including of pancreatic cancers (Lohr, M. et al., Cancer Res. 2001, 61:550-555; Yasutome, M. et al., Clin. Exp. Metastasis 2005, 22:461-473), and of colon cancers (Levy, L., and Hill, C. S., Molec. Cell. Biol. 2005, 25:8108-8125). A reduced expression of SMAD4 in tumors has been associated with poor prognosis for patient survival, particularly in patients with SMAD4-deficient pancreatic adenocarcinomas (Liu, F., SMAD4/DPC4 and Pancreatic Cancer Survival, Clin. Cancer Res.2001, 7:3853-3856; Tascilar, M. et al., SMAD4/DPC4 and Pancreatic Cancer Survival, Clin. Cancer Res. 2001, 7:4115-4121; Toga, T. et al., The Dissociated Expression of Protein and Messenger RNA of DPC4 in Human Invasive Ductal Carcinoma of the Pancreas and their Implication for Patient Outcome, Anticancer Res.2004, 24:1173-1178). The mechanism of the tumor suppressive activity of the SMAD4 gene product is poorly understood, but it is thought that it may act as a “switch” regulating the growth-suppressive and growth- activating activities of certain components of the TGF-β signaling pathway (for reviews, see Akhurst, A. J., J. Clin. Invest. 2002, 109:1533-1536; Bachman, K. E., and Park, B. H., Curr. Opin. Oncol.2004, 17:49-54; Bierie, B., and Moses, H. L., Nature Rev. Cancer 2006, 6:506-520). As described in Table 1, a list of high impact mutations in SMAD4 was discovered to result in sensitivity of a subject to TGFβr1 inhibitor mutations. The SMAD4 mutations are denoted by amino acid positions and mutation type in Table 1 (PMID 29625053, https://pubmed.ncbi.nlm.nih.gov/29625053/). Table 1 further shows loss-of-function SMAD4 mutations (G386 frame-shift mutation (G386fs)) observed in patient tumor sample. Table 1

Therapeutic Methods and Uses The present invention provides methods and uses that are useful for treating cancer. Some embodiments provided herein result in one or more of the following effects: (1) inhibiting cancer cell proliferation; (2) inhibiting cancer cell invasiveness; (3) inducing apoptosis of cancer cells; (4) inhibiting cancer cell metastasis; (5) inhibiting angiogenesis; or (6) overcoming one or more resistance mechanisms relating to a cancer treatment. In one aspect, the invention relates to the discovery that there is a relationship between the expression levels of the tumor suppressor gene SMAD4 (also known as JIP, DPC4, MADH4 or MYHRS) and the responsiveness of a patient population to TGFβr1nhibitors, particularly in cancer cells from such patient population. Specifically, it has been found that tumor cells that display decreased levels of expression of SMAD4 or SMAD4-deficient tumor cells have a higher propensity to be responsive to TGFβr1 inhibitors, in particular to PF-06952229, compared to tumor cellshat display higher expression levels of SMAD4. In other embodiments, the invention also provides methods using identification ofhis differential expression of a SMAD4 gene in determining the invasive and/or metastatic potential of tumor cells and in identifying those tumor cells, such as certain cancers (e.g., prostate cancer), that may be more likely to rapidly progress and which should be aggressively treated in a patient. The invention also provides methods ofdentifying those tumors in which the cells making up the tumor may be more likely to respond to treatment with a TGFβr1 inhibitor. The invention also provides methods of diagnosis and treatment/prevention of tumor metastasis. Such methods include, for example, determining the levels of expression of theumor suppressor gene SMAD4 by the tumor cells, wherein a reduced expression of SMAD4 indicates that the tumor cell is more likely to be responsive to TGFβr1 inhibitors.n certain such embodiments, the level of expression of SMAD4 in cells of a tumor, e.g., a prostate cancer tumor, is determined in tissue sections obtained from a patient sufferingrom such a tumor, wherein a decrease in the expression of SMAD4 in the tumor cells relative to that in non-tumor tissue samples (ideally, from the same organ in the same patient) indicates that the tumor is more likely to be responsive to TGFβr1 inhibitors. Inhis way, appropriate and aggressive protocols for treating such tumors in a patient can rapidly be identified and implemented, thereby providing an increased likelihood of positive treatment outcomes for cancer patients. The invention thus provides methods for determining the responsiveness of tumor cells to TGFβr1 inhibitors by examining the expression of TGFβ canonical singling pathways, and SMAD4 by the tumor cells, as well as methods of diagnosis and treatment/predicting /prevention of tumor progression using TGFβr1 inhibitors, particularly in cancer cells with SMAD4 mutations. In one aspect, the invention provides a of treating cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a TGFβr1 inhibitor, wherein the cancer comprises a loss or reduction of canonical TGFβ signaling. In some embodiments of this aspect, the loss or reduction of canonical TGFβ signaling is caused by a mutation, reduced expression or deletion of a SMAD4 gene or combinations thereof. In some embodiments of this aspect, the loss or reduction of canonical TGFβ signaling is caused by a mutation of a SMAD4 gene. In a preferred embodiment of each of the foregoing, the mutation of SMAD4 is a loss of function mutation, a low expression mutation and/or a loss of copy number mutation. In one such embodiment, the mutation of the SMAD4 gene results is a premature stop codon. In some embodiments, the reduced expression of the SMAD4 gene is due to a RNA expression level or a DNA mutation which may result in lower expression of SMAD4. In some embodiments, the loss of SMAD4 expression or function results from epigenetic regulation such as DNA methylation and histone modifications. In another embodiment, the mutation of the SMAD4 gene results in a frame-shift mutation. In a preferred embodiment, the mutation of the SMAD4 gene is c.-430C>T or c.1155delA (deletes A) , or combinations thereof. In some such embodiments, the mutation of the SMAD4 gene is a loss of function mutation. In one embodiment, the loss of function mutation of the SMAD4 gene is G386fs, - 301fs, A199Sfs, A309Sfs*25, A532Pfs*5 or C443*, or combinations thereof. In one such embodiment, the loss of function mutation of the SMAD4 gene is G386fs. In further embodiments of each of the foregoing, the mutation of the SMAD4 gene interferes with a SMAD4 protein binding to a SMAD2 or SMAD3 protein. In some embodiments, loss or reduction of canonical TGFβ signaling is caused by a mutation, reduced expression or deletion of a SMAD2 or SMAD3 gene, or combinations thereof. In some such embodiments, the loss or reduction of canonical TGFβ signaling is caused by a mutation of a SMAD2 or SMAD3 gene. In some embodiment, the mutation or deletion of the SMAD2 or SMAD3 gene is aoss of function mutation, a low expression mutation and/or a loss of copy number mutation. In yet another such embodiment, the mutation of the SMAD2 or SMAD3 genenterferes with a SMAD2 or SMAD3 protein binding to a SMAD4 protein. In some embodiments of the each of the foregoing, the TGFβr1 inhibitor is selectedrom the group consisting of galunisertib, LY2109761, SB525334, SP505124, GW788388, LY364947, RepSox, SD-208, vactosertib, LY3200882 and 4-(2-(5-chloro-2-luorophenyl)-5-isopropylpyridin-4-ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide PF-06952229), or a pharmaceutically acceptable salt thereof. In a preferred embodiment of each of the foregoing, the TGFβr1 inhibitor is 4-(2- 5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N-(1,3-dihydroxypropan-2- yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the invention also provides a method of treating cancer in a subject, comprising administering to the subject in need thereof aherapeutically effective amount of a TGFβr1 inhibitor, wherein the TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N-(1,3-dihydroxypropan-2- yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof, and wherein the cancer comprises a loss or reduction of canonical TGFβ signaling. In some embodiments of each of the methods and uses herein, the method further comprises administering to the subject an amount of one or more anti-cancer agents; wherein the amounts of the TGFβr1 inhibitor and the one or more anti-cancer agentsogether are effective in treating cancer. In some such embodiments, the one or more anti-cancer is selected from the group consisting of a further TGFβr1 inhibitor, an anti-tumor agent, an anti-androgen and an anti-angiogenic agent. In one aspect, the invention provides a method of treating cancer in a subject comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; c. selecting the subject for treatment with a TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product, is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population; and d. administering to the selected subject an effective amount of the TGFβr1 inhibitor, thereby treating cancer in the subject. In a preferred embodiment, the invention also provides a method of treating cancer in a subject comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; c. selecting the subject for treatment with a TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product, is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population; and d. administering to the selected subject an effective amount of the TGFβr1 inhibitor, thereby treating cancer in the subject, wherein the TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4- ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof. In a further aspect, the invention provides a method of selecting a subject having cancer for treatment with a TGFβr1 inhibitor, comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; and c. selecting the subject for treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population. In a preferred embodiment, the invention also provides a method of selecting a subject having cancer for treatment with a TGFβr1 inhibitor, comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; and c. selecting the subject for treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population, wherein the TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4- ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof. In another aspect, the invention provides a method of predicting whether a subject having cancer will respond to treatment with a TGFβr1 inhibitor, comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; c. selecting the subject for treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population; and d. predicting the subject will respond to treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population. In a preferred embodiment, the invention also provides a method of predicting whether a subject having cancer will respond to treatment with a TGFβr1 inhibitor, comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; c. selecting the subject for treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population; and d. predicting the subject will respond to treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population, wherein the TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin- 4-ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof. In some embodiments of the each of the foregoing, the biological sample comprises cancer tissue, cancer cells or circulating tumor DNA. In further embodiments of each of the foregoing, the cancer tissue is obtained from a biopsy. In some embodiments of the each of the foregoing, the biological sample is a bodily fluid. In some such embodiments, the bodily fluid is selected from the group consisting of blood, lymph, urine, saliva, fluid from ductal lavage, and nipple aspirate. In some embodiments, the cancer is a solid tumor such as sarcomas and carcinomas. In some embodiments, the cancer that may be treated is a liquid tumor such as leukemia or lymphoma. Examples of cancers that may be treated by methods of thenvention include, but are not limited to, prostate cancer, breast cancer, ovarian cancer,ung cancer, colon cancer, brain cancer, gastric cancer, liver cancer, thyroid cancer, endometrial cancer, gallbladder cancer, kidney cancer, adrenocortical cancer, sarcoma, skin cancer, head and neck cancer, leukemia, bladder cancer, colorectal cancer, hematopoietic cancer and pancreatic cancer. In a preferred embodiment, the cancer is a prostate cancer. In yet another preferred embodiment, the prostate cancer is hormone dependent prostate cancer. In one preferred embodiment, the prostate cancer is a metastatic castration resistant prostate cancer (mCRPC). In yet another preferred embodiment, the prostate cancer is hormone dependent prostate cancer. In a preferred embodiment, the prostate cancer is advanced prostate cancer. In yet another preferred embodiment, the prostate cancer is metastatic, non-metastatic, locally advanced, advanced hormone sensitive, advanced castration resistant, or recurrent. In yet another preferred embodiment, the prostate cancer is mCRPC or prostate adenocarcinoma. In some embodiments, the breast cancer is breast carcinoma (ER negative or ER positive), primary breast ductal carcinoma, mammary adenocarcinoma, mammary ductal carcinoma (ER positive, ER negative or HER2 positive), HER2 positive breast cancer,uminal breast cancer or triple negative breast cancer (TNBC). In some embodiments,he breast cancer is unclassified. In some embodiments, the triple negative breast cancers a basal-like TNBC, a mesenchymal TNBC (mesenchymal or mesenchymal stem-like), an immunomodulatory TNBC, or a luminal androgen receptor TNBC. In some embodiments, the ovarian cancer is ovary adenocarcinoma. In some embodiments, the lung cancer is lung carcinoma, non-small lung carcinoma, adenocarcinoma, mucoepidermoid, anaplastic, large cell, or unclassified. In some embodiments, the colon cancer is colon adenocarcinoma, colon adenocarcinoma from a metastatic site lymph node, metastatic colorectal cancer, or colon carcinoma. In some embodiments, the brain cancer is glioblastoma, astrocytoma, meduloblastoma, meningioma or neuroblastoma. In some embodiments, gastric cancer is stomach cancer. In some embodiments, liver cancer is hepatocellular carcinoma, hepatoblastoma or cholangiocarcinoma. In some embodiments, liver cancer is hepatitis B virus derived.n some embodiments, liver cancer is virus negative. In some embodiments, thyroid cancer is papillary thyroid carcinoma, follicularhyroid cancer or medullary thyroid cancer. In some embodiments, endometrial cancer is high grade endometrial cancer, uterine papillary serous carcinoma (UPSC) or uterine clear cell carcinoma (UCCC). In some embodiments, gallbladder cancer is gallbladder adenocarcinoma or squamous cell gallbladder carcinoma. In some embodiments, kidney cancer is renal cell carcinoma or urothelial cell carcinoma. In some embodiments, adrenocortical cancer is adrenal cortical carcinoma. In some embodiments, sarcoma is synovial sarcoma, osteosarcoma, habdomyosarcoma (RMS), fibrosarcoma or Ewing’s sarcoma. In some embodiments, skin cancer is basal cell carcinoma, squamous carcinoma or melanoma. In some embodiments, head and neck cancer is oropharyngeal cancer, nasopharyngeal cancer, laryngeal cancer and cancer of the trachea. In some embodiments, the leukemia is acute promyelocytic leukemia, acuteymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenouseukemia (CML), acute myeloid leukemia (AML), mantle cell lymphoma or multiple myeloma. In some embodiments, the leukemia is acute promyelocytic leukemia, acuteymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, mantle cell lymphoma or multiple myeloma. In some embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin'symphoma (NHL), e.g., B-cell lymphomas, including diffuse large B-cell lymphoma DLBCL), follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphomas; nodal marginal zone B-cell lymphoma; nodal marginal zone B-cell lymphoma; splenic marginal zone B-cell lymphoma; Burkitt lymphoma; lymphoplasmacytic lymphoma Waldenstrom macroglobulinemia); hairy cell leukemia; primary central nervous system CNS) lymphoma and T-cell lymphomas. Examples of T-cell lymphomas include T-cellymphomas, precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cellymphomas (such as cutaneous T-cell lymphomas (mycosis fungoides, Sezary syndrome, among others); adult T-cell leukemia/lymphoma; angioimmunoblastic T-cellymphoma, extranodal natural killer/T-cell lymphoma; nasal type; enteropathy-associatedntestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL); and peripheral T-cell lymphoma. The invention additionally provides a method for treating a tumor comprising contacting the tumor with an effective amount of one or more compounds of the invention, or a salt thereof. In one aspect of the method, a compound or salt thereof is administeredo a subject in need of tumor treatment. Exemplary tumors are derived from carcinomas of the breast, prostate, ovary, lung, or colon. In one aspect, the treatment results in a eduction of the tumor size. In another aspect, the treatment slows or prevents tumor growth and/or metastasis. The invention further provides methods for treating a hematopoietic malignancy comprising administering an effective amount of one or more compounds of the inventiono a subject in need thereof. In some embodiments, the hematopoietic malignancy is acute promyelocytic leukemia. Any of the methods of treatment provided herein may be used to treat a primaryumor. Any of the methods of treatment provided herein may also be used to treat a metastatic cancer (that is, cancer that has metastasized from the primary tumor). Any ofhe methods of treatment provided herein may be used to treat cancer at an advanced stage. Any of the methods of treatment provided herein may be used to treat cancer at aocally advanced stage. Any of the methods of treatment provided herein may be used toreat early-stage cancer. Any of the methods of treatment provided herein may be usedo treat cancer in remission. In some of the embodiments of any of the methods ofreatment provided herein, the cancer has reoccurred after remission. In some embodiments of any of the methods of treatment provided herein, the cancer is progressive cancer. Any of the methods provided herein may be used wherein the cancer is a resistant, or relapsed cancer to one or more prior therapies administered to treat the cancer. In some embodiments, the cancer is resistant to chemotherapy and/or radiotherapy. Any of the methods of treatment provided herein may be used to treat an individual e.g., human) who has been diagnosed with or is suspected of having cancer. In some embodiments, the subject may be a human who exhibits one or more symptoms associated with cancer. In some embodiments, the subject may have advanced disease or a lesser extent of disease, such as low tumor burden. In some embodiments, the subject is at an early stage of a cancer. In some embodiments, the subject is at an advanced stage of cancer. In some of the embodiments of any of the methods ofreatment provided herein, the subject may be a human who is genetically or otherwise predisposed (e.g., has one or more so-called risk factors) to developing cancer who has or has not been diagnosed with cancer. In some embodiments, these risk factors include, but are not limited to, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposure. In some embodiments, the subjects at risk for cancer include, e.g., those having relatives who have experienced this disease and those whose risk is determined by analysis of genetic or biochemical markers. In some embodiments, the subject does not have type I diabetes. In some embodiments, the subject does not have type II diabetes with sustained hyperglycemia or type II diabetes with hyperglycemia for prolonged duration (e.g., for several years). Any of the methods of treatment provided herein may be practiced in an adjuvant setting. In some embodiments, any of the methods of treatment provided herein may be used to treat a subject who has previously been treated for cancer, e.g., with one or more other therapies such as radiation, surgery or chemotherapy. Any of the methods ofreatment provided herein may be used to treat a subject who has not previously beenreated for cancer. Any of the methods of treatment provided herein may be used to treat a subject at risk for developing cancer, but who has not been diagnosed with cancer. Any of the methods of treatment provided herein may be used as a first line therapy. Any ofhe methods of treatment provided herein may be used as a second line therapy. Any of the methods of treatment provided herein in one aspect reduce the severity of one or more symptoms associated with cancer by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding symptom in the same subject prior to treatment or compared to the corresponding symptom in other subjects not receiving a compound or composition of the invention. Any of the methods of treatment provided herein may be used to treat, stabilize, prevent, and/or delay any type or stage of cancer. In some embodiments, the subject is at least about any of 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years old. In some embodiments, one or more symptoms of the cancer are ameliorated or eliminated. In some embodiments, the size of a tumor, the number of cancer cells, or the growth rate of a tumor decreases by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. In some embodiments, the cancer is delayed or prevented. In a preferred embodiment, the method of the invention further comprises one or more anti-cancer agents, e.g., a further TGFβr1 inhibitor, an anti-tumor agent, an anti- androgen and/or an anti-angiogenic agent, for use in the manufacture of a medicament.n some such embodiments, the anti-tumor agent is mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, radiation, cell cyclenhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxics or anti-hormones. In some such embodiments, the anti-androgen inhibitor is selected from the group consisting of enzalutamide (Xtandi^®), apalutamide (ERLEADA^®), darolutamide (NUBEQA^®), bicalutamide (CASODEX^®) and flutamide (Eulexin^®). In some such embodiments, the anti-angiogenic agent is Fumagillin, which is known as 2,4,6,8- decatetraenedioic acid; mono[3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methyl-2-butenyl)oxi-ranyl]-1-oxaspiro[2.5]oct-6-yl]ester, (2E,4E,6E,8E)-(9CI); Shikonin, which is also known as 1,4-naphthalenedione, 5,8-dihydroxy-2-[(1R)-1-hydroxy-4- methyl-3-pentenyl]-(9CI); Tranilast, which is also known as benzoic acid, 2-[[3-(3,4- dimethoxyphenyl)-1-oxo-2-propenyl]amino]-(9CI); ursolic acid; suramin; thalidomide or enalidomide (REVLIMID ^®). In yet another preferred embodiment, the method of thenvention further comprises chemotherapy, surgery or radiation therapy, or combinationshereof. Pharmaceutical Compositions and Routes of Administration The pharmaceutical composition of the present invention may comprise a TGFβr1nhibitor and at least one excipient. As used herein, the term “excipient” refers to a pharmaceutically acceptable ingredient that is commonly used in pharmaceuticalechnology for the preparation of solid oral dosage formulations. The intended function of an excipient is to act as the carrier (vehicle or basis) or as a component of the carrier ofhe active substance(s) and, in so doing, to contribute to product attributes such as stability, biopharmaceutical profile, appearance and patient acceptability and/or to the ease with which the product can be manufactured. Examples of categories of excipients include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. The amount of each excipient used may vary within ranges conventional in the art. The following references which are all hereby incorporated by reference disclose techniques and excipients usedo formulate oral dosage forms. See The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et. al., Eds., American Pharmaceuticals Association (2003); and Remington: The Science and Practice of Pharmacy, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2000). Suitable excipients include magnesium carbonate, magnesium stearate, talc,actose, lactose monohydrate, sugar, pectin, dextrin, starch, tragacanth, microcrystalline cellulose, methyl cellulose, sodium carboxymethyl cellulose, corn starch, colloidal anhydrous Silica, titanium dioxide, a low-melting wax, cocoa butter, and the like. In some embodiments, the pharmaceutical composition comprises at least one excipient. The pharmaceutical composition may of the present invention may be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained releaseormulation, solution or suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream, or for rectal administration as a suppository. Exemplary parenteral administration forms include solutions or suspensions of an active compound in a sterile aqueous solution, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms may be suitably buffered, if desired. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise amounts. Pharmaceutical compositions suitable for the delivery of the therapeutic agents ofhe combination therapies of the present invention, and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in ‘Remington’s Pharmaceutical Sciences’, 19th Edition (Mack Publishing Company, 1995), the disclosure of which is incorporated herein by reference in its entirety. The agents of the combination therapies of the invention may be administered orally. Oral administration may involve swallowing, so that the agent enters the gastrointestinal tract, or buccal or sublingual administration may be employed by whichhe agent enters the blood stream directly from the mouth. Formulations, dosage unit forms or pharmaceutical compositions suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, powders, granules, aqueous and nonaqueous oral solutions and suspensions, lozenges (including liquid-filled), troches, hard candies, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, injectable solutions, chews, multi- and nano-particulates, solid solution, liposome, films (including muco-adhesive), ovules, sprays, liquid formulations and parenteral solutions packaged in containers adapted for subdivision into individual doses. Liquid formulations include suspensions, solutions, syrups and elixirs. Suchormulations may be used as fillers in soft or hard capsules and typically include a carrier,or example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquidormulations may also be prepared by the reconstitution of a solid, for example, from a sachet. Parenteral formulations include pharmaceutically acceptable aqueous or nonaqueous solutions, dispersion, suspensions, emulsions, and sterile powders for the preparation thereof. Examples of carriers include water, ethanol, polyols (propylene glycol, polyethylene glycol), vegetable oils, and injectable organic esters such as ethyl oleate. Fluidity can be maintained by the use of a coating such as lecithin, a surfactant, or maintaining appropriate particle size. Exemplary parenteral administration formsnclude solutions or suspensions of the compounds of the invention in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosageorms can be suitably buffered, if desired. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules. Preferred materials, therefor,nclude lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active compoundherein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof. Therapeutic agents of the combination therapies of the present invention may also be used in fast-dissolving, fast-disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986 by Liang and Chen (2001), the disclosure of which is incorporated herein by reference in its entirety. Methods of preparing various pharmaceutical compositions with a specific amount of active compound are known, or will be apparent, to those skilled in this art. For examples, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975). For tablet dosage forms, the agent may make up from 1 wt% to 80 wt% of the dosage form, more typically from 5 wt% to 60 wt% of the dosage form. In addition to the active agent, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch and sodium alginate. Generally, the disintegrant may comprise from 1 wt% to 25 wt%, preferably from 5 wt% to 20 wt% of the dosage form. Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such asactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate. Tablets may also optionally include surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents are typically in amounts of from 0.2 wt% to 5 wt% of the tablet, and glidants typically from 0.2 wt% to 1 wt% of the tablet. Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally are present in amounts from 0.25 wt%o 10 wt%, preferably from 0.5 wt% to 3 wt% of the tablet. Other conventional ingredients include anti-oxidants, colorants, flavoring agents, preservatives and taste-masking agents. Exemplary tablets may contain up to about 80 wt% active agent, from about 10 wt% to about 90 wt% binder, from about 0 wt% to about 85 wt% diluent, from about 2 wt% to about 10 wt% disintegrant, and from about 0.25 wt% to about 10 wt% lubricant. Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tableting. The final formulation may include one or moreayers and may be coated or uncoated; or encapsulated. The formulation of tablets is discussed in detail in “Pharmaceutical Dosage Forms: Tablets, Vol.1,” by H. Lieberman and L. Lachman, Marcel Dekker, N.Y., N.Y., 1980 (ISBN 0-8247-6918-X), the disclosure of which is incorporated herein by reference in its entirety. Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Suitable modified release formulations are described in U.S. Patent No. 6,106,864. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles may be found in Verma et. al., Current Status of Drug Delivery Technologies and Future Directions, Pharmaceutical Technology On-line, 2001, 25(2), 1-14. The use of chewing gum to achieve controlled release is described in WO 2000/035298. The disclosures of these references are incorporated herein by reference in their entireties. Pharmaceutical compositions may comprise one or more pharmaceutical acceptable carriers or diluents and may be manufactured in conventional manner by mixing one or both combination partners with a pharmaceutically acceptable carrier or diluent. Examples of pharmaceutically acceptable diluents include, but are not limited to,actose, dextrose, mannitol, and/or glycerol, and/or lubricants and/or polyethylene glycol. Examples of pharmaceutically acceptable binders include, but are not limited to, magnesium aluminum silicate, starches, such as corn, wheat or rice starch, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and, if desired, pharmaceutically acceptable disintegrators include, but are not limited to, starches, agar, alginic acid or a salt thereof, such as sodium alginate, and/or effervescent mixtures, or adsorbents, dyes, flavorings and sweeteners. It is also possible to use the compounds of the present invention in the form of parenterally administrable compositions or in the form of infusion solutions. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting compounds and/or emulsifiers, solubilisers, salts for regulating the osmotic pressure and/or buffers. The effective dosage of each of agents employed in the combination of thenvention may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the condition being treated, the severity of the condition being treated. Thus, the dosage regimen of the combination of the invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed. A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentration of drug within the range that yields efficacy requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug. For purposes of the present invention, a therapeutically effective dose will generally be a total daily dose administered to a host in single or divided doses. The compound of formula (I) may be administered to a host in a daily dosage range of, for example, from about 0.05 to about 50 mg/ kg body weight of the recipient, preferably about 0.1-25 mg/kg body weight of the recipient, more preferably from about 0.5 to 10 mg/kg body weight of the recipient. Agent (b) may be administered to a host in a daily dosage range of, for example, from about 0.001 to 1000 mg/kg body weight of the ecipient, preferably from 1.0 to 100 mg/kg body weight of the recipient, and most preferably from 1.0 to 50 mg/kg body weight of the recipient. Dosage unit compositions may contain such amounts of submultiples thereof to make up the daily dose. Kits The present invention is also directed to a kit for assaying a biological sample to determine a SMAD4 gene deletion or mutation in the biological sample, as disclosed herein. In a specific aspect, the invention provides a kit for assaying a biological sampleo determine a SMAD4 gene deletion or mutation in the biological sample, comprising airst set of probes for detecting the expression level of the SMAD4 gene in the biological sample. In another aspect, the invention provides a kit for assaying a biological sample to determine a SMAD4 gene deletion or mutation in the biological sample, comprising a first set of probes for detecting the activity level of a SMAD4 gene encoded product in the biological sample. In some embodiments of each of the foregoing, the kit of the invention further comprises a second set of probes for detecting the expression level of a set of normalization genes in the tumor sample These kits of the invention may optionally further comprise at least one additional container which may contain, for example, a reagent (such as a buffered salt solution) for delivering the TGFβr1 inhibitor (e.g., PF-06952229) to a test sample such as an organ,issue or cell sample from a patient; at least one additional container containing, for example, a reagent (including but not limited to the nucleic acid reagents described above) suitable for determining the expression of SMAD4 in a biological sample; one or more detergents, lysis agents, or other solutions suitable for preparation of a biological sample for testing. In yet another aspect, the kit of the present invention may comprise a “packagensert” or “instructions for use,” wherein the package insert comprises instructions forreating a subject for cancer using the kit. “Instructions for use” typically includes information, such as dosage, administrationnstructions, e.g., a tangible expression describing the technique to be employed in usinghe kit to affect a desired outcome, such as to decrease or kill a tumor. Such dosage and administration instructions can be of the kind that are provided to a doctor, for example by a drug product label, or they can be of the kind that are provided by a doctor, such as nstructions to a patient. Optionally, the kit also contains other useful components, such as, diluents, buffers, syringes, IV bags and lines, needles catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily ecognized by those of skill in the art. In a preferred embodiment, the present invention provides a kit for assaying a biological sample to determine a SMAD4 gene deletion or mutation in the biological sample, comprising a first set of probes for detecting the expression level of the SMAD4 gene in the biological sample. In a preferred embodiment, the present invention provides a kit for assaying a biological sample to determine a SMAD4 gene deletion or mutation in the biological sample, comprising a first set of probes for detecting the activity level of a SMAD4 gene encoded product in the biological sample. In a preferred embodiment of each of the aspects described herein, the invention provides a second a second set of probes for detecting the expression level of a set of normalization genes in the tumor sample. In some embodiments of the each of the foregoing, the kits as described herein may be useful in treating cancer, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, brain cancer, gastric cancer, liver cancer, thyroid cancer, endometrial cancer, gallbladder cancer, kidney cancer, adrenocortical cancer, sarcoma, skin cancer, head and neck cancer, leukemia, bladder cancer, colorectal cancer, hematopoietic cancer and pancreatic cancer. In some embodiments, the prostate cancer is hormone dependent prostate cancer. In some embodiments, the prostate cancer is hormone dependent prostate cancer. Other suitable additional components of such kits of the invention will be familiaro those of ordinary skill in the art. These and other aspects of the invention, including the exemplary preferred embodiments listed below, will be apparent from the teachings contained herein. EXAMPLES Example 1: Whole Exome and Transcriptome Sequencing Tumor biopsy from cancer patients with metastatic castration resistant prostate cancer (mCRPC), squamous cell cancer of the head and neck, melanoma, mesothelioma, metastatic pancreatic cancer, colorectal cancer, renal cell carcinoma, and hepatocellular cancer were obtained from a phase 1 trial of PF-06952229 NCT03685591). Tumor tissues were excised and incubated in 10% neutral bufferedormalin between 12-24 hours before paraffin-embedding using standard embedding procedure. Whole exome (DNA) and whole transcriptome (RNA) next generation sequencing (NGS) of the formalin-fixed, paraffin-embedded (FFPE) tumor samples was conducted at Personalis Inc. (Menlo Park, CA, USA). Dual DNA and RNA extractionsrom ten 4 micron-thick tumor sections were performed using Qiagen AllPrep DNA/RNA kit (Qiagen, Germantown, MD, USA) per manufacturer’s protocol. Sequencing libraries were prepared using KAPA Stranded RNA-Seq Library Preparation Kit and KAPA HiFi DNA Polymerase (Roche, Wilmington, MA, USA) for RNA and DNA, respectively. Exomearget capture enrichment was performed using Agilent SureSelect Clinical Research Exome (SSCR) (Agilent Technologies Inc. Santa Clara, CA, USA) according to manufacturers’ recommendations. Additional supplementation with Personalis’ Accurate and Content Enhanced Augmented Exome (ACE) proprietary target probes synthesized by Agilent was performed to enhance coverage in difficult to sequence regions within sets of biomedically and medically relevant genes. Details regarding Personalis’ ACE assay design are described further in Patwardhan, A., Harris, J., Leng, N. et al. Achieving high- sensitivity for clinical applications using augmented exome sequencing. Genome Med 2015, 7, 71. Sequencing of 250 bp library was performed on NovaSeq 600 (Illumina, San Diego, CA, USA) sequencers with single lane, paired-end 2 x 150 bp read lengths andllumina’ s proprietary reversible terminator-based method. For DNA sequencing, DNA extracted from tumor tissues were sequenced to an average depth of coverage of 200X across the 69.4 Mb ACE assay genomic footprint. For RNA sequencing, RNA extractedrom tumor specimens were sequenced to an average output of 50 million paired end eads (total of 100 million reads). Tumor samples were further analyzed through Personalis somatic DNA pipeline for small variant calling (SNVs, InDels), and calculation of tumor mutational burden (TMB). TMB is defined as the total number of non- synonymous somatic mutations, and the number of non-synonymous somatic mutations per mega base (Mb). For RNA Sequencing, tumor samples were analyzed through Personalis’ somatic RNA pipeline for small variant calling (SNVs, insertion/deletions InDels), and fusions) and expression (gene and mutant variant level) data. Analysis of tumor DNA and RNA through whole exome and whole transcriptome sequencing from the patient described in Example 2 revealed a number of somatic variations. Among those found were mutations causing loss of function/reduced expression in genes involved in canonical TGFβ signaling pathway. These mutationsncluded two mutations in SMAD4, c.430C>T, c.1155delA (deletes Adenosine) (Table 2). Additional mutations in protein members of the TGFβ canonical signaling were alsodentified in SMAD2, SMAD3, SMAD6, and SMAD9. The expected impact of the SMAD4 mutation was considered to be the highest causing a frameshift preventing protein production and loss of function. This loss of function mutation in SMAD4 is expected tonterrupt canonical TGFβ signaling, causing tumor promotion and potentially making a patient responsive to an inhibitor of TGFβr1. Table 2

The letter ‘c’ refers to cDNA. A minus sign means the mutation occurs upstream of the translational start site. For example, “c.-430C>T” refers a C to T mutation at 430 nucleotides upstream to the translational start site of SMAD4. Similarly, “c.484C>T” referso a C to T mutation at nucleotide position 484 of the SMAD6 gene. Example 2: Response of to a Treatment with PF-06952229 In the phase 1 trial of PF-06952229, a patient with prostate cancer was treated with a dose of 375 mg of PF-06952229 twice a day as a monotherapy. The patient had been pretreated with 8 lines of prior therapy as listed in Table 3. The patient was progressed on or after all prior treatments including standard and experimental therapies in both the adjuvant setting and advanced metastatic castrate esistant setting. Accordingly, the patient was determined to be resistant to availableherapy by the treating physician. Table 3

Upon entering the Phase 1 study of PF-06952229 (NCT03685591), the patient had an elevated prostate specific antigen (PSA, a soluble marker of prostate cancer) level of 125 ng/ml blood. After approximately 8 weeks of treatment with PF-06952229, the patient’s PSA level dropped from 125 ng/ml in the blood to 27.3 ng/ml. The patient’s PSA continued to drop and entered the normal range at 3.3 ng/ml after 12 weeks of therapy Table 4 and Figure 1). Sixteen weeks after initiating the therapy, the patient had a CT scan to assess tumor burden. The results confirmed that the patient had a partial esponse to therapy with a reduction in a target lesion as shown in Table 5 (target and non-target Tumor Lesions CT Scan results (Figure 2)), and stable non-target lesions Table 6). The patient has maintained a partial response with a complete reduction of arget lesion (Tables 4 and 5). The patient’s PSA has remained in the normal range. The patient remains on treatment and continues to benefit from therapy with PF-06952229. Table 4

Table 5

Table 6

Monotherapy treatment with PF-06952229 caused a reduction in tumor burden. Accordingly, the patient with metastatic castrate resistant prostate cancer who was esistant prior therapies was shown to benefit from administration of PF-06952229. The sustained clinical benefit observed with PF-06952229 is attributed to the inhibition of TGFβr1. The sensitivity of the patient to inhibition of TGFβr1 by PF-06952229 implies hat the patient’s tumor was benefiting from TGFβr1 signaling due to the loss of function mutation in SMAD4 preventing full canonical signaling.

Claims

Claims 1. A method of treating cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a TGFβr1 inhibitor, whereinhe TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4-ylamino)-N- 1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof, and wherein the cancer comprises a loss or reduction of canonical TGFβ signaling.

2. The method of claim 1, wherein the loss or reduction of canonical TGFβ signaling is caused by a mutation, reduced expression or deletion of a SMAD4 gene.

3. The method of claim 2, wherein the loss or reduction of canonical TGFβ signaling is caused by a mutation of a SMAD4 gene.

4. The method of claim 3, wherein the mutation of the SMAD4 gene resultsn a frame-shift mutation.

5. The method of claim 3, wherein the mutation of the SMAD4 gene is c.- 430C>T or c.1155delA, or combinations thereof.

6. The method of claim 3, wherein the mutation of the SMAD4 gene is a loss of function mutation.

7. The method of claim 6, wherein the loss of function mutation of the SMAD4 gene is G386fs, -301fs, A199Sfs, A309Sfs*25, A532Pfs*5 or C443, or combinations thereof.

8. The method of claim 7, wherein the loss of function mutation of the SMAD4 gene is G386fs.

9. The method of any one of claims 1 to 8, wherein the method further comprises administering to the subject an amount of one or more anti-cancer agents; wherein the amounts of the TGFβr1 inhibitor and the one or more anti-cancer agentsogether are effective in treating cancer.

10. A method of treating cancer in a subject comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; c. selecting the subject for treatment with a TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product, is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population; and d. administering to the selected subject an effective amount of the TGFβr1 inhibitor, thereby treating cancer in the subject, wherein the TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4- ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof.

11. A method of selecting a subject having cancer for treatment with a TGFβr1 inhibitor, comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; and c. selecting the subject for treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population, wherein the TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin-4- ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof.

12. A method of predicting whether a subject having cancer will respond toreatment with a TGFβr1 inhibitor, comprising: a. providing a biological sample from the subject; b. determining the expression level of a SMAD4 gene or the activity level of a SMAD4 gene encoded product in the biological sample; c. selecting the subject for treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population; and d. predicting the subject will respond to treatment with the TGFβr1 inhibitor when: (i) the expression level of the SMAD4 gene is downregulated relative to the expression levels in a non-tumor biological sample from the subject or relative to a median expression level in a control population; and/or (ii) the activity level of the SMAD4 gene encoded product is less relative to the activity level of the SMAD4 gene encoded product in a non-tumor biological sample from the subject or relative to a median activity level in a control population, wherein the TGFβr1 inhibitor is 4-(2-(5-chloro-2-fluorophenyl)-5-isopropylpyridin- 4-ylamino)-N-(1,3-dihydroxypropan-2-yl)nicotinamide (PF-06952229) having the structure:

or a pharmaceutically acceptable salt thereof.

13. The method of any one of claims 1 to 12, wherein the cancer is selectedrom the group consisting of prostate cancer, breast cancer, ovarian cancer, lung cancer, colon cancer, brain cancer, gastric cancer, liver cancer, thyroid cancer, endometrial cancer, gallbladder cancer, kidney cancer, adrenocortical cancer, sarcoma, skin cancer, head and neck cancer, leukemia, bladder cancer, colorectal cancer, hematopoietic cancer and pancreatic cancer.

14. The method of claim 13, wherein the cancer is a resistant or relapsed cancer.

15. The method of claim 13 or 14, wherein the cancer is prostate cancer.

16. The method of claim 15, wherein the prostate cancer is castration resistant prostate cancer.

17. The method of claim 16, wherein the castration resistant prostate cancers metastatic castration resistant prostate cancer.

18. The method of any one of claims 1 to 17, wherein the subject is a human.

19. A kit for assaying a biological sample to determine a SMAD4 gene deletion or mutation in the biological sample, comprising a first set of probes for detecting the expression level of the SMAD4 gene in the biological sample.

20. A kit for assaying a biological sample to determine a SMAD4 gene deletion or mutation in the biological sample, comprising a first set of probes for detecting the activity level of a SMAD4 gene encoded product in the biological sample.