Co-targeting NCOA3 and PD-1 for cancer therapy

Abstract

The disclosure provides methods for predicting and / or determining the aggressiveness and drug responsiveness of a subject's cancer based on the level of NCOA3 expression or copy number. The disclosure also provides methods sensitizing a cancer to anti-PD-1 and / or anti-PDL1 therapy comprising inhibiting the expression or activity of NCOA3 or its binding partners.

Description

Attorney docket No.00010-020WO1 CO-TARGETING NCOA3 AND PD-1 FOR CANCER THERAPY CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 from Provisional Application Serial No. 63 / 537,041, filed September 7, 2023, the disclosures of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The disclosure provides methods for treating a subject with cancer by targeting NCOA3 (or one of its binding partner) or NCOA3 (or one of its binding partners) and PD-1. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] Accompanying this filing is a Sequence Listing entitled, “00010-020WO1.xml” created on September 5, 2024 and having 6418 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated by reference in its entirety for all purposes. BACKGROUND

[0004] Cancers, such as Melanoma, are life threatening often with a poor prognosis. One reason for poor prognosis is that cancer cells are able to hide from the immune system. Immunotherapy with immune checkpoint blockade (ICB) agents has transformed cancer therapy, including melanoma, especially using monoclonal antibodies targeting programmed death 1 (PD1) or programmed death ligand 1 (PDL1) (aka CD274). However, acquired or intrinsic resistance to immunotherapy is present in a significant proportion of tumors and is a major clinical problem.

[0005] Tumors that express higher levels of PDL1 have higher response rates to PD1 blockade. Strategies that (re-)sensitize to PD1 blockade would be of significant clinical benefit. In addition, a proportion of tumors simply do not respond to ICB, referred to as immunologically “cold” tumors, either with a minimal pre-existing immune infiltrate, with absent PDL1 expression, or with suppressed IFN gamma signaling. Strategies that convert immunologically cold tumors into hot ones would be of significant clinical value in order to sensitize to PD1 blockade. SUMMARYAttorney docket No.00010-020WO1

[0006] The disclosure provides a method of determining whether a cancer will be responsive to an anti-PDL1 or anti-PD-1 therapy, the method comprising detecting in tumor cells obtained from a subject the expression or activity of NCOA3 above a normal control level. In one embodiment, if the level of NCOA3 expression or activity is increased relative to a normal control, then the subject is non-responsive to anti-PDL1 or anti-PD-1 therapy. In still another or further embodiment, the NCOA3 levels are determined on a microarray or by using fluorescence in situ hybridization. In still another embodiment, the method further comprises administering an inhibitor of NCOA3 expression or activity to the subject. In still a further embodiment, the inhibitor of NCOA3 is an inhibitory nucleic acid. In still a further embodiment, the inhibitory nucleic acid is an siRNA or shRNA. In another embodiment, the inhibitor of NCOA3 is a small molecule inhibitor. In a further embodiment, the small molecule inhibitor is bufalin. In yet another embodiment, an inhibitor of a binding partner of NCOA3, wherein such binding partner can be an estrogen receptor or retinoic acid receptor, would be used alone or with anti-PD1 or anti-PD-L1 therapy.

[0007] The disclosure also provides a method comprising, measuring the level of NCOA3 in a subject having or suspected of having cancer, administering an NCOA3 expression or activity inhibitor the subject if the level of NCOA3 is higher than a normal control, and then administering an anti-PD-1 and / or anti-PDL1 inhibitor to the subject.

[0008] The disclosure provides a method of determining whether a cancer will be responsive to an anti-PDL1 or anti-PD-1 therapy, the method comprising detecting in tumor cells obtained from a subject in need of cancer therapy, the expression or activity of a NCOA3 above a normal control level, and administering an agent that inhibits NCOA3 expression, activity or a binding partner to a subject with NCOA3 above a normal control level. In one embodiment, if the level of NCOA3 copy number, expression or activity is increased relative to a normal control, then the subject is non-responsive to anti-PDL1 or anti-PD-1 therapy. In another or further embodiment, the NCOA3 level is determined on aAttorney docket No.00010-020WO1 microarray or by fluorescence in situ hybridization. In another embodiment, the method further comprises administering an inhibitor of NCOA3 expression or activity to the subject. In a further embodiment, the inhibitor of NCOA3 is an inhibitory nucleic acid. In still a further embodiment, the inhibitory nucleic acid is an siRNA or shRNA. In yet another embodiment, the inhibitor of NCOA3 is a small molecule inhibitor. In a further embodiment, the small molecule inhibitor is bufalin.

[0009] The disclosure also provides a method comprising, measuring the level of NCOA3 in a subject having or suspected of having cancer, administering an NCOA3 expression or activity inhibitor to the subject if the level of NCOA3 is higher than a normal control, and then administering an anti-PD-1 and / or anti- PDL1 inhibitor to the subject.

[0010] The disclosure also provides a method of treating a cancer, the method comprising administering an agent that inhibits the expression or activity of NCOA3 and contacting the subject with a PD1- and / or PDL-1 antagonist. In one embodiment, the agent is an siRNA. In another embodiment, the agent is a small molecule agent. In a further embodiment, the small molecule agent is bufalin. In another embodiment, the cancer is melanoma. In further embodiment, the agent is administered topically. In another embodiment, the PD1- and / or PDL-1 antagonist is an antibody. In another embodiment, the agent inhibits ESR1. In a further embodiment, the agent is selected from (R)-6-{2-{ethyl[4-(2- ethylaminoethyl)benzyl]amino}-4-methoxyphenyl}-5,6,7,8- tetrahydronaphthalen-2-ol, fulvestrant, toremifene, and AZD9496. In another embodiment, the agent inhibits RARA. In a further embodiment, the agent is selected from the group consisting of AGN 194301, AGN 194574, ER50891, LEI 35, Ro 41- 5253 and BMS 195614.

[0011] The disclosure provides for one or more embodiments set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS

[0012] Figure 1 shows NCOA3 silencing in human melanoma cells upregulates PDL1 expression.Attorney docket No.00010-020WO1

[0013] Figures 2 shows bufalin treatment upregulates PDL1 expression in multiple cell lines.

[0014] Figure 3 shows Western Blots of PDL1 in the various cell lines when treated with bufalin.

[0015] Figure 4 shows NCOA3 gene silencing in B16F10 melanoma cells upregulates expression of PDL1 and IFN gamma pathway genes.

[0016] Figure 5 shows in vivo effects of anti-tumor activity of combined NCOA3 and PD1 blockade in B16F10 melanoma cells.

[0017] Figure 6 shows that over expression of Estrogen receptor 1 (ESR1) cDNA downregulates PDL1 RNA expression in melanoma cells.

[0018] Figure 7 shows that overexpression of ESR1 or RARA cDNA downregulates PD-L1 protein expression in melanoma cells.

[0019] Figure 8 shows overexpression of RARA cDNA downregulates PD-L1 RNA expression in melanoma cells.

[0020] Figure 9A-B: FISH analysis of NCOA3 copy number in a matched specimen from the same melanoma patient before (A) and after (B) treatment with PD-1 blockade.

[0021] Figure 10A-B shows (A) Representative microscopy of C8161.9 nuclei constructed from Z-stacks of immunofluorescent detection of NCOA3 and ESR1, with foci reflecting areas of co- localization. (B) Western blot analysis of co-immunoprecipitation using anti-NCOA3 antibody to detect the binding of ESR1 and an anti-ESR1 antibody to detect the binding of NCOA3 in C8161.9 melanoma cells, with isotype and lysate representing negative and positive controls.

[0022] Figure 11A-B shows ChIP-quantitative PCR analyses showing binding of ESR1 to the estrogen receptor 1 (ER1) and estrogen receptor 2 (ER2) elements in the human PD-L1 / CD274 promoter in C8161.9 (A) and A375 (B) melanoma cells. ChIP primers for the GAPDH promoter were used as a positive control.

[0023] Figure 12A-C shows quantitative immunofluorescence analysis examining expression of PD-L1 / CD274 expression following transfection of siRNA targeting human NCOA3 RNA into A375 (A), D04 (B), and C8161.9 (C) human melanoma cells.

[0024] Figure 13A-B shows luciferase activity (in relative light units, or RLU) following co-transfection of (i) a plasmid encoding the luciferase gene driven by the human PD-L1 / CD274Attorney docket No.00010-020WO1 promoter and either ESR1 cDNA or RARA cDNA in C8161.9 (A) or A375 (B) human melanoma cells.

[0025] Figure 14A-D shows luciferase activity (in relative light units, or RLU) following transfection of a plasmid encoding the luciferase gene driven by the human PD-L1 / CD274 promoter and treatment with either fulvestrant or AGN194310 in A375 (A and B, respectively) or C8161.9 (C and D, respectively) human melanoma cells.

[0026] Figure 15 shows tumor volume of C57BL / 6 mice bearing YUMM1.7 murine melanoma tumors treated with (i) isotype antibody; (ii) anti-PD-1 antibody; (iii) fulvestrant; and (iv) combination of fulvestrant plus anti-PD-1 antibody. DETAILED DESCRIPTION

[0027] As used herein and in the appended claims, the singular forms "a,” "an,” and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a probe" includes a plurality of such cells and reference to "the cell" includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are now described.

[0029] All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which are described in the publications, which might be used in connection with the description herein. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that hasAttorney docket No.00010-020WO1 been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.

[0030] The terms "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of ±10% or less, +1-5% or less, ±1% or less, and ±0.1% or less of and from the specified value. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically contemplated.

[0031] “Administering” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. For example, the administration of an anti-PD-1 antibody includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Non-parenteral routes include oral, topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or for one or more extended periods.

[0032] A "biological sample" includes skin and dermal sample, sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include skin samples, samples of mucosal surfaces, blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, lymph and tongue tissue, cultured cells, e.g.,Attorney docket No.00010-020WO1 primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most commonly a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; or a rodent, e.g., guinea pig, rat, mouse, or rabbit.

[0033] A "biopsy" refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the disclosure. The biopsy technique applied will depend on the tissue type to be evaluated (e.g., skin, mucosal surface, etc.), the size and type of the tumor (e.g., solid or suspended, blood or ascites), among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy. An "excisional biopsy" refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An "incisional biopsy" refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor. A diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core-needle biopsy" of the tumor mass, or a "fine-needle aspiration biopsy" which generally contains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al., eds., 16th ed., 2005, Chapter 70, and throughout Part V.

[0034] Reference throughout this specification to "one embodiment", "an embodiment," "an exemplary embodiment," means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, appearances of the phrases "in one embodiment," "in another embodiment," or "an exemplary embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments.

[0035] As used herein, an "effective amount" is an amount sufficient to inhibit growth, proliferation, migration orAttorney docket No.00010-020WO1 invasiveness of a cancer cell in a subject suffering from a cancer. One skilled in the art can readily determine an effective amount of an NCOA3 inhibitor (or that of a binding partner), an anti-PD-1 and / or an anti-PDL1 agent to be administered to a given subject, by taking into account factors, such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.

[0036] “Nuclear Receptor Coactivator 3” or “NCOA3” refers to a member of the steroid receptor coactivator protein family in humans. Accession numbers for representative nucleic acids encoding NCOA3 include: NM_008679, BC092516, and BC088343, among others. Accession numbers for representative NCOA3 proteins include: CAI42141, CAC17693, CAB40662, AAH88343, and NP_032705, among others. The contents of the foregoing accession numbers are incorporated herein by reference. NCOA3 mediates many of its biological effects by virtue of interacting with a family of nuclear hormone receptors, including, but not limited to, estrogen receptor or retinoic acid receptor, to regulate expression of numerous target genes.

[0037] The terms "overexpress," "overexpression" or "overexpressed" interchangeably refer to a protein or nucleic acid that is transcribed or translated or present at an increased copy number at a detectably greater level, usually in a cancer cell, in comparison to a normal cell, a nevus, or a primary cancer. The term includes overexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a normal cell. Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e., ELISA, immunohistochemical techniques). Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell. In certain instances, overexpression is 1-fold, 2-fold, 3-fold, 4-fold or more higher levels of transcription or translation in comparison to a normal cell.Attorney docket No.00010-020WO1 Overexpression can also include any expression in a sample cell when compared to the absence of expression in a normal cell.

[0038] “Programmed Death-1” or “PD-1” refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. U64863 (or UniProt Accession No. Q15116), the sequence and data associated with the accession number is incorporated herein by reference.

[0039] “Programmed Death Ligand-1” (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulate T cell activation and cytokine secretion upon binding to PD-1. The term “PD-L1” as used herein includes human PD-L1 (hPD- L1), variants, isoforms, and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under UniProt Accession No. Q9NZQ7, the sequence and data associated with the accession number is incorporated herein by reference.

[0040] The terms "treat", "treating" and "treatment", as used herein, refer to ameliorating symptoms associated with a disease or condition, for example, a melanoma, including preventing or delaying the onset of the disease symptoms, and / or lessening the severity or frequency of symptoms of the disease or condition. The terms "subject" and "individual" are defined herein to include animals, such as mammals, including but not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent, or murine species. In a preferred embodiment, the mammal is a human.

[0041] Melanoma is a form of cancer that begins in melanocytes, the cells that produce melanin pigment. While frequently occurring on the skin, melanoma can also occur in the eye and rarely in the membranes of the nasal passages, oral, pharyngeal mucosa, vaginal and anal mucosa. The American Joint Committee on Cancer (AJCC) has devised a system for classifying melanomas into 4 stages (with manyAttorney docket No.00010-020WO1 substages) based on pathological criteria and survival rates. Melanoma may arise from moles or benign nevi on the skin and progress through the stages defined by the AJCC staging system (see, e.g., Harrison's Principles of Internal Medicine, Kasper et al., 16th ed., 2005, for additional background). Melanoma cells are highly resistant to chemotherapy, resulting in low response rates to the administering of chemotherapeutic reagents (La Porta CA., Curr Cancer Drug Targets 9:391-7 (2009)). In addition, melanoma cells are common and represent a major cause of skin disease and surgery.

[0042] NCOA3 is activated in melanoma by virtue of copy number elevation. The disclosure demonstrates that NCOA3 silencing resulted in significant suppression of melanoma cell growth and metastasis in vivo. Bufalin, identified in toad venom extract, has been identified as a small molecule inhibitor of NCOA3. The disclosure shows that bufalin treatment results in NCOA3 suppression, with significant anti-tumor activity in vivo, identifying NCOA3 as a rational therapeutic for melanoma therapy. Moreover, several NCOA3 binding partners can be targeted by small molecule inhibitors.

[0043] Programmed death-ligand 1 (PD-L1) and programmed cell death-1 (PD-1) are transmembrane proteins that play a role in cancer immune escape. PD-L1 expressed on cancer cells engages with programmed cell death-1 (PD-1) on immune cells, contributing to cancer immune escape. For multiple cancer types, the PD-1 / PD-L1 axis is the anti-cancer immune response. In this context, blocking PD-1 / PD-L1 could restore T cells from exhausted status and eradicate cancer cells. However, only a subset of PD-L1 positive patients benefits from α-PD-1 / PD-L1 therapies; in part due to expression of the PD-L1 on cancer cells. PD-L1 expression is regulated by various factors, leading to the diverse significances of PD-L1 positivity. Knockdown of NCOA3 levels is shown herein to increase the expression of PD-L1 in, e.g., melanoma cells.

[0044] The disclosure provides a method of identifying cancer cells that have a resistance to PD-L1 therapy, the method comprising measuring the expression level or copy number of NCOA3 to determine if NCOA3 levels are greater than a normal wild-typeAttorney docket No.00010-020WO1 level (e.g., a benign nevi), wherein an elevated level is indicative of a cancer cell that is unlikely or will not respond to anti-PD-L1 therapy. In some instances, NCOA3 can be considered an immune check point inhibitor resistant gene as elevated levels of NCOA3 decrease the ability that anti-PD-L1 therapy or an immune checkpoint inhibitor will be effective.

[0045] The disclosure demonstrates that NCOA3 overexpression results in the reduced expression of PD-L1 and moreover, that inhibition of NCOA3 by use of small molecule inhibitors or gene silencing upregulates expression of PD-L1. For example, NCOA3 gene silencing upregulates PDL1 expression in human melanoma cells (FIG. 1), suggesting that NCOA3 targeting can sensitize cancer cells to PD1-based therapy. Moreover, the disclosure demonstrates that bufalin targeting of human melanoma cell lines upregulates PDL1 expression (FIG. 2 and FIG. 3).

[0046] The data shows NCOA3 gene silencing upregulates PDL1 expression and promotes a “warm” microenvironment, with upregulation of several IFN gamma pathway genes (FIG. 4). In vivo studies of B16F10 melanoma cells showed the anti-tumor activity of combined NCOA3 gene silencing with PD1 antibody (FIG. 5).

[0047] Estrogen receptor alpha (ERα), also known as NR3A1 (nuclear receptor subfamily 3, group A, member 1), is one of two main types of estrogen receptor, a nuclear receptor (mainly found as a chromatin-binding protein) that is activated by the sex hormone estrogen. In humans, ERα is encoded by the gene ESR1 (Estrogen Receptor 1). ERα is the main estrogen receptor in the human epidermis.

[0048] The data show that two NCOA3 binding partners, ESR1 and RARA (retinoic acid receptor alpha), each negatively regulate PDL1 expression in melanoma cells (FIGs. 6-8). Taken together, these studies indicate the utility of co-targeting PD1 / PDL1 and NCOA3, its binding partners (e.g., ESR1 and / or RARA) and combinations thereof as a rational approach for melanoma therapy.

[0049] In one embodiment, subjects with melanoma can be treated by reducing the expression of or inhibiting the activity of NCOA3. In a further embodiment, the subjects can be further treated with an anti-PD-1 or anti-PD-L1 therapy. In a further embodiment,Attorney docket No.00010-020WO1 subjects with cancer, such as melanoma, can be treated by reducing the expression of NCOA3 in conjugation with administering one or more additional therapeutic agents (e.g., agents that target PD-1 or PD-L1). Examples of therapeutic agents include, but are not limited to, anticancer agents, de-methylating agents, alkylating agents, anti-metabolite agents, mitotic inhibitors, tyrosine kinase inhibitors, topoisomerase inhibitors, cancer immunotherapy monoclonal antibodies (e.g., anti-PDL1 antibodies), anti-tumor antibiotic agents, and targeted anti-cancer agents. In a particular embodiment, subjects with cancer, such as melanoma, can be treated by reducing the expression of NCOA3 in combination with the administration of an anti-PD-1 or anti-PDL1 antibody. In one embodiment, the method comprises reducing expression of NCOA3 with an siRNA (e.g., shRNA or another inhibitory nucleic acid). In still another embodiment, the subject is treated with a molecule that inhibits NCOA3 activity such as bufalin.

[0050] In still another embodiment, the subject can be treated with an agent that inhibits binding of ESR1 and / or RARA with NCOA3 to inhibit NCOA3 activity. The subject can then be treated with an anti-PD1 and / or anti-PDL1 therapy. In still another embodiment, the subject can be treated with an NCOA3 activity or expression inhibitor, an agent that inhibits ESR1 and / or RAR or inhibits the binding of ESR1 and / or RARA with NCOA3, and an anti-PD1 and / or anti-PDL1 inhibitor. In another embodiment, a method of treating a cancer using check-point inhibitors (e.g., anti-PD1 or anti-PDL1 therapeutics) comprising inhibiting an NCOA3 binding partner (e.g., ESR1 and / or RARA), such that PD-L1 expression is elevated, thereby generating a “warm” environment for treating with and anti-PD1 or anti-PDL1 therapeutic.

[0051] In another embodiment, the disclosure provides a method of treating a cancer, the method comprising administering an effective amount of an agent that inhibits NCOA3 activity followed by, or currently with, administration of an anti-PD-1 and / or anti- PDL1 antibody.

[0052] Although melanoma is exemplified herein, cancers that may be treated by compositions comprising nucleic acid inhibitors or small molecule inhibitors of NCOA3, ESR1 and / or RARA, with orAttorney docket No.00010-020WO1 without PD-1 or PD-L1 antagonists (e.g., anti-PD-1 or anti-PD-L1 antibodies) include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may be comprised of non-solid tumors (such as leukemias and lymphomas) or may be solid tumors. Types of cancers treated with the agent or composition of the disclosure include carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers alike may be treated in accordance with the methods disclosed herein.

[0053] Examples of tumors / cancers which may be treated include, ovarian, breast (including HER2 and metastatic), colorectal, colon, renal, rectal, pancreatic, prostate, stomach, gastrointestinal, gastric, stomach, esophageal, bile duct, lung (including small cell and non-small cell lung tumors; adenocarcinoma of the lung and squamous carcinoma of the lung), liver, epidermoid tumors, squamous tumors such as head and neck tumors, epithelial squamous cell cancer, thyroid, cervical, neuroendocrine tumors of the digestive system, neuroendocrine tumors, cancer of the peritoneum, hepatocellular cancer, hepatoblastoma, HPCR, glioblastoma, bladder cancer, hepatoma, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, bone cancer, soft tissue sarcoma (including embryonal and alveolar rhabdomyosarcoma, GIST, alveolar soft part sarcoma and clear cell sarcoma), cholangiocarcinoma, bile cancer, gallbladder carcinoma, myeloma, vulval cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, retinal, hematopoietic cancer, androgen-dependent tumors, androgen-independent tumors, Other examples include Kaposi's sarcoma, synovial sarcoma, vasoactive intestinal peptide secreting tumor, CNS neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas, and cerebral metastases, melanoma, rhabdomyosarcoma, glioblastoma, including glioblastoma multiforme, EMB, RMS, ALV, medulloblastoma, ependymoma, Wilm's cancer, Ewing's cancer, osteosarcoma, PNT, rhabdoid, rhabdomyosarcoma, retinoblastoma, adrenal cortical cancer, adrenalAttorney docket No.00010-020WO1 cancer, and leiomyosarcoma. In a certain embodiment, the cancer to be treated is melanoma.

[0054] In a particular embodiment, the disclosure provides a method of treating cancer, such as melanoma, in a subject, the method comprising administering an effective amount of an agent that reduces the expression and / or activity of NCOA3 or a binding partner of NCOA3 (e.g., ESR1 and / or RARA). In a further embodiment, the subject is then treated with a PD-1 and / or PD-L1 antagonist (e.g., an anti-PD-1 and / or anti-PD-L1 antibody or fragment thereof). In another embodiment, the agent is a shRNA transcribed downstream of a polymerase II or III promoter. In one embodiment, the shRNA has a sequence of 5’-GAAUAAUGCACUUCUUAGA-3’ (SEQ ID NO:1). In another embodiment, the agent is an oligonucleotide that is an siRNA, an antisense oligonucleotide or the like. In another embodiment, the agent is a small molecule or chemical compound such as bufalin, fulvestrant and / or AGN194310.

[0055] Transducing viral (e.g., retroviral, adenoviral, lentiviral and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al., Current Eye Research 15:833-844 (1996); Bloomer et al., Journal of Virology 71:6641-6649 (1997); Naldini et al., Science 272:263-267 (1996); and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319 (1997)). For example, a polynucleotide encoding an shRNA can be cloned into a retroviral vector and its expression can be driven from a suitable promoter or from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, (1990); Friedman, Science 244:1275-1281 (1989); Eglitis et al., BioTechniques 6:608- 614 (1988); Tolstoshev et al., Current Opinion in Biotechnology 1:55-61 (1990); Sharp, The Lancet 337:1277-1278 (1991); Cornetta et al., Nucleic Acid Research and Molecular Biology 36(31):1-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood CellsAttorney docket No.00010-020WO1 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995)). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson et al., U.S. Pat. No.5,399,346). Non-viral approaches can also be employed for the introduction of an inhibitory NCOA3 oligonucleotide (e.g., an shRNA) to inhibit expression of NCOA3. For example, a oligonucleotide or polynucleotide comprising an inhibitory nucleic acid that inhibits NCOA3 can be introduced into a cell by administering the nucleic acid in the presence of cationic lipid (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413 (1987); Ono et al., Neuroscience Letters 17:259 (1990); Brigham et al., Am. J. Med. Sci. 298:278 (1989); and Staubinger et al., Methods in Enzymology 101:512 (1983)); asialoorosoinucoid-polylysine conjugation (Wu et al. Journal of Biological Chemistry 263:14621 (1988); Wu et al., Journal of Biological Chemistry 264:16985 (1989); or by micro-injection under surgical conditions (Wolff et al., Science 247:1465 (1990).

[0056] Polynucleotides comprising NCOA3 inhibitors and / or agents that decrease or inhibit the expression or activity of NCOA3 may be administered as part of a pharmaceutical composition. The pharmaceutical composition is preferably sterile and contains a therapeutically effective amount of a polynucleotide / oligonucleotide molecule and / or an agent that inhibits the expression or activity of NCOA3 in a unit of weight or volume suitable for administration to a subject.

[0057] The therapeutic polynucleotide molecule comprising NCOA3 inhibitors and / or agents that decrease or inhibit the expression or activity of NCOA3 or its binding partners may be administered with a pharmaceutically-acceptable carrier, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a cancer.

[0058] “Carrier” as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages andAttorney docket No.00010-020WO1 concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and / or nonionic surfactants such as TWEEN , polyethylene glycol (PEG), and PLURONICS .

[0059] Polynucleotides comprising NCOA3 inhibitors and / or agents that decrease or inhibit the expression or activity of NCOA3 may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers comprising NCOA3 inhibitors and / or agents that decrease or inhibit the expression or activity of NCOA3, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ɣ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolicAttorney docket No.00010-020WO1 acid enable release of molecules for over 100 days, certain hydrogels release of molecules for shorter time periods.

[0060] In another embodiment, the pharmaceutical compositions comprising NCOA3 inhibitors and / or agents that decrease or inhibit the expression or activity of NCOA3 or its binding partners are administered in conjunction with other therapeutic agents. “Conjunction”, with respect to administering other therapeutic agents, refers to agents that may be administered prior to, concurrently, or subsequent to pharmaceutical compositions comprising NCOA3 inhibitors and / or agents that decrease or inhibit the expression or activity of NCOA3 or its binding partners. In one embodiment, the other therapeutic agent is a PD-L1 and / or PD-1 antagonist (e.g., an anti-PD-L1 and / or anti-PD-1 antibody).

[0061] In a particular embodiment, the pharmaceutical compositions comprising NCOA3 inhibitors and / or agents that decrease or inhibit the expression or activity of NCOA3 are administered in conjunction with radiotherapy, chemotherapy, photodynamic therapy, surgery or other immunotherapy, to a patient who has a hyperproliferative disorder, such as cancer or a tumor. In one example, a pharmaceutical composition of the disclosure is administered to a subject in conjunction with an anti-PD-L1 antibody, an anti-PD-1 antibody, or both an anti-PD-L1 antibody and an anti-PD-1 antibody. The pharmaceutical compositions of the disclosure may be administered in conjugation with one or more other prophylactic or therapeutic agents, including but not limited to cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti- angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, agents that promote proliferation of hematological cells, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, anti-cancer antibodies, or other therapeutic agents.

[0062] Examples of chemotherapeutic agents include, but are not limited to, platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; proteins such as arginine deiminase and asparaginase; alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN ); alkyl sulfonates such as busulfan,Attorney docket No.00010-020WO1 improsulfan and piposulfan; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; folic acid replenisher such as frolinic acid; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes, e.g. paclitaxel (TAXOL , Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE , Rhne- Poulenc Rorer, Antony, France); topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (such as Tomudex); additional chemotherapeutics including aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene;Attorney docket No.00010-020WO1 edatraxate; defofamine; demecolcine; diaziquone; difluoromethylornithine (DMFO); elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK ; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacyto sine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; retinoic acid; esperamicins; and capecitabine.

[0063] Administration of the pharmaceutical compositions disclosed herein may begin after the subject is determined to have a cancer or suspected of having a cancer. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

[0064] Methods well known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" Ed. A. R. Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or polyoxyethylene- polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systemsAttorney docket No.00010-020WO1 for inhibitory nucleic acid molecules include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

[0065] In addition to the therapeutic aspects of inhibiting NCOA3 expression and / or activity, NCOA3 can also be used as a diagnostic and prognostic marker to predict resistance to anti-PD1- and / or PDL1-based therapy. In a certain embodiment, the expression level of NCOA3 is determined from one or more biological samples from a subject. The biological sample can be a tissue, blood, or other biological sample known to one of skill in the art. In one example, a tissue sample, such as a tissue biopsy, can be removed from a subject in accordance with a method known to one of skill in the art. In another example, a blood sample can be removed from a subject, and individual components of the blood sample can be isolated for extraction of nucleic acids or proteins by standard techniques.

[0066] In a particular embodiment, one or more control samples are obtained from one or more benign nevi (when melanoma is suspected) or from a tissue or subject that does not have the cancer. Examples of nevi include, but are not limited to, compound nevus, spitz nevus, halo nevus, junctional nevus, pseudomelanoma, blue nevus, congenital melanocytic nevus, balloon cell nevus, dysplastic nevus / dysplastic nevus syndrome, acral nevus, Becker’s nevus, Benign melanocytic nevus, and nevus spilus. The copy number or expression level of NCOA3 in the control samples is measured, and in one embodiment, the mean expression level for NCOA3 serves as the control expression level for NCOA3. In a particular embodiment, the one or more control sample(s) and the test sample(s) are obtained from the same subject. In an alternate embodiment, the one or more control sample(s) are from multiple subjects, which may or may not include the test subject.

[0067] The expression level or copy number of NCOA3 can be quantitated and the level compared to control values. For example,Attorney docket No.00010-020WO1 the expression level of NCOA3 in a test sample can be measured and the level determined using various diagnostic methods (e.g., ELISA, PCR, and the like). Thus, it can be determined if the level of NCOA3 is greater than the expression level of the NCOA3 in a control sample. As used herein, the expression of NCOA3 is "over- expressed" when the amount of NCOA3 expression in a test sample from a subject is greater than the amount of the expression level of NCOA3 in a control sample by a statistically significant amount (e.g., 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or greater compared to control; or any value between any of the foregoing number percentages).

[0068] The level of NCOA3 in a sample can be measured by using any technique that is suitable for detecting DNA copy number, RNA expression levels or protein levels in a biological sample. Suitable techniques for determining RNA expression levels in cells from a biological sample are well known to those of skill in the art. Examples of such techniques include, but are not limited to, Northern blot analysis, RT-PCR, microarrays, in situ hybridization. In a particular embodiment, a high-throughput system, for example, a microarray, is used to measure the expression level of a plurality of genes.

[0069] In a certain embodiment, the level of NCOA3 is detected using Northern blot analysis. For example, total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labeled DNA or RNA probes complementary to the RNA in question.

[0070] Suitable probes for Northern blot hybridization of NCOA3 can be produced from the nucleic acid sequences of NCOA3. Methods for preparation of labeled DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook etAttorney docket No.00010-020WO1 al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11.

[0071] In one example, the nucleic acid probe can be labeled with, e.g., a radionucleotide, such as H, P, P, C, or S; a heavy metal; or a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g., biotin, avidin or an antibody), a fluorescent molecule, a chemiluminescent molecule, or an enzyme. Probes can be labeled to high specific activity by nick translation, random priming, or other methods known to one of skill in the art. For example, by replacing preexisting nucleotides with highly radioactive nucleotides according to the nick translation method, it is known to prepare P-labeled nucleic acid probes with a specific activity well in excess of 10 cpm / microgram.

[0072] Autoradiographic detection of hybridization can then be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides a measurement of NCOA3 transcript levels. In another embodiment, NCOA3 gene transcript levels can be quantified by computerized imaging systems, such the Molecular Dynamics 400-B 2D Phosphorimager available from Amersham Biosciences, Piscataway, N.J.

[0073] In another embodiment, the random-primer method can be used to incorporate an analogue, for example, the dTTP analogue 5- (N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate, into the probe molecule. The biotinylated probe oligonucleotide can be detected by reaction with biotin-binding proteins, such as avidin, streptavidin, and antibodies (e.g., anti- biotin antibodies) coupled to fluorescent dyes or enzymes that produce color reactions.

[0074] In a further embodiment, determining the levels of NCOA3 copy number or expression can be accomplished using the technique of in situ hybridization. This technique requires fewer cells than the Northern blotting technique, and involves depositing whole cells onto a microscope cover slip and probing the nucleic acid content of the cell with a solution containing radioactive or otherwise labeled nucleic acid (e.g., cDNA or RNA) probes. ThisAttorney docket No.00010-020WO1 technique is particularly well-suited for analyzing tissue biopsy samples from subjects. The practice of the in situ hybridization technique is described in more detail in U.S. Pat. No. 5,427,916, the disclosure of which is incorporated herein by reference.

[0075] The relative number of NCOA3 gene transcripts in cells can also be determined by reverse transcription of NCOA3 gene transcripts, followed by amplification of the reverse-transcribed transcripts by polymerase chain reaction (RT-PCR). The levels of NCOA3 gene transcripts can be quantified in comparison with an internal standard, for example, the level of a "housekeeping" gene present in the same sample. A suitable "housekeeping" gene for use as an internal standard includes, e.g., myosin or glyceraldehyde-3- phosphate dehydrogenase (G3PDH). The methods for quantitative RT- PCR and variations thereof are within the skill in the art. In another embodiment, a high throughput stem loop real-time quantitative polymerase chain reaction (RT-qPCR) is used to detect NCOA3 expression. See Mestdagh et al., Nucleic Acid Research 36(21) (2008)).

[0076] In some instances, it may be desirable to simultaneously determine the expression level of a plurality of different cancer markers in a sample. In another embodiment, an oligolibrary, in microchip format (i.e., a microarray), may be constructed containing a set of probe oligodeoxynucleotides that are specific for a set of cancer marker genes. Using such a microarray, the expression level of multiple cancer genes in a biological sample can be determined by reverse transcribing the RNAs to generate a set of target oligodeoxynucleotides, and hybridizing them to probe oligodeoxynucleotides on the microarray to generate a hybridization, or expression, profile. The hybridization profile of the test sample can then be compared to the pre-determined expression level of a control sample to determine which cancer gene markers have an altered expression level in cancer cells. As used herein, "probe oligonucleotide" or "probe oligodeoxynucleotide" refers to an oligonucleotide that is capable of hybridizing to a target oligonucleotide. "Target oligonucleotide" or "target oligodeoxynucleotide" refers to a molecule to be detected (e.g., via hybridization). By "NCOA3-specific probe oligonucleotide" orAttorney docket No.00010-020WO1 "probe oligonucleotide specific for an NCOA3" is meant a probe oligonucleotide that has a sequence selected to hybridize to a specific NCOA3 gene sequence, or to a reverse transcript of the specific NCOA3 gene sequence.

[0077] An "expression profile" or "hybridization profile" of a particular sample is essentially a fingerprint of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell. That is, normal tissue may be distinguished from a cancer tissue, and within a cancer tissue, different prognosis states (good or poor long term survival prospects, for example) may be determined. By comparing expression profiles of a cancer tissue in different states, information regarding which genes are important (including both up- and down- regulation of genes) in each of these states is obtained. The identification of sequences that are differentially expressed in a cancer tissue or normal tissue, as well as differential expression resulting in different prognostic outcomes, allows the use of this information in a number of ways. For example, a particular treatment regime may be evaluated (e.g., to determine whether a chemotherapeutic drug acts to improve the long-term prognosis in a particular patient). Similarly, diagnosis may be done or confirmed by comparing patient samples with the known expression profiles. Furthermore, these gene expression profiles (or individual genes) allow screening of drug candidates that suppress the cancer expression profile or convert a poor prognosis profile to a better prognosis profile.

[0078] The microarray can be prepared from gene-specific oligonucleotide probes generated from known normal and cancer gene sequences, including NCOA3. In one embodiment, the array contains two different oligonucleotide probes for each sequence to be identified. The array may also contain controls, such as one or more mouse sequences differing from human orthologs by only a few bases, which can serve as controls for hybridization stringency conditions. tRNAs from both species may also be printed on the microchip, providing an internal, relatively stable, positiveAttorney docket No.00010-020WO1 control for specific hybridization. One or more appropriate controls for non-specific hybridization may also be included on the microchip.

[0079] The microarray may be fabricated using techniques known in the art. For example, probe oligonucleotides of an appropriate length are 5'-amine modified at position C6 and printed using commercially available microarray systems, e.g., the GENEMACHINE, OMNIGRID 100 MICROARRAYER and AMERSHAM CODELINK activated slides. Labeled cDNA oligomer corresponding to the target RNAs is prepared by reverse transcribing the target RNA with labeled primer. Following first strand synthesis, the RNA / DNA hybrids are denatured to degrade the RNA templates. The labeled target cDNAs thus prepared are then hybridized to the microarray chip under hybridizing conditions, e.g., 6 x SSPE / 30% formamide at 25 °C for 18 hours, followed by washing in 0.75 x TNT at 37 °C for 40 minutes. At positions on the array where the immobilized probe DNA recognizes a complementary target cDNA in the sample, hybridization occurs. The labeled target cDNA marks the exact position on the array where binding occurs, allowing automatic detection and quantification. The output consists of a list of hybridization events, indicating the relative abundance of specific cDNA sequences, and therefore the relative abundance of the corresponding complementary cancer gene, in the patient sample. According to one embodiment, the labeled cDNA oligomer is a biotin- labeled cDNA, prepared from a biotin-labeled primer. The microarray is then processed by direct detection of the biotin-containing transcripts using, e.g., STREPTAVIDIN-ALEXA647 conjugate, and scanned utilizing conventional scanning methods. Image intensities of each spot on the array are proportional to the abundance of the corresponding cancer gene marker in the patient sample.

[0080] In addition to use for quantitative expression level assays of a specific cancer gene (e.g., NCOA3), a microchip containing cancer-specific probe oligonucleotides corresponding to a substantial portion of all cancer-associated genes, preferably the entire genome of cancer associated genes, may be employed to carry out gene expression profiling, for analysis of expressionAttorney docket No.00010-020WO1 patterns. Distinct cancer signatures can be associated with established disease markers, or directly with a disease state.

[0081] According to the expression profiling methods described herein, total RNA from a sample from a subject suspected of having a cancer (e.g., melanoma) is quantitatively reverse transcribed to provide a set of labeled target oligodeoxynucleotides complementary to the RNA in the sample. The target oligodeoxynucleotides are then hybridized to a microarray comprising cancer-specific probe oligonucleotides to provide a hybridization profile for the sample. The hybridization profile comprises the signal from the binding of the target oligodeoxynucleotides from the sample to the cancer- specific probe oligonucleotides in the microarray. The profile may be recorded as the presence or absence of binding (signal vs. zero signal). The profile recorded can include the intensity of the signal from each hybridization. The profile is compared to the hybridization profile generated from a control sample. An alteration in the signal is indicative of a chemotherapy response in the subject.

[0082] Other techniques for measuring gene expression are also within the skill in the art, and include various techniques for measuring rates of RNA transcription and degradation, including RNase Protection Assays, Nuclear run-ons, slot blotting, etc.

[0083] In another embodiment, the disclosure provides a method for prognosticating the presence of a cancer in a subject. The method comprises the step of determining whether or not NCOA3 levels are elevated or suppressed in a biological sample from the subject, relative to the levels of NCOA3 from one or more control samples.

[0084] In yet another embodiment, the disclosure provides a method of determining the progression of cancer in a subject. The method comprises the step of measuring the NCOA3 levels from biological samples taken from a patient having a cancer at various time points, such that the change in the level of NCOA3 between samples from the different time points indicates the progression or recovery from the cancer in the subject. In a particular embodiment, if the level of NCOA3 remains high or is increasing between earlier and later time points indicates that the subject’sAttorney docket No.00010-020WO1 cancer is progressing to later stages. In an alternate embodiment, if the level of NCOA3 is decreasing between earlier and later timepoints indicates that the subject’s cancer is in the process of remission. In a further embodiment, said cancer is melanoma.

[0085] The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used. EXAMPLES

[0086] Cell culture. C8161.9 human melanoma cells were grown in DMEM / Ham’s F12 containing 5% FBS and 1X pen / strep. B16F10, D04 LOX, and A375 melanoma cells were cultured in RPMI medium 1640 containing 5% FBS plus 1X pen / strep.

[0087] Transfections and generation of stable transformants. A shRNA NCOA3 target set (RHS4533-NM_006534) (Dharmacon) and lentiviruses including the anti-luciferase (luc) shRNA were prepared. Subconfluent C8161.9 and D04 cells were infected with each harvested lentivirus in the presence of 8µg / ml of polybrene, and selected in 1.5µg / ml, 1µg / ml or 0.5µg / ml of puromycin at 48h post-infection, respectively. The consequences of NCOA3 gene silencing were confirmed using a second siRNA targeting a distinct sequence in NCOA3 mRNA. siRNAs were transfected at 100nM with Lipofectamine 2000 and the assays performed at 48h post- transfection.

[0088] Mouse NCOA3 shRNA: V2LMM_24526 (ShRNA 1) Clone Id: V2LMM_24526 Mature Antisense: TTCATTTCAAGTGCCTGCC (SEQ ID NO:2) V1LMM_30108 (ShRNA 2) Clone Id:V2LMM_30108 Mature Antisense: TTGAACATCATCATCACTG (SEQ ID NO:3) V3LMM_462343 (Sh RNA 3) Clone Id: V3LMM_462343 Mature Antisense: GTATCTATATTGACAACCT (SEQ ID NO:4)

[0089] Validated NCOA3 siRNA was purchased from Lifetech (#4427038), and GFP siRNA duplex oligo was purchased from Integrated DNA Technologies:Attorney docket No.00010-020WO1

[0090] 5'-GCUACGUCCAGGAGCGCACCCUCAA-3' (SEQ ID NO:5);

[0091] 3'-CGAUGCAGGUCCUCGCGUGGGAGAA-5' (SEQ ID NO:6).

[0092] To overexpress pcDNA, human ESR1 cDNA or human RARA cDNA in A375 or C8161.9 cells, lipofectamine 2000-mediated transfections (9µL) of (3µg) plasmids expressing control pcDNA or human ESR1 cDNA, or human RARA cDNA, were carried out following manufacturer instructions (Thermo Fisher Scientific).

[0093] Quantitative real-time polymerase chain reaction (qRT- PCR) analysis. The TaqMan probes for human genes used were as follows (Thermo Fisher Scientific): NCOA3 (Hs00180722_m1), PD- L1 / CD274 (Hs00204257 m1), and HPRT1 (Hs03929098_m1). All reactions were run in triplicate and the expression level of each gene was normalized to the human HPRT1 gene before comparisons. Data was analyzed using the 2−ΔΔCT method.

[0094] Western analysis. Membranes were first incubated overnight at 4°C with the following antibodies: NCOA3 (#sc-9119 at 1:1000 dil.; Santa Cruz Biotechnology), human PD-L1 (Protein-Tech, Cat #28076-1-AP at 1:1000 dil.,; mouse anti-PD-L1 (BioRad at 1:1000 dilution), GAPDH (#MAB374 at 1:1000 dil.; EMD Millipore) and then with their respective horseradish peroxidase-labeled secondary antibodies for 1h at room temperature: goat anti-mouse HRP (#170– 6516 at 1:1000 dil.; Biorad) and bovine anti-rabbit HRP (#172–1019 at 1:1000 dil.; Biorad). Binding was detected by using luminol reagent (sc-2048; Santa Cruz Biotechnology). For the bufalin experiments, various melanoma cell lines were treated with DMSO or varying concentrations of bufalin for 24h before the RNA isolation or protein extraction.

[0095] Immunofluorescence and FISH. Expression of various proteins was assessed using immunofluorescence performed cultured on coverslips. Antibodies against NCOA3 (#sc-9119 at 1:1000 dil.; Santa Cruz Biotechnology), or PDL1 (BioRad, Cat#RM320 at 1:500 dil.) were utilized. DAPI staining was used as counterstain. Images were taken at fixed exposures with a Zeiss Axio Image Z2 microscope and the fluorescence intensities of at least 200 cells quantified using Axiovision software. The mean pixel intensities were used for statistical analysis using Microsoft Excel and Data Desk. For FISH analysis, BAC clones RP11-456N23 (20q13.12), RP5-1049G16Attorney docket No.00010-020WO1 (20q13.12), representing the NCOA3 locus, and RP4–610C12 (20q11.1) for the centromere of chromosome 20, were used (February 2009 freeze of the University of California, Santa Cruz Genome Browser, [http:][ / / ]genome.ucsc.edu; brackets are used to avoid hyperlinks). All clones were obtained from the Children’s Hospital of Oakland Research Institute (CHORI). BAC DNAs were prepared and labeled. The quality and mapping of all probes were verified by hybridization to normal metaphase spreads in combination with a commercially available centromeric probe for chromosome 20 (Empire Genomics), before analysis. Hybridization on cell lines, tissue and metaphase preparations was performed as described (25). Images were taken with a Zeiss Axio Imager Z2 controlled by Axiovision software.

[0096] Given the demonstration of elevated NCOA3 copy number in melanoma, a matched tissue cohort was obtained of 13 patients undergoing PD-1-based therapy in whom both pre- and post-treatment tissue for molecular analysis. Fluorescence in situ hybridization (FISH) analysis of NCOA3 copy number in the matched tissues was performed. Copy number was defined as % of cells harboring 3 or more copies of NCOA3. An increased NCOA3 copy number was observed in 11 of the 13 post-treatment samples when compared with the pre- treatment tissue (P=.008, Wilcoxon matched pairs, signed-ranks test). Taken together, these analyses provide support that NCOA3 levels are elevated (and possibly selected for) in metastatic melanoma tumors following progression on PD-1 blockade, providing further support for blocking NCOA3 (or its binding partners) to sensitize (or re-sensitize) tumors to PD-1 or PD-L1 blockade.

[0097] Animal studies. All animal care was in accordance with institutional guidelines. Groups of at least ten C57BL / 6 mice were injected s.c. with 75,000 YUMM1.7 cells, or 100,000 B16F10 melanoma cells stably expressing anti-NCOA3 shRNA or control shRNA until the tumors reached a mean tumor volume of at least 75mm. The mice were then subjected to various treatments, including i.p. injections with isotype antibody (control), anti-PD1 antibody (at a dose of 0.2mg per mouse three times weekly for the duration of the experiment), and / or with fulvestrant (at a dose of 25 mg / kg once weekly).Attorney docket No.00010-020WO1

[0098] Statistical methods. All quantified data represent an average of at least triplicate samples or as indicated. Statistical significance was determined by the Student’s t-test, randomization test, Chi-square test, Mann-Whitney test, ANOVA, directional Le test or Kolmogorov-Smirnov test, and p values <0.05 were considered significant. In the in vivo drug efficacy studies, sample sizes were determined prospectively, using a type I error rate of 0.05 and power of 0.8 to detect differences in means of at least 30%. In all experiments, * denotes p<0.05 versus control and error bars represent the Standard Error of the Mean.

[0099] NCOA3 and RARA co-localize and physically interact in melanoma cells. An immunofluorescence analysis was performed to demonstrate the co-localization of NCOA3 and ESR1 in melanoma cells (FIG. 10A). Subsequently, a co-immunoprecipitation (CoIP) analysis was performed to demonstrate the physical interaction between NCOA3 and ESR1 (FIG. 10B). This interaction supports the therapeutic targeting of either NCOA3 or ESR1 as a rational therapeutic strategy for melanoma.

[0100] A chromatin immunoprecipitation (ChIP) study was performed using an antibody targeting ESR1 and a DNA sequence encompassing the human PD-L1 / CD274 promoter to show that ESR1 physically interacts with the PD-L1 / CD274 promoter in A375 and C8161.9 human melanoma cells (FIG. 11A-B). These studies show the binding of ESR1 to the human PD-L1 / CD274 promoter, directly supporting its potential ability to regulate the transcriptional activity of PD-L1 / CD274.

[0101] Transfection of a short interfering RNA (siRNA) targeting human NCOA3 results in increased PD-L1 / CD274 protein expression in multiple melanoma cell lines, as determined by quantitative immunofluorescence analysis (FIG. 12A-C). These results demonstrate that blocking NCOA3 expression can promote PD- L1 / CD274 protein expression, potentially sensitizing melanoma cells to PD-1-based immune checkpoint blockade (ICB).

[0102] A cDNA plasmid construct that contained the firefly luciferase reporter gene driven by the human PD-L1 / CD274 promoter was co-transfected along with cDNA encoding either human ESR1 or RARA in C8161.9 (FIG. 13A) and A375 (FIG. 13B) human melanomaAttorney docket No.00010-020WO1 cells. The data show that transfection of ESR1 or RARA cDNA each dramatically represses luciferase expression driven by the human PD-L1 / CD274 promoter. These studies provide evidence that regulating ESR1 or RARA gene expression can directly regulate PD- L1 / CD274 promoter activity, providing a mechanistic basis for its regulation of PD-L1 / CD274 expression.

[0103] Conversely, studies were performed using small molecule inhibitors of ESR1 (e.g., fulvestrant) or RARA (AGN194310). Drug treatment, along with transfection of the plasmid containing the luciferase cDNA driven by the human PD-L1 / CD274 promoter, resulted in significant upregulation of luciferase expression in A375 and C8161.9 human melanoma cells (FIG. 14A-D). These studies indicate that pharmacologic targeting of ESR1 or RARA can upregulate human PD-L1 / CD274 promoter activity, resulting in increased levels of PD- L1 / CD274 expression, thereby potentially sensitizing to PD-1-based immune checkpoint blockade.

[0104] An in vivo study in the YUMM1.7 murine melanoma model examining the anti-tumor activity of the following four interventions was performed: (i) isotype antibody (as a negative control); (ii) anti-PD-1 antibody; (iii) fulvestrant, an FDA- approved estrogen receptor antagonist; and (iv) combination of anti-PD-1 antibody and fulvestrant, following subcutaneous inoculation of YUMM1.7 melanoma cells into C57BL / 6 mice. The combination of fulvestrant and anti-PD-1 antibody showed significant suppression of tumor volume when combined with either drug alone or control isotype antibody (FIG. 15). These results provide direct in vivo evidence that pharmacologic targeting of ESR1 can potentiate the anti-tumor activity of PD-1-based immune checkpoint blockade, supporting the combinatorial activity of targeting NCOA3 or its binding partners along with PD-1 blockade in melanoma therapy.

[0105] A number of embodiments have been described herein. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

Attorney docket No.00010-020WO1 WHAT IS CLAIMED IS:

1. A method of determining whether a cancer will be responsive to an anti-PDL1 or anti-PD-1 therapy, the method comprising detecting in tumor cells obtained from a subject in need of cancer therapy, the expression or activity of a NCOA3 above a normal control level.

2. The method according to claim 1, wherein if the level of NCOA3 copy number, expression or activity is increased relative to a normal control, then the subject is non-responsive to anti-PDL1 or anti-PD-1 therapy.

3. The method of claim 1 or 2, wherein the NCOA3 levels are determined on a microarray or by fluorescence in situ hybridization.

4. The method of claim 1, further comprising administering an inhibitor of NCOA3 expression or activity to the subject.

5. The method of claim 4, wherein the inhibitor of NCOA3 is an inhibitory nucleic acid.

6. The method of claim 5, wherein the inhibitory nucleic acid is an siRNA or shRNA.

7. The method of claim 4, wherein the inhibitor of NCOA3 is a small molecule inhibitor.

8. The method of claim 7, wherein the small molecule inhibitor is bufalin.

9. A method comprising, measuring the level of NCOA3 in a subject having or suspected of having cancer, administering an NCOA3 expression or activity inhibitor to the subject if the level of NCOA3 is higher than a normal control, and then administering an anti-PD-1 and / or anti-PDL1 inhibitor to the subject.Attorney docket No.00010-020WO1 10. A method of treating a cancer, the method comprising administering an agent that inhibits the expression or activity of NCOA3 and contacting the subject with a PD1- and / or PDL-1 antagonist.

11. The method of claim 10, wherein the agent is an siRNA.

12. The method of claim 10, wherein the agent is a small molecule agent.

13. The method of claim 12, wherein the small molecule agent is bufalin.

14. The method of claim 10, wherein the cancer is melanoma.

15. The method of claim 14, wherein the agent is administered topically.

16. The method of claim 10, wherein the PD1- and / or PDL-1 antagonist is an antibody.

17. The method of claim 10, wherein the agent inhibits ESR1.

18. The method of claim 17, wherein the agent is selected from (R)-6-{2-{ethyl[4-(2-ethylaminoethyl)benzyl]amino}-4- methoxyphenyl}-5,6,7,8-tetrahydronaphthalen-2-ol, fulvestrant, toremifene, and AZD9496.

19. The method of claim 10, wherein the agent inhibits RARA.

20. The method of claim 19, wherein the agent is selected from the group consisting of AGN 194301, AGN 194574, ER50891, LEI 35, Ro 41-5253 and BMS 195614.

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