COMPOSITIONS AND METHODS FOR TREATING AR- AND/OR LNCRNA-

MEDIATED DISEASES

Cross-Reference to Related Applications

This application claims the benefit of U.S. Provisional Application No. 62/557,997, filed on September 13, 2017; and U.S. Provisional Application No. 62/699,960, filed on July 18, 2018; the entire contents of each applications are incorporated herein in their entirety by this reference. Statement of Rights

This invention was made with government support under grant numbers

5R01CA185151-04, R01CA140986, R35GM122532, lZIABCOl 1585, T32AI007386, T32CA009156, and 4R01DK102165-04, awarded by the National Institutes of Health. The U.S. government has certain rights in the present invention.

Background of the Invention

Long non-coding RNAs (IncRNAs) are spliced, capped, and generally

polyadenylated transcripts of >200 nucleotides. Many IncRNAs have been demonstrated to play critical roles in tissue physiology, disease processes, immune regulation and cancer. Remarkably, despite their established importance, the fundamental mechanism of action of IncRNAs has been poorly studied; only a few of the predicted >90,000 human IncRNAs have been well characterized. Despite this, it is believed that many IncRNAs function through direct interactions with protein partners, acting to scaffold the formation of ribonucleoprotein (RNP) complexes required for regulation of critical cellular processes. Well-characterized examples of scaffolding IncRNAs include XIST, which regulates X- chromosome inactivation through association with histone modification complexes, and HOTAIR, an oncogenic IncRNA that coordinates gene expression patterns partially through interactions with the poly comb repressive complex (Hajjari, M. and A. Salavaty, Cancer Biol Med, 2015. 12(1): p. 1-9; Cerase, A., et al., Genome Biol, 2015. 16: p. 166).

Scaffolding IncRNAs may also bind and regulate the activity of transcription factors. For example, the IncRNAs SLNCR1 and HOTAIR, and possibly SRAl and

PCGEM1, bind to the androgen receptor (AR) protein to modulate its downstream transcriptional activity (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37; Zhang, A., et al, Cell Rep, 2015. 13(1): p. 209-21; Agoulnik, I.U. and N.L. Weigel, Steroids, 2009. 74(8): p. 669-74; Prensner, J.R., et al, Oncotarget, 2014. 5(6): p. 1434-8; Yang, L., et al, Nature, 2013. 500(7464): p. 598-602). Interestingly, in contrast to canonical AR activation, in which an androgenic ligand, such as dihydrotestosterone (DHT), binds to AR to elicit nuclear localization and protein dimerization in a head-to-tail formation, IncRNA-mediated AR function occurs in an androgen-independent manner (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37; Zhang, A., et al, Cell Rep, 2015. 13(1): p. 209-21 ; Yang, L., et al, Nature, 2013. 500(7464): p. 598-602). In this way, RNA-driven AR function is fine- tuned to modulate expression of only a subset of genes, as opposed to global, androgen- driven AR activation. Although AR has been implicated in multiple cancers, including prostate, bladder, kidney, lung, liver, pancreatic, thyroid, and breast cancers, the

fundamental mechanisms of AR activation in many of these cancers is not well-established (Chang, C, et al., Oncogene, 2014. 33 : p. 3225-3234; Kanda, T., et al, World J

Gastroenterol, 2014. 20: p. 9229-9236; Stanley, J.A., et al, J Steroid Biochem Mol Biol, 2012. 130: p. 105-124). RNA-driven AR function is believed to function largely in an oncogenic manner (Zhang, A., et al, Cell Rep, 2015. 13(1): p. 209-21; Yang, L., et al, Nature, 2013. 500(7464): p. 598-602). For example, it has been revealed that SLNCR1 increases AR occupancy at the promoter of the gene encoding the matrix metalloproteinase MMP9, increasing MMP9-mediated melanoma invasion (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Thus, understanding of lncRNA-driven, androgen-independent regulation of AR activity, including RNA-mediated activation, may explain many unresolved questions regarding AR's oncogenic or tumor suppressive functions and is an intriguing and clinically-relevant area of research.

In addition to AR, SLNCR1 also binds to the transcription factor brain-specific homeobox protein 3a (Brn3a), and all three components are required for increased melanoma invasion (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). A highly conserved region of SLNCR1 (SLNCRl ^coas ), spanning nucleotides (nts) 372-672, is necessary and sufficient to mediate increased melanoma invasion. The scaffolding model for IncRNA function posits that IncRNAs bind multiple protein partners concurrently. In agreement with this model, Brn3a and AR bind to adjacent sequences on SLNCRl ^com , with Brn3a binding to SLNCRl ^462'512 , while AR binds to SLNCRl ⁵⁶ ^ ^{631 '} .

The predicted or confirmed AR-binding IncRNAs (SLNCR1, HOTAIR, SRA1 and PCGEM1) contain a highly conserved region of approximately 28 nts (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). This conserved sequence is located within the segment oiSLNCRl, HOTAIR, and PCGEM1 required for AR-lncRNA association (SLNCRl ⁵⁶ * ^'631 , HOTAIR ^1'360 , and PCGEM ^2lAm ), indicating that AR interacts with RNA in a sequence- specific manner (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37; Yang, L., et al, Nature, 2013. 500(7464): p. 598-602; Zhang, A., et al, Cell Rep, 2015. 13(1): p. 209-21). Moreover, HOTAIR and PCGEM1 mediate ligand-independent AR transcriptional activity, indicating that these interactions may play an important role in AR-driven, androgen- independent cancers (Yang, L., et al, Nature, 2013. 500(7464): p. 598-602; Zhang, A., et al, Cell Rep, 2015. 13(1): p. 209-21).

The world-wide incidence of melanoma has been on the rise for the past 30 years.

In the United States, there are -73,000 new cases diagnosed and -10,000 deaths annually attributed to melanoma (Siegel, R.L., Miller, K.D., and A. Jemal, CA: a cancer journal for clinicians, 2015. 65: p. 5-29). Of these deaths, approximately two-thirds will occur in males, the result of a well-established sex bias disfavoring males in melanoma etiology (Bidoli, E., et al, BMC Cancer, 2012. 12: p. 443; de Vries, E., et al, Annals of oncology : official journal of the European Society for Medical Oncology / ESMO, 2007. 18: p. 1110- 1116; Fisher, D.E., and A.C. Geller, JAMA Dermatology, 2013. 149: p. 903-904; Geller, A.C., et al, JAMA, 2002. 288: p. 1719-1720; Joosse, A., et al, The Journal of Investigative Dermatology, 2011. 131 : p. 719-726; Schwartz, J.L., et al, Cancer, 2002. 95: p. 1562-1568; Swetter, S.M., et al, Archives of Dermatology, 2009. 145: p. 488-490). In addition to a significant survival advantage compared to males (38%), females demonstrate fewer metastases, a longer delay before relapse, and higher curable rates, strongly indicating a biological basis for the observed sex bias (Gamba, C.S., et al, JAMA Dermatology, 2013. 149: 912-920; Joosse, A., et al, Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology, 2012. 30: p. 2240-2247; Joosse, A., et al, The Journal of Investigative Dermatology, 2011. 131 : p. 719-726).

AR regulates tumorigenesis in many human cancers, including prostate, breast, kidney, lung, bladder and liver (Chang, C, et al, Oncogene, 2014. 33 : p. 3225-3234). AR may function as a tumor suppressor or oncogene, likely dependent on cellular context and the presence or absence of AR-modulating factors. For example, AR activity in prostate cancer cells may be modulated by RNAs, including the IncRNA HOTAIR (Zhang, A., et al, Cell Rep, 2015. 13(1): p. 209-21), or by proteins, such as the transcriptional repressor REl- Silencing Transcription Factor (REST, also called Neuron-Restrictive Silencer Factor, RSF) (Svensson, C, et al, Nucleic Acids Res, 2014. 42(2): p. 999-1015). Early studies suggested that AR has oncogenic functions in melanoma, and that differences in AR function or expression might explain the observed melanoma gender differences (de Vries, E., et al, Annals of oncology : official journal of the European Society for Medical Oncology / ESMO, 2008. 19: p. 583-589; Joosse, A., et al, The Journal of Investigative Dermatology, 2011. 131 : p. 719-726; Li, W.Q., et al., Journal of Clinical Oncology :

Official Journal of the American Society of Clinical Oncology, 2013. 31 : p. 4394-4399; Micheli, A., et al, European Journal of Cancer, 2009. 45: p. 1017-1027; Morvillo, V., et al, Pigment Cell Res, 1995. 8: p. 135-141; Morvillo, V., et al, Melanoma Research, 2002. 12: p. 529-538; Spanogle, J.P., et al, Journal of the American Academy of Dermatology, 2010. 62: p. 757-767). In direct support of an oncogenic function for AR, AR was shown recently to increase melanoma invasion through transcriptional upregulation of the matrix metalloproteinase MMP9 (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37).

Interestingly, this regulation occurs independently of canonical AR activation, in which an androgen (such as testosterone) binds to the receptor to elicit downstream transcriptional patterns. Instead, AR-mediated invasion requires a novel IncRNA SLNCR, specifically the abundant SLNCR1 isoform that directly binds to and recruits AR to the MMP9 promoter.

LncRNAs are transcripts of >200 nucleotides that lack an open-reading frame and exhibit cell-type and tissue-specific expression. LncRNAs are important regulators of tissue physiology and disease processes, and may function as either oncogenes or tumor suppressors (Li, J., Xuan, Z., and C. Liu, International Journal of Molecular Sciences, 2013. 14: p. 18790-18808; Serviss, J.T., Johnsson, P., and D. Grander, Frontiers in Genetics, 2014. 5: p. 234). While the fundamental mechanism of many IncRNA remains unknown, many (like SLNCR) function through direct interactions with proteins. Using a highly- sensitive technique developed for the identification of RNA-associated transcription factors called RATA (RNA-associated transcription factor array), SLNCR was shown to bind to multiple transcription factors, possibly regulating their downstream transcriptional activities (Schmidt K, et al, J Biol Methods, 2017. 4(1): p. e67; Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). In addition to AR and Brn3a, both of which are required for SLNCR1- mediated regulation of MMP9, early growth response 1 (EGR1) was identified as a candidate ,SZNCR7-interacting transcription factor (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). EGR1 is a zinc-finger transcription factor implicated in many human cancers, likely functioning as a tumor suppressor. In prostate cancer, EGR1 is a critical regulator of AR function (Yang, S.Z., and S.A. Abdulkadir, Journal of Biological Chemistry, 2003. 278(41): p. 39906-11). In melanoma, EGR1 has been implicated in apoptosis, cell growth, and fibronectin matrix synthesis (Ahmed, M.M., et al, The Journal of Biological Chemistry, 1996. 271 : p. 29231-29237; Gaggioli, C, et al, Oncogene, 2005. 24: p. 1423-1433;

Muthukkumar, S., et al, Mol Cell Biol, 1995. 15: 6262-6272; Sells, S.F., et al, Mol Cell Biol, 1995. 15: p. 682-692). Other roles for EGRl in melanomagenesis, including biological consequences of possible physical interactions with SLNCR and/or AR have not been described.

Currently, it is nearly impossible to predict IncRNA function from sequence analysis alone because so little is known about the role of IncRNA sequence and structural motifs relative to interactions with other proteins and nucleic acids. This general lack of understanding of the mechanisms of IncRNA activity hinders the development and potential therapeutic impact of modalities and strategies that target IncRNAs. Thus, there is a critical need for studies of the fundamental biology of IncRNAs and identification of functional IncRNA motifs. Establishing sequence and/or structural parameters to enable prediction of IncRNA-protein binding activity will significantly advance our ability to identify and characterize functional IncRNAs, and to design novel IncRNA-targeting therapeutics.

Summary of the Invention

The present invention is based, at least in part, on the discovery that AR interacts with lnc RNA in a sequence-specific manner, and that single-stranded nucleic acids designed to sterically block the interaction between AR and such IncRNA without significantly altering the expression of AR and/or IncRNA can be applied to effectively modulate the activity of AR or IncRNA.

The present invention is also based, at least in part, on the discovery that the N- terminal, regulatory domain (NTD) of AR (AR NTD) interacts with an unstructured, pyrimidine-rich RNA in a sequence-specific manner. A novel AR-binding motif in

HOXA11 AS-203 is described herein such that this IncRNA also functions in complex with AR. In-cell SHAPE reveals that SLNCRFs AR-binding motifs are found in a region of high structural flexibility, and that SLNCRFs AR binding motifs display in-cell protections consistent with protein binding. Mutational analysis confirms that SLNCRFs AR-binding motifs are required for SLNCRl- and AR-mediated melanoma invasion. Furthermore, designed single-stranded oligos to sterically block the interaction of AR and SLNCRl show that these oligos negate ,SZNCR7-mediated melanoma invasion. It is revealed herein how characterizing IncRNA structure and protein interactions enables identification of novel IncRNA function and supports design of novel IncRNA-targeting therapeutics.

The present invention is further based, at least in part, on the discovery that AR directly binds many ffiNCR-regulated genes in a ligand-independent manner in cancer cells. It is discovered herein that SLNCR directly recruits AR to EGR1 -bound chromatin. AR and SLNCR act as a transcriptional switch, reversing EGR1 -mediated upregulation of the - suppressor gene. These results indicate that SLNCR, AR and EGR1

form a novel regulatory triad that regulates cancer proliferation. These data demonstrate that a comprehensive evaluation of AR function in melanoma is critical to understanding the mechanistic underpinnings of the melanoma sex bias.

In one aspect, a single-stranded nucleic acid that binds to androgen receptor (AR) or a long non-coding RNA (IncRNA), and blocks interaction between AR and the IncRNA, wherein the single-stranded nucleic acid does not significantly reduce the expression of AR or the IncRNA, is provided.

Numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the single-stranded nuclei acid binds to AR to block the interaction of AR with the IncRNA. In another embodiment, the single-stranded nucleic acid localizes at least partially to the nucleus. For example, the single-stranded nucleic acid localizes to the nucleus. In still another embodiment, the single-stranded nucleic acid is at least 1 1 nucleotides (nt) in length (e.g., 17 nt in length). In yet another embodiment, the single-stranded nucleic acid dose not comprise a high level of secondary structure. In another embodiment, the single-stranded nucleic acid comprises at least one UCUCCU or UCUCCA motif. In still another embodiment, the single-stranded nucleic acid comprises two or more motifs selected from the group consisting of UCUCCU and UCUCCA motifs. The single-stranded nucleic acid may further comprise an internal sequence between these motifs. Such internal sequence may be 9 nt in length. In yet another embodiment, the single-stranded nucleic acid is at least 20 nt in length, and the pyrimidine content within a 17-20 nt region of the single-stranded nucleic acid that comprises the UCUCCU or UCUCCA motif is at least 65% (e.g., more than 75%). In another embodiment, the IncRNA is selected from the group of SLNCR, HOXAl l-AS-203, SRA1, HOTAIR, and PCGEM1. For example, the the IncRNA is SLNCR. In still another embodiment, the single-stranded nucleic acid is selected from the group of MIMIC 1 (SEQ ID NO: 23), MFMIC 2 (SEQ ID NO: 24), MUT L (SEQ ID NO:4), MUT R (SEQ ID NO:5), MUT I (SEQ ID NO : 8), MUT PT (SEQ ID NO : 7), WT- 17 ((SEQ ID NO : 9), WT-20 (SEQ ID NO: 10), SRA1 (SEQ ID NO:20), and WT-11 (SEQ ID NO: 17).

In one embodiment, the single-stranded nucleic acid binds to the IncRNA to block the interaction of AR with the IncRNA. In another embodiment, the nucleic acid contains at least one AGGAGA or UGGAGA motif. In still another embodiment, if the IncRNA contains two consecutive motifs selected from the group consisting of UCUCCU and

UCUCCA motifs, the single-stranded nucleic acid binds to both motifs simultaneously with an appropriate internal sequence between the sequences that bind to the two motifs. In yet another embodiment, the IncRNA is selected from the group of SLNCR, HOXAl l-AS-203, SRA1, HOTAIR, and PCGEM1. For example, the the IncRNA is SLNCR. In another embodiment, the single-stranded nucleic acid localizes at least partially to the nucleus. In still another embodiment, the single-stranded nucleic acid is at least 20 nt in length (e.g., 28 nt in length). In yet another embodiment, the single-stranded nucleic acid dose not comprise a high level of secondary structure. In another embodiment, the single-stranded nucleic acid comprises an AGGAGA motif and a UGGAGA motif. In still another embodiment, the single-stranded nucleic acid comprises a GGAGA motif and a UGGAGA motif. In yet another embodiment, the single-stranded nucleic acid comprises an

AGGAGA motif and a UGGAG motif. In another embodiment, the single-stranded nucleic acid comprises a GGAGA motif and a UGGAG motif. In still another embodiment, the two motifs are linked by an internal sequence. Such internal sequence may be 9 nt in length. In yet another embodiment, the internal sequence has more than 75% (e.g., 100%) sequence identity to the sequence AGACCAGGG. In another embodiment, the single- stranded nucleic acid is at least 30 nt in length, and the purine content within a 30 nt region of the single- stranded nucleic acid that comprises the AGGAGA or UGGAGA motif is at least 65% (e.g., more than 75%). In still another embodiment, the single-stranded nucleic acid is ANTISENSE 1 (SEQ ID NO: 25) or ANTISENSE 2 (SEQ ID NO: 26).

In one embodiment, the single-stranded nucleic acid comprises at least one chemical modification. In another embodiment, the single-stranded nucleic acid penetrates the cellular membrane of a cell in the absence of a delivery vehicle. In still another embodiment, the chemical modification extends the half-life of the single-stranded nucleic acid in a cell. For example, the chemical modificaiton may be any chemical modificaiton of nucleic acid known in the art, such as one selected from the group consisting of a FANA chemical modification, a fluorinated nucleoside, a DNA nucleotide, a 2'-0-methylated nucleoside, a phosphorothioate bond and a cholesterol moiety. In certain embodiment, the nucleic acid comprises a FANA chemical modification.

In one embodiment, a pharmaceutical composition comprising the single-stranded nucleic acid and a pharmaceutically acceptable agent selected from the group consisting of excipients, diluents, and carriers, is provided. In another embodiment, the pharmaceutical composition comprises the single-stranded nucleic acid at a purity of at least 75%. In still another embodiment, the pharmaceutical composition further comprises a nuclear receptor targeting drug. The nuclear receptor targeting drugs, described herein, may be luteinizing hormone-releasing hormone (LHRH) analogs, androgen receptor inhibitors, anti -androgens, hormone blocking drugs, nuclear receptor agonists, nuclear receptor antagonists, selective receptor modulators, selective androgen receptor modulators (SARMs), selective estrogen receptor modulators (SERMs), selective progesterone receptor modulators (SPRMs), selective glucocorticoid receptor agonists (SEGRAs), or selective glucocorticoid receptor modulators (SEGRMs). For example, the nuclear receptor target drug may be selected from the group consisting of leuprolide (Lupron®, Eligard®), goserelin (Zoladex®), triptorelin (Trelstar®), histrelin (Vantas®), degarelix (Firmagon®), bicalutamide

(Casode®), enzalutamide (Xtandi®), flutamide (Eulexin®), nilutamide (Nilandron®), ketoconazole (Nizoral®), abiraterone (Zytiga®), dexamethasone, megestrol acetate

(Megace®), medroxyprogesterone acetate (MP A), ethisterone, norethindrone acetate, norethisterone, norethynodrel, ethynodiol diacetate, norethindrone, norgestimate, norgestrel, levonorgestrel, medroxyprogesterone acetate, desogestrel, etonogestrel, drospirenone, norelgestromin, desogestrel, etonogestrel, gestodene, dienogest,

drospirenone, elcometrine, nomegestrol acetate, trimegestone, tanaproget, BMS948, mifepristone, 4-hydroxytamoxifen, CF PAl, Cyproterone acetate (Androcur®, Cyprostat®, Siterone®), chlormadinone acetate (Clordion®, Gestafortin®, Lormin®, Non-Ovlon®, Normenon®, Verton®), 17-hydroxyprogesterone (17-OHP), THC, clotrimazole, PK11195 [l-(2-chlorophenyl)-N-methyl-N-(l-methylpropyl)-3-isoquinoli necarboxamide], meclizine, androstanol, CITCO [6-(4-chlorophenyl)imidazo [2,l-b][l,3] thiazole-5-carbaldehyde O- (3,4-dichlorobenzyl) oxime], zearalenone (ZEN), T0901317, S07662, enobosarm, BMS- 564,929, LGD-4033, AC-262,356, JNJ-28330835, LGD-2226, LGD-3303, S-40503, S-23, clomifene, femarelle, ormeloxifene, raloxifene, tamoxifen, toremifene, lasofoxifene, ospemifene, afimoxifene, arzoxifene, bazedoxifene, gulvestrant (Faslodex®, ICI- 182780), CDB-4124, asoprisnil, proellex, mapracorat (BOL-303242-X, ZK 245186), fosdagrocorat (PF-04171327), ZK 216348, and 55D1E1.

In one embodiment, a vector comprising the single-stranded nucleic acid is provided. The vector may be an expression vector. In another embodiment, a host cell transfected with such expression vector is provided. In still another embodiment, a method of producing a single-stranded nucleic acid comprising culturing the host cell in an appropriate culture medium to, thereby, produce the single- stranded nucleic acid, is provided. The host cell could be a bacterial cell or a eukaryotic cell. In yet another embodiment, a method of producing a single-stranded nucleic acid comprising transcribing the vector in vitro under an appropriate reaction condition, and isolating the single-stranded nucleic acid from the reaction mixture, is provided.

In another aspect, a method of treating a subject afflicted with an AR- and/or

IncRNA-mediated disease is provided, the method comprising administering to the subject a therapeutically effective amount of a single-stranded nucleic acid that binds to androgen receptor (AR) or a long non-coding RNA (IncRNA), and blocks interaction between AR and the IncRNA, wherein the single-stranded nucleic acid does not significantly reduce the expression of AR or the IncRNA.

As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the AR- and/or IncRNA-mediated disease is androgen-independent. In another embodiment, the AR- and/or lncRNA- mediated disease is selected from the group of cancer, inflammatory disease, metabolic diseases, spontaneous abortion, alopecia, Alzheimer disease, androgen-insensitivity syndrome, heart disease (e.g., artiosclerosis, coronary artery disease, and cardiomyopathy), azoospermia, cholelithiasis, cognition disorders, cryptorchidism, depressive disorder, diabetes mellitus, diabetic retinopathy, sex development disorders, bone fractures, hyperglycemia, hypertension, hypospadias, hypertrophy, hyperandrogenism, infertility, klinefelter syndrome, liver cirrhosis, liver disease, lupus, migraine disorder, Menkes Kinky Hair Syndrome, muscular atrophy, nerve degeneration, obesity, oligospermia, osteoarthritis, osteoporosis, Paget Disease, Polycystic Ovary Syndrome, Primary Ovarian Insuffiency, Schizophrenia, spinal fractures, thrombocytosis, thyroid diseases, Tourette syndrome, Turner syndrome, and autoimmune disease. The cancer, described herein, may be, e.g., melanoma, lung adenocarcinoma, lung squamous cell carcinoma, cervical cancer, ovarian cancer, uterine cancer, pancreatic cancer, colorectal cancer, lower grade glioma, glioblastoma multiforme, breast cancer, endometrial cancer, prostate cancer, testicular cancer, thyroid cancer, osteosarcoma, esophageal cancer, liver cancer or bladder cancer. For example, the cancer is melanoma. The inflammatory disease, described herein, may be, e.g., acne vulgaris, asthma, and rheumatoid arthritis. The autoimmune disease described herein, may be, e.g., lupus, type I diabetes, and multiple sclerosis. In still another embodiment, the single-stranded nucleic acid inhibits the growth and/or metastasis the cancer.

In still another aspect, a method of inhibiting proliferation or invasion of cancer cells is provided, the method comprising contacting the cancer cells with a single-stranded nucleic acid that binds to androgen receptor (AR) or a long non-coding RNA (IncRNA), and blocks interaction between AR and the IncRNA, wherein the single-stranded nucleic acid does not significantly reduce the expression of AR or the IncRNA.

As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the single-stranded nucleic acid is administered in a pharmaceutically acceptable formulation. In another embodiment, the IncRNA is selected from the group of SLNCR, HOXA1 l-AS-203, SRA1, HOTAIR and PCGEM1. For example, the IncRNA is SLNCR. In still another embodiment, the single- stranded nucleic acid is selected from the group consisting of MTMIC 1, MTMIC 2,

ANTISENSE 1, and ANTISENSE 2. In yet another embodiment, the single-stranded nucleic acid inhibits a SLNCR-regulated gene. The SLNCR-regulated gene, described herein, may be, e.g., MMP9, p21, CXCL2, JUN, STAT3, IL24, or MCAM. In another embodiment, the method further comprises admisnistering a nuclear receptor targeting drug. The nuclear receptor targeting drugs, described herein, may be luteinizing hormone- releasing hormone (LHRH) analogs, androgen receptor inhibitors, anti -androgens, hormone blocking drugs, nuclear receptor agonists, nuclear receptor antagonists, selective receptor modulators, selective androgen receptor modulators (SARMs), selective estrogen receptor modulators (SERMs), selective progesterone receptor modulators (SPRMs), selective glucocorticoid receptor agonists (SEGRAs), or selective glucocorticoid receptor modulators (SEGRMs). For example, the nuclear receptor target drug may be selected from the group consisting of leuprolide (Lupron®, Eligard®), goserelin (Zoladex®), triptorelin

(Trelstar®), histrelin (Vantas®), degarelix (Firmagon®), bicalutamide (Casode®), enzalutamide (Xtandi®), flutamide (Eulexin®), nilutamide (Nilandron®), ketoconazole (Nizoral®), abiraterone (Zytiga®), dexamethasone, megestrol acetate (Megace®), medroxyprogesterone acetate (MP A), ethisterone, norethindrone acetate, norethisterone, norethynodrel, ethynodiol diacetate, norethindrone, norgestimate, norgestrel,

levonorgestrel, medroxyprogesterone acetate, desogestrel, etonogestrel, drospirenone, norelgestromin, desogestrel, etonogestrel, gestodene, dienogest, drospirenone, elcometrine, nomegestrol acetate, trimegestone, tanaproget, BMS948, mifepristone, 4- hydroxytamoxifen, CF PA1, Cyproterone acetate (Androcur®, Cyprostat®, Siterone®), chlormadinone acetate (Clordion®, Gestafortin®, Lormin®, Non-Ovlon®, Normenon®, Verton®), 17-hydroxyprogesterone (17-OHP), THC, clotrimazole, PK11195 [l-(2- chlorophenyl)-N-methyl-N-(l-methylpropyl)-3-isoquinolinecarb oxamide], meclizine, androstanol, CITCO [6-(4-chlorophenyl)imidazo [2,l-b][l,3] thiazole-5-carbaldehyde O- (3,4-dichlorobenzyl) oxime], zearalenone (ZEN), T0901317, S07662, enobosarm, BMS- 564,929, LGD-4033, AC-262,356, JNJ-28330835, LGD-2226, LGD-3303, S-40503, S-23, clomifene, femarelle, ormeloxifene, raloxifene, tamoxifen, toremifene, lasofoxifene, ospemifene, afimoxifene, arzoxifene, bazedoxifene, gulvestrant (Faslodex®, ICI- 182780), CDB-4124, asoprisnil, proellex, mapracorat (BOL-303242-X, ZK 245186), fosdagrocorat (PF-04171327), ZK 216348, and 55D1E1. In still another embodiment, the subject is a mammal (e.g., human). In yet another embodiment, the mammal is an animal model of an AR- and/or lncRNA-mediated disease.

In yet another aspect, a cell-based method for identifying a single-stranded nucleic acid that binds to AR or a IncRNA and blocks interaction between AR and the IncRNA is provided, the method comprising: a) contacting a cell expressing AR and/or IncRNA with a test single-stranded nucleic acid that binds to AR or the IncRNA; and b) determining that the single-stranded nucleic acid blocks interaction between AR and the IncRNA, wherein the single-stranded nucleic acid does not significantly reduce the expression of AR or the IncRNA, optionally further determining that the single-stranded nucleic acid treats an AR- and/or lncRNA-mediated disease.

As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the said cells are isolated from an animal model of an AR- and/or IncRNA-mediated disease, or a human patient afflicted with the AR- and/or IncRNA-mediated disease. In another embodiment, the step of contacting occurs in vivo, ex vivo, or in vitro. In still another embodiment, the method further comprisies determining the ability of the test single-stranded nucleic acid to bind to either AR or IncRNA before or after determining the effect of the test single-stranded nucleic acid on the interaction between AR and the IncRNA. In yet another embodiment, the method further comprises determining the effect of the test single-stranded nucleic acid on the level of expression of AR or IncRNA before or after determining the effect of the test single- stranded nucleic acid on the interaction between AR and the IncRNA. In another embodiment, the method further comprises determining the activity of AR or IncRNA in the disease cell. The activity of AR or IncRNA may be assessed by determining the magnitude of cellular proliferation, cell death, cellular migration, replication, induction of angiogenesis, cellular invasion/metastasis, immune response, immune evasion, or transcription of AR- or lncRNA-regulated genes.

In another aspect, an in vitro cell-free method for identifying a single-stranded nucleic acid that binds to AR or a IncRNA and blocks interaction between AR and the IncRNA is provided, the method comprising a) contacting a mixture of AR protein and IncRNA with a test single-stranded nucleic acid that binds to AR or the IncRNA; and b) determining that the single- stranded nucleic acid blocks interaction between AR and the IncRNA, wherein the single-stranded nucleic acid does not significantly reduce the expression of AR or the IncRNA, optionally further determining that the single-stranded nucleic acid treats an AR- and/or IncRNA-mediated disease.

As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, in one embodiment, the method further comprises determining the ability of the test single-stranded nucleic acid to bind to either AR or IncRNA before or after determining the effect of the test single-stranded nucleic acid on the interaction between AR and the IncRNA. In another embodiment, the IncRNA is a full-length RNA, or functional variants or fragments thereof. In still another embodiment, AR is a full-length protein, or functional variants or fragments thereof. In yet another embodiment, AR is an N-terminal region of AR protein having the amino acid sequence of SEQ ID NO: 27. Brief Description of Figures

FIG. 1A - FIG. 1C show REMSA-based identification of minimal requirements for AR-SLNCR1 interaction. FIG. 1 A shows sequences of biotinylated probes used in these assays. The highly-predicted secondary structure of WT-41 is shown below. For REMSA results shown in FIG. IB - FIG. 1C, the indicated probes (from FIG. 1 A) were incubated with increasing concentrations of either full-length AR (FIG. IB) or a truncated protein corresponding to AR' s N-terminal region (FIG. 1C), resolved on a 5% TBE gel and probed using Streptavidin-URP following transfer to a negatively charged membrane. The lines indicate either unbound labeled probe or the slower migrating RNA/protein complexes.

FIG. 2 A - FIG. 2C show that AR's N-terminus specifically binds to biotinylated probes corresponding to WT-41, WT-28, and WT-20 as comfirmed by competition assays. FIG. 2A shows schematic of AR's protein domains. NTD = N-terminal domain, DBD = DNA binding domain, and LBD = ligand binding domain. The recombinant N-terminal AR protein used in these studies (amino acids 2-556) is denoted. For REMSA results shown in FIG. 2B - FIG. 2C, probes were incubated with 600 nM of recombinant (FIG. 2B) full- length AR protein, or (FIG. 2C) N-terminal AR protein. Where indicated, 10 μΜ of unlabeled RNA competitor corresponding to WT-41 was added prior to addition of biotinylated probes. REMSAs were completed as in FIG. 1A-FIG. 1C.

FIG. 3A - FIG. 3G show that AR binds to single-stranded RNA in a sequence specific manner. Sequences of biotinylated probes used in these assays are shown in FIG. 3A and FIG. 3C. Nucleotides that are mutated compared to wild-type SLNCR1 are highlighted in red, bonded font. The predicted AR binding motifs are highlighted in the grey boxes. The yellow box denotes the non-endogenous AR binding motif in MUT PT. FIG. 3B and FIG. 3E show REMSA of the indicated probes incubated with 600 nM of recombinant N-terminal AR, as in FIG. IB and FIG. 1C. FIG. 3D shows highly-predicted secondary structure of stem-loop probe with the non-endogenous nucleotides denoted by the red bar in (FIG. 3C). FIG. 3F shows the measurement of binding affinity by surface plasm on resonance (SPR) of the indicated concentrations of either WT-41 (left panel) or WT-20 (right panel) to full-length recombinant AR. RU, resonance units. FIG. 3G shows the consensus motif identified among AR-binding RNA sequences.

FIG. 4A - FIG. 40 show that AR does not bind promiscuously to pyrimidine-rich RNAs, and that AR and Brn3a binding sites reside within an unstructured region of SLNCR1 in vivo. FIG. 4A-40 further show the SHAPE-guided modeling of the SLNCR1 RNA structure. Sequences of biotinylated probes used in these assays are shown in FIG. 4A. FIG. 4B shows REMSA of the indicated probes incubated with 600 nM of recombinant N-terminal AR., as in FIG. IB and FIG. 1C. FIG. 4C-4E show the normalized SHAPE reactivity profiles for WM1976 cell SLNCRJ ^403'780 RNA from (FIG. 4C) cell-free extracted nuclear RNA, (FIG. 4D) in-cell total cellular RNA, and (FIG. 4E) cell-free extracted cytoplasmic RNA. Colored bars with associated errors represent SHAPE reactivities (see scale). Purple and dark blue rectangles mark Brn3a and AR interaction sites, respectively. Grey and light blue rectangles represent sites of significant in-cell SHAPE and DMS protection, respectively. Grey bars below zero indicate residues with high background (and are thus classified as no-data). No data are collected for 5' and 3 ' nucleotides where RT and PCR primers bind. FIG. 4F-4H show the SHAPE-guided modeling reveals SLNCRJ ^403'780 has limited stable RNA structure. FIG. 4F shows the SHAPE profile for Ul snRNA.

Colored arcs represent SHAPE-modeled base pairs with associated pairing probability. Entropy values (bottom) are local 50 nucleotide windowed medians. The horizontal dotted line marks the low entropy threshold 0.03. Purple shading indicates regions where windowed-median SHAPE and entropy values both fall below predetermined thresholds (0.4 SHAPE, 0.03 entropy) and grey shading marks expanded motif boundaries. FIG. 4G shows the comparison of increases in mutation rates (proportional to SHAPE reactivities) for SLNCR1 ⁴⁰³ -™ ⁰ and Ul snRNA from nuclear extracts. On the X-axis, 5NIA, DMSO, and Denatured refer to the nucleotide mutation rates observed after MaP RT and sequencing of 5NIA treated, untreated, and denaturing control sample RNA, respectively. FIG. 4H shows the SHAPE profile for SLNCRl ^{403'1 0} IncRNA. SHAPE reactivities and Shannon entropies are plotted on the same scale as in panel A. Vertical dashed lines highlight a region of especially high SHAPE reactivity and high entropy. FIG. 4I-4J show that SHAPE-MaP accurately models structure and protein-binding of Ul snRNA. FIG. 4I-4J show the normalized SHAPE reactivity profiles for Ul small nuclear RNA from (FIG. 41) cell-free extracted nuclear RNA (WM1976 cells) or (FIG. 4J) in-cell total RNA. Colored bars with associated errors represent SHAPE reactivities (see scale). 70K, Ul A, and Sm protein interaction sites on Ul, backed by crystal structure data, are highlighted above. Grey rectangles above 0 represent sites of significant in-cell SHAPE protection. Grey bars below zero indicate residues with high background (and are thus classified as no-data). No data are collected for 5' and 3' nts where RT and PCR primers bind. FIG. 4K shows the colored arcplot of modeled base-pairs with labeled probabilities (bottom) aligned to the modeled minimum free energy secondary structure (top). All correct duplexes are observed in the model (black), and only one extra duplex is predicted (grey) where normally Ul RNA is engaged by proteins in vivo. FIG. 4L shows the comparison of SHAPE-MaP and DMS- MaP mutation rates. Differences in mutation rates of SLNCR1 ^403'120 between treated and untreated cell-free (nucleus) and in-cell SHAPE and DMS-MaP experiments on WM1976 cells are shown. Mutation rate differences have been averaged over a 10 nucleotide window and 5NIA rates were scaled up by the DMS/5NIA median ratio for ease of comparison. FIG. 4M-40 show the minimum free energy and alternative models of SLNCRl ^{403'1 0} . FIG. 4M shows the colored arcplot of modeled base-pairs with labeled probabilities (bottom) aligned to the modeled minimum free energy secondary structure (top) of SLNCRl ^{403'1 0} . Low SHAPE, low entropy regions relative to the global

SLNCR403-780 median, which do not conform to the thresholds described in the text, are highlighted in purple, and grey shading marks expanded motif boundaries. Secondary structure projections of the (FIG. 4N) minimum free energy model and (FIG. 40) arcplot- supported alternative structures are represented with associated SHAPE reactivities and in- cell reagent protections. SSI and SS2 denote low SHAPE, low Shannon regions 1 and 2 that are maintained in both models. All expanded low SHAPE, low entropy structures are shaded gray.

FIG. 5A - FIG. 5C show that mutation of SLNCRFs AR binding motif negates SLNCR1 -mediated invasion. FIG. 5 A shows schematic of the SLNCR1 sequences expressed in (FIG. 5B), zoomed in on the region containing site-directed mutations within the AR binding motif. FIG. 5B shows matrigel invasion assays of A375 cells transfected with either an empty vector or a vector expressing the indicated SLNCR1 sequence.

SLNCRl ^coas corresponds to SLNCR1 nts 372-672. Invasion is calculated as the percent of invading cells compared to mobile cells as counted in 8 fields of view. Top panels show representative images of the indicated invading and mobile cells. Quantification from 3 independent replicates, represented as mean + SD, is shown at the bottom. FIG. 5C shows that the SLNCR1 expression vectors express similar levels of SLNCR1. RT-qPCR data is represented as the fold change compared to A375 vector control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the Student's t-test: * p-value < 0.05, ** p-value < 0.005, ns = not significant. FIG. 6A - FIG. 6D show that AR directly binds to a sequence corresponding to IncRNA HOXAl 1 -AS 1-203, and possibly SRA1. Sequences of probes corresponding to the regions of the indicated IncRNAs predicted to bind to AR are shown in FIG. 6A. FIG. 6B shows REMSA of the indicated probes incubated with 600 nM of recombinant N-terminal AR, as in FIG. IB and FIG. 1C. FIG. 6C shows schematic of the genomic loci of HOXAl 1 gene (blue) and its cognate antisense RNAs (green). The HOXAl IAS transcript variant 203 contains the AR binding sequence and is highlighted in red. Transcript variants are based on Ensembl genome assembly: GRCh38.pl0 (GCA 000001405.25). FIG. 6D shows sequence of HOXAl 1 AS-203 variant, with the sequence corresponding to the probe used in (FIG. 6 A; highlighted in yellow).

FIG. 7A - FIG. 7B show that AR's N-terminal region likely directly binds to SRA1 and HOXAl 1 AS-203. For REMSA shown in FIG. 7A, the biotinylated probe

corresponding to a region of SRA1 (FIG. 6 A) was incubated with 600 nM of recombinant N-terminal AR protein. In lane 3, 10 μΜ of unlabeled RNA competitor corresponding to WT-41 was added prior to addition of biotinylated probes. REMSA was completed as in FIG. IB and FIG. 1C. FIG. 7B shows that HOXAl 1 AS-203 is expressed in AR-expressing tissues. Expression of HOXAl 1AS-203 was quantified using RT-qPCR with isoform specific primers, represented as the fold change compared to skeletal muscle expression, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. AR mRNA expression is represented as the average RPKM from GTEx RNA- seq, as presented in The Human Protein Atlas.

FIG. 8 shows that FANA-modified oligos are gymnotically delivered to melanoma short-term cultures. The indicated melanoma short term cultures were seeded in a 24-well plate, and Alexa-labeled scrambled FANA-modified oligos were added 24 hours later at the indicated concentrations. Cells were incubated for 4 days, and Alexa 647 signal was quantified using FACs.

FIG. 9A - FIG. 9C show that sterically-blocking the AR-SLNCR1 interaction negates or prevents ,SZNCR7-mediated melanoma invasion. FIG. 9A - FIG. 9B show that steric-blocking FANA-modified oligos block the interaction of AR and SLNCR1. RNA electromobility shift assay (REMSA) using a biotinylated RNA probe corresponding to the region of SLNCR1 that binds to AR (UAUUUUUCCCUCUCCACCCU). RNA binding reactions were assembled in REMSA binding buffer (Thermo Scientific) with 0.5 nM of biotinylated RNA probe, 2 μg of yeast tRNA, 600 nM of N-terminal AR protein (corresponding to amino acids 2-556) where indicated, and 10 μΜ of the indicated FANA- modified oligos where indicated. FANA-oligos were designed to either bind to AR (AR binders, or MTMIC), or are the reverse compliment (ANTISENSE) to SLNCRl and bind to the IncRNA. Reactions were incubated at room temperature for 25 minutes, 5x loading buffer was added, and samples were resolved on a 5% TBE gel in 0.5X TBE buffer.

Following electrophoresis, the gel was transferred to Amersham Hybond-N+ membrane (GE Healthcare), crosslinked, and probed using the LightShift® Chemiluminescent RNA EMSA Kit (Thermo Scientific). dsRNA = double stranded RNA, formed upon addition of the reverse compliment FANA oligos. FIG. 9C shows that FANA-modified oligos block SLNCRl -mediated melanoma invasion. Matrigel invasion assays of A375 cells transfected with either an empty vector or a vector expression SLNCRl. The indicated FANA-modified oligos were added 24 hours post-transfection, at a final concentration of 500 nM. Invasion is calculated as the percent of invading cells compared to mobile cells as counted in 8 fields of view. Top panels show representative images of the indicated invading and mobile cells. Quantification from 3 independent replicates, represented as mean + SD, is shown at the bottom. Significance compared to the vector and scramble only control was calculated using the Student's t-test: * p-value < 0.05, ** p-value < 0.005, *** p-value < 0.0005, ns = not significant.

FIG. 10A - FIG. 10D show that sterically-blocking the AR-SLNCR1 interaction negates SLNCRl -mediated MMP9 upregulation. Relative (FIG. 1 OA) SLNCRl or (FIG. 10B) MMP9 expression in A375 cells transfected with either an empty or SLNCR1- expressing plasmid, and following addition of the indicated FANA-modified oligos. RT- qPCR data is represented as the fold change compared to A375 vector and scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance in FIG. 10B was calculated using the Student's t-test compared to vector/scramble only control: * p-value < 0.05, n.s. = not significant. FIG. 10C-10D show that mutation of SLNCRL s AR motifs 1 and 2 negates SLNCRl -mediated MMP9 upregulation. Relative (FIG. IOC) SLNCRl or (FIG. \0Ό) MMP9 expression in A375 cells transfected with plasmids expressing either wild-type or mutant ,SZNCR7-expressing plasmids. RT-qPCR data is represented as the fold change compared to A375 vector control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance in FIG. 10D was calculated using the Student's t-test compared to vector only control: ** p-value < 0.005, **** p-value < 0.001, n.s. = not significant. FIG. 11 shows that AR's N-terminal region likely requires at least 11 nucleotides for RNA binding. For REMSA shown in FIG. 11, either 11 nucleotide (WT-11) or 10 nucleotide (WT-10) biotinylated probes corresponding to a region of SLNCR1 was incubated with 600 nM of recombinant N-terminal AR protein. REMSA was completed as in FIG. IB and FIG. 1C.

FIG. 12A - FIG. 12L show that SLNCR is expressed in a gender- and stage-specific manner, regulates melanoma cell proliferation, and increase melanoma invasion. FIG. 12A shows box plots of SLNCR expression from 150 TCGA melanomas categorized by patient gender and stage of melanoma. Data are represented as mean + SEM. Significance was calculated using the one-way analysis variance (ANOVA). FIG. 12B shows box plots of SLNCR expression from male (left panel) or female (right panel) melanomas pre- and post- lymph node metastasis. Data are represented as mean + SEM. Significance was calculated using the two-tailed Student's t-test: * p-value < 0.05, ** p-value < 0.005, n.s. = not significance. FIG. 12C shows Kaplan-Meier survival analysis high- or \ow-SLNCR expressing TCGA male or female melanomas, defined by the median SLNCR expression (RPKM = 14.1). FIG. 12D shows representing expression of AR mRNA in male or female melanomas exhibiting low (left panel) or high (right panel) expression of SLNCR. FIG. 12E shows Heat map of differentially expressed genes upon knockdown of SLNCR in the MSTC WM1976. The shading represents the log2 fold change compared to scramble siRNA control. Genes are clustered with Euclidean distance and average linkage clustering. FIG. 12F shows Gene Ontology biological process enriched in differential expressed genes represented in (FIG. 12E). FIG. 12G shows that the indicated MSTCs were seeded in 96- well plates, transfected with the indicated siRNAs and cell proliferation was quantified at the indicated time points using WST-1 proliferation reagent. Each assay was repeated 2-3 times, and shown is one representative assay. Error bars represent the mean ± SD of 3 technical replicates. Significance was calculated using the two-way analysis variance (ANOVA), with the Dunnett test for multiple comparison testing. * p-value < 0.05, ** p- value < 0.005, *** p-value < 0.0005, **** p < 0.0001. FIG. 12H shows the Tukey box plots of SLNCR expression in TCGA melanomas (n=172) exhibiting low (<1) or high (>1) primary mitotic growth rates. Significance was calculated using a Mann-Whitney test: * p < 0.05. FIG. 12I-12L show that SLNCR J, but not SLNCR2 nor SLNCR3, increase melanoma invasion. FIG. 121 shows schematic of the exons (numbered) of the 3 SLNCR isoforms previously identified in melanomas (not drawn to scale). Denoted is the sequence required for AR binding, and the regions targeted by the siRNAs used in this study. FIG. 12J shows quantification from 3 independent replicates, represented as mean ± SD, of matrigel invasion assays of A375 cells transfected with the indicated empty or SLNCR- expressing plasmid. Invasion is calculated as the percent of invading cells compared to mobile cells as counted in 8 fields of view. FIG. 12K shows relative MMP9 (left) or SLNCR (right) expression in A375 cells transfected with the indicated empty or SLNCR- expressing plasmid. RT-qPCR data is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the two-tailed Student's t-test: ** p < 0.005. FIG. 12L shows RNA immunoprecipitations from HEK293T cells transfected with GFP- tagged AR, using a-AR antibody or a matched IgG nonspecific control. Left panel shows the western blot analysis of input or bound proteins following IP with either IgG or a-AR (AR) antibodies. Right panel shows the relative enrichment of the indicated transcript measured via RT-qPCR compared to IgG nonspecific control.

FIG. 13A - FIG. 13F. FIG. 13 A shows box plot of SLNCR expression from pre- and post-metastatic melanomas. Data are represented as mean + SEM. Significance was calculated using the two-tailed Student's t-test: n.s. = not significance. FIG. 13B shows box plot of relative AR protein expression, determined via reverse phase protein array (RPPA), in male and female TCGA melanomas. Data are represented as mean ± SEM. Significance was calculated using the two-tailed Student's t-test: * p-value < 0.05. FIG. 13C-13F show that siRNA-mediated knockdown of SLNCR does not affect melanoma apoptosis. FIG. 13C shows relative SLNCR expression in the indicated cells transfected with either scramble or -SZNCR-targeting siRNAs. RT-qPCR data is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the two-tailed Student's t- test: * p-value < 0.05, ** p-value < 0.005, *** p-value < 0.0005, n.s. = not significance. FIG. 13D shows the populations of apoptotic cells in A375, WM1976, and WM858 cells transfected with either scramble or siRNAs by FACS. Specifically, FIG. 13D shows representative scatter plots of annexin V and 7-AAD staining, as measured via fluorescence-activated cell sorting (FACS) analysis, of the indicated melanoma cells transfected with the indicated siRNAs. Cells are classified as "viable" (Q4, bottom left), "apoptotic" (Ql, top left), or "necrotic" (Q4, top right). FIG. 13E shows the Scatter plot of the log2 fold change of genes significantly dysregulated (p < 0.01) upon knockdown of both SLNCR1 and SLNCR (all isoforms). Genes that are dysregulated in an opposing manner by SLNCR1 versus SLNCR are labeled and denoted in red. Pearson's correlation (r) was calculated using GraphPad Prism. FIG. 13F shows the scatter plot of primary melanoma mitotic growth rate versus Log2 SLNCR expression for 172 melanomas from the TCGA (mitotic growth rate was available for only 172 melanomas).

FIG. 14A - FIG. 14B show that SLNCR and AR cooperatively regulate melanoma proliferation in a hormone-independent manner. The indicated cells were either transfected with the indicated siRNAs (FIG. 14 A) or FANA-modified oligos were added to cell media (FIG. 14B) 24 hours after cells were seeded in 96-well plates. Cell proliferation was quantified using WST-1 reagent, as in FIG. 12B. Error bars represent the mean ± SD of 3 technical replicates. Significance was calculated using the two-way analysis of variance (ANOVA), with the Dunnett test for multiple comparison testing, n.s. = not significant, **** ₌ p < o.oooi .

FIG. 15A - FIG. 15D show that AR increases melanoma cell proliferation. FIG. 15 A shows that vehicle-control or the indicated concentration of flutamide was added twenty-four hours after the indicated cells were seeded in 96-well plates. Cell proliferation was quantified using WST-1 reagent, as in FIG. 12B. FIG. 15B shows relative AR expression in the indicated cells transfected with either scramble or _^ SZNCR-targeting siRNAs. FIG. 15C shows western blot of A375 cell lysates following transfection with the indicated siRNAs. Left panel: representative blot probed with a-AR and a-GAPDH antibodies. Right panel: quantification from three independent replicates, normalized to GAPDH. FIG. 15 D shows relative SLNCR expression in the indicated cells following addition of the indicated FANA-modified oligos. RT-qPCR data ((FIG. 15 A) and (FIG. 15C)) is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions.

Significance was calculated using the Student's t-test: * p-value < 0.05, ** p-value < 0.005, *** p-value < 0.0005.

FIG. 16A - FIG. 16C show that AR is bound at the promoters of a number of -SZNCR-regulated genes. Left panel of FIG. 16A shows Venn diagram representing the number of active genes (i.e. AR bound genes) in A375 cells transfected with either an empty or ,SZNCR-expressing plasmid. Right panel of FIG. 16A shows plot of tag densities for vector or ,SZNCR7-expressing cells. FIG. 16B shows Venn diagram representing AR active genes (as determined via AR ChlP-Seq of either vector or ,SZNCR-expressing cells), SLNCR differentially expressed (DE) genes (as determined via RNA-seq), and the genes that are both AR-bound and -SZNCR-regulated. FIG. 16C shows the Integrated Genome Viewer plots displaying AR ChlP-seq read intensities from vector control (light blue, top track), ,SZNCR7-expressing cells (dark blue, middle track) or input control (grey, bottom track) corresponding to the indicated genomic loci. Numbers on the top left indicate plot height for tracks (significant enrichment, Binomial Test p<0.0001).

FIG. 17A - FIG. 17C show that SLNCR and AR regulate many overlapping genes, including genes implicated in melanoma proliferation. FIG. 17A shows relative expression of the indicated genes from indicated cells transfected with either scramble, SLNCR-, or AR-targeting siRNAs. RT-qPCR data is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the Student's t-test: * p- value < 0.05, ** p-value < 0.005, *** p-value < 0.0005. FIG. 17B shows scatterplots of SLNCR expression and expression of the indicated genes from TCGA patient melanomas (n = 150). Nonparametric two-tailed Spearman correlation coefficients and significances are shown. FIG. 17C shows heat map of -SZNCR-regulated genes (as in FIG. 12A) that are also AR-bound. The shading represents the log2 fold change compared to scramble siRNA control. Genes are clustered with Euclidean distance and average linkage clustering. Red arrows depict genes that are implicated in cell proliferation.

FIG. 18A - FIG. 18H show that SLNCR and AR inhibit expression of

CDKNJA/p2\ independently of p53. FIG. 18A and 18B show that the knockdown of SLNCR or AR increase CDKNIA levels. FIG. 18 A, 18B and 18E show relative expression of the indicated genes from indicated cells transfected with either scramble, SLNCR-, or AR-targeting siRNAs. RNA was isolated 72 hours post transfection with 10 nM of the indicated siRNAs. RT-qPCR data is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the Student's t-test: * p-value < 0.05, ** p- value < 0.005, *** p-value < 0.0005. FIG. 18C, 18D and 18F show in western blot data that knockdown of SLNCR or AR increase p21 protein levels independently of p53. 72 hours post-transfection of the indicated cells with 10 nM of the indicated siRNAs, lysate was prepared using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific), according to manufacturer's instructions. Samples were separated on BioRad Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels and transferred to LF-PVDF using the mixed MW protocol on the BioRad Transblot Turbo. The following antibodies were used: Cell Signaling P21 Wafl/Cipl (12D1) at 1 : 1000, Cell Signaling GAPDH (14C10) at 1 : 5000, LICOR secondary at 1 : 10,000. Protein normalization is represented as a fold change of p21 levels, normalized to GAPDH levels, and was performed using ImageJ software. Bars represent mean ±SD from 3 independent biological replicates. FIG. 18E shows that AR and SLNCR inhibit CDKNIA expression independently of p53. Relative expression of the indicated transcripts 72 hours post-transfection of the p53 -deficient SK- MEL-28 melanoma cell line with 10 nM of either scramble or SLNCR or AR targeting siRNAs, as in FIG. 18A and 18B. FIG. 18F shows that AR and SLNCR inhibit p21 expression independently of p53. Same as in FIG. 18C and 18D, using p53 -deficient SK- MEL-28 melanoma cell line. Significance was calculated using the Student's t-test: * p < 0.05, ** p < 0.005, *** p < 0.0005. FIG. 18G shows knockdown of CDKNIA in A375 cells. Left panel shows relative CDKNIA expression following transfection of the indicated siRNAs, represented as the fold change compared to scramble control, normalized to GAPDH. Middle panel shows western blot of A375 cell lysates following transfection with the indicated siRNAs probed with a-p21 and a-GAPDH antibodies. Right panel shows quantification from three independent replicates, normalized to GAPDH. FIG. 18H shows cell proliferation quantified using WST-1 reagent following transfection with the indicated siRNAs. Significance was calculated using the two-way analysis of variance (ANOVA), with the Dunnett test for multiple comparison testing, **** p < 0.0001. RT-qPCR data are represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the Student's t-test: * p < 0.05, ** p < 0.005, *** p < 0.0005.

FIG. 19A - FIG. 19D show SLNCR and AR do not directly regulate expression of TP53. FIG.19A and 19B show relative expression of TP 53 from indicated cells transfected with either scramble, SLNCR-, or AR-targeting siRNAs. RNA was isolated 72 hours post transfection with 10 nM of the indicated siRNAs. RT-qPCR data is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the Student's t-test and was determined to not be significant: no indication indicates difference was not significant, ** p-value < 0.005. FIG. 19C shows that knockdown of SLNCR or AR in A375 melanoma cells does not affect p53 expression. FIG. 19C shows western blots of the indicated cells transfected with the indicated siRNAs were performed as in FIG. 18C, 18D, and 18F. Santa Cruz p53 (DO-1) sc-126 was used at 1 :200. Protein levels were quantified using ImageJ, and are presented as a fold change of p21 levels, normalized to GAPDH levels. Bars represent mean ±SD from 3 independent biological replicates. FIG. 19D shows that knockdown of SLNCR does not affect AR expression. Relative expression of AR 72 hours post-transfection of the indicated cells with 10 nM of either scramble or SLNCR targeting siRNAs is shown. RT-qPCR data are represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the Student's t- test and is only indicated where determined to be significant: ** p < 0.005.

FIG. 20A - FIG. 20D. FIG. 20A shows that SLNCR regulates cell cycle

progression, and SLNCR knockdown induces G2 cell cycle arrest. Indicated cells were seeded in a 96-well plate, transfected with 10 nM of the indicated siRNAs, and incubated in a humidified chamber for 72 hours. Cells were then fixed in cold 70% ethanol for 2 hours, incubate in LifeTech PI/RNaseA solution for 30 minutes at 37 °C, and analyzed on FACs machine Fortessa X-20. Cell populations were analyzed using FlowJo software, and significance was calculated using GraphPad Prism software. Top panel: Cell populations of one representative analysis in WM858 cells. Bottom panel: Bars represent the average percent of total cells in the indicated stage of the cell cycle, and error bars represent SD from 3 independent replicates. Significance was calculated using the two-tailed Student's t- test via GraphPad Prism, n.s. = not significant, * p-value < 0.05. FIG. 20B and FIG. 20C show that SLNCR regulates activity of multiple transcription factors. For FIG. 20B, FWM1976 cells were seeded in 6-well dishes, transfected with either scramble or si-SLNCR (1) siRNAs, and 48 or 72 hours post transfections cells were fractionated using NE-PER fractionation kit. Approximately 10 μg of the nuclear fraction for input directly in

Signosis' Transcription Factor Activation Array I. The ratio of relative luminescence units (RLUs) corresponding to the indicated transcription factor signals of si-SLNCR (1) versus scramble control. Bars represent mean ± SD from 2 independent biological replicates. FIG. 20C shows that transcription factor network enriched among genes that are

significantly dysregulated upon SLNCR knockdown in WM1976, that are not bound by AR as determined via AR ChlP-seq. Analysis was performed by MetaCore (Thompson

Reuters), against a set of genes normally expressed in human tissue. FIG. 20D shows the transcription factor activity in WM1976 nuclear lysate following transfection with either scramble or si-SLNCR (1) siRNA measured by Signosis' Transcription Factor Activation Array I. The raw relative luminescence units (RLUs) are shown for all transcription factor probes included in the array.

FIG. 21A - FIG. 21B show that SLNCR does not regulate cell cycle in A375 cells, and AR does not regulate cell cycle progression in multiple melanoma cells. Cell cycle analysis of the indicated cells transfected with 10 nM of the indicated siRNAs was performed as done in FIG. 20A.

FIG. 22A - FIG. 22H show that AR-bound, S NCR-regulated genes contain an enriched motif with high similarity to the -SZNCR-bound transcription factor EGRl, and that co-binding of -SZNCR-associated transcription factors EGRl and AR is enriched on SJNCR-regulated genes. FIG. 22A shows that TOMTOM analysis of AR ChlP-seq peaks found within 10,000 basepairs of -SZNCR-regulated genes (as determined via RNA-seq) identified a significant enrichment of a motif (bottom) resembling the EGRl DNA binding motif (top) (p-value 2.24e-05). FIG. 22B shows that that EGRl is significantly enriched in SLNCR1 immunoprecipitate. FIG. 22B shows western blot analysis of total A375 lysate ('Input') or immunoprecipitate enriched following incubation with in vitro transcribed, biotinylated full-length SLNCR1 ('Bound'). AR and the S6 Ribosomal Protein serve as positive controls, while GAPDH serves as a negative control. Western bot was performed as in FIG. 18C, 18D, and 18F. Cell Signaling Egrl (44D5) was used at 1 : 1000, and Cell Signaling S6 Ribosomal Protein (5G10) was used at 1 : 1000. For REMSA results shown in FIG. 22C shows that EGRl directly binds to SLNCR1. FIG. 22C, in vitro transcribed, biotinylated full length SLNCR1 was incubated with the indicated concentration of recombinant EGRl corresponding to amino acids 282-433 (Aviva Systems Biology, catalogue number OPCD02876). Where indicated, 10 μΜ of unlabeled RNA competitor corresponding to full length SLNCR1 was added prior to addition of biotinylated SLNCR1. REMSAs were completed as in FIG. 1A-FIG. 1C. For FIG. 22D, TOMTOM analysis identified a significant enrichment of a motif in EGRl ChlP-seq peaks (bottom) showing significant similarity to the consensus EGRl DNA binding motif (top panel). FIG. 22E is a venn diagram representating the genes significantly differentially expressed upon SLNCR knockdown (SLNCR DE genes or SLNCR DEGs) in WM1976 cells (pink), and genes bound by either AR (blue) or EGRl (green) within 10,000 bp of the annotated gene in A375 cells (Fisher Exact p<0.0001). FIG. 22F shows RNA immunoprecipitations from A375 cells using a-EGRl antibody or a matched IgG nonspecific control. Left panel shows western blot analysis of input and either bound or flow-through (F.T.) samples following immunoprecipitation with the indicated antibody. Right panel shows relative enrichment of SLNCR measured via RT-qPCR compared to input after normalization to the indicated transcript. FIG. 22G shows the Integrated Genome Viewer plot displaying AR (blue) and EGRl (green) ChlP-seq read intensities for the indicated transcripts. AR ChlP-seq reads are from the sample ectopically expressing SLNCRl. Numbers on the top left indicate plot height for each track. FIG. 22H shows the Integrated Genome Viewer plot displaying EGRl ChlP-seq read intensities for the indicated transcripts. Numbers on the left indicate plot height.

FIG. 23A - FIG. 23C show that the majority of EGRl bound genes are also bound by AR, and that AR and EGRl cobind EGRl consensus DNA motifs in melanoma cells. FIG. 23 A shows that the REST motif (top) is significantly enriched in AR ChlP-seq peaks both in the presence (SLNCRl) or absence (vector) of ectopic SLNCRl in A375 cells, as determined via TOMTOM analysis. The Venn diagram in FIG. 23B represents the degree of overlap between total sequences bound by AR or EGRl (active regions) as determined via ChlP-seq in A375 cells. Specifically, the Venn diagram represents the total regions bound by either AR in A375 cells transfected with vector (light blue) or SLNCR1- expressing plasmid (blue), or bound by EGRl (green). AR ChlP-seq was performed both in the presence (SLNCRl) or absence (vector) of ectopic SLNCRl. FIG. 23C is the same as in FIG. 23B, but combining all AR bound regions from both ChlP-seq experiments (vector and SLNCRl), and limiting the analysis to only DNA sequences found within 10,000 bp of an annotated gene. Specifically, the Venn diagram represents the genes bound by either AR (blue) or EGRl (green) within 10,000 bp of an annotated gene in A375 cells.

FIG. 24 A - FIG. 24D show that EGRl increases expression of p21, and the EGRl DNA binding site is required for AR and -SZNCR-mediated repression of p21. FIG. 24A- 24C show that knockdown of EGRl decreases CDKN1A levels independent of p53. FIG. 24A and 24B show that relative expression of EGRl or CDKN1A from indicated cells transfected with either scramble, EGRl -targeting siRNAs. RNA was isolated 72 hours post transfection with 10 nM of the indicated siRNAs. RT-qPCR data is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Significance was calculated using the

Student's t-test: * p-value < 0.05, ** p-value < 0.005 and *** p-value < 0.0005. FIG. 24C shows western blots of the indicated cells transfected with the indicated siRNAs were performed as in FIG. 18C, 18D, and 18F. Specifically, the left panel shows the representative western blot analysis of A375 (top) or SK-MEL-28 (bottom) cell lysates probed for EGRl, GAPDH, or p21 levels. Middle and right panels show the protein levels that were quantified using ImageJ, and are presented as relative expression of the indicated protein, normalized to GAPDH levels. Bars represent mean ±SD from 3 independent biological replicates. FIG. 24D shows that the mutation of the EGRl DNA binding site negates AR- and ^NCR-mediated CDKNIA regulation. FIG. 24D shows the schematic of the promoter region of CDKNIA cloned upstream of a luciferase reporter. The wild-type EGRl binding was mutated as shown below, and both the wild-type and mutant sequences of EGR/AR binding site are shown below, with mutated bases in red. Mutation of the EGRl binding site prevents AR- and ,SZNCR-mediated regulation of p21. A375 cells were transfected with a p21 -firefly (FL) reporter plasmid, a CMV-RL (renilla luciferase) control, and the indicated siRNAs. Specifically, A375 cells were transfected with the indicated siRNAs, and 24 hours later were subsequently transfected with the wildtype (top panel) or mutant (bottom panel) CDKNIA firefly (FL) reporter plasmid and a CMV-RL (renilla luciferase) control. Luciferase activity was measured 24 hours post-transfection. Relative FL activity was calculated as a fold-change compared to vector and scramble only control cells, after normalization to RL activity. Shown is one representative assay from at least fourindependent biological replicates. Error bars represent standard deviation from three technical replicates. Significance was calculated using the Student's t-test: * p-value < 0.05, ** p-value < 0.005, *** p-value < 0.0005, ns = not significant.

FIG. 25A - FIG. 25E show that AR and -SZNCR-mediated regulation of p21 does not require p53 or androgens, and AR and p21 show gender-specific expression. FIG. 25 A shows that EGRl does not regulate TP 53 expression. Knockdown of EGRl does not affect TP53 levels. Relative expression of TP53 72 hours post-transfection of either A375 (left) or SK-MEL-28 (right) cells with 10 nM of either scramble or EGRl -targeting siRNAs. RT- qPCR of the indicated cells transfected with scramble or EGRl targeting siRNAs. RNA was isolated 72 hours post transfection with 20 nM of the indicated siRNAs. RT-qPCR data is represented as the fold change compared to scramble control, normalized to

GAPDH. Error bars represent standard deviations calculated from 3 reactions.

Significance was calculated using the Student's t-test and was determined to not be significant. FIG. 25B and 25C present results from luciferase assays, as in FIG. 24D. FIG. 25B shows that AR and ,SZNCR-mediated regulation of the CDKNIA does not require the presence of androgens (i.e. effects are observed even when cells are hormone-derpived: grown in phenol-red free media containing charcoal stripped FBS). Same as in FIG. 22C, using A375 cells grown in hormone-starved conditions. Significance was calculated using the Student's t-test: * p < 0.05, ** p < 0.005, *** p < 0.0005, ns = not significant. FIG. 25C are one representative assay where knockdown of SLNCR or AR increases expression from the CDKN1A promoter (replicating results seen at the endogenous promoter). The discrepancy seen in these assays (i.e. either increased expression, seen here, or decreased expression, seen in FIG. 24D) likely result from altered stoichiometry of the transcription factors, IncRNA, and DNA due to variations in transfection between individual

experiments. FIG. 25D showst hat female p-53 deficient melanomas express significantly higher levels of p21 than males. P21 protein expression was taken from The Cancer Genome Atlas (TCGA), from only p-53 deficient (i.e. p53 mutant) melanomas as determined via RNA-seq. Significance was calculated using Student's t-test. FIG. 25E shows gender specific expression of AR and p21. Box plot of relative p21 (left) and AR (right) expression, but for only p53-deficient melanomas, is shown. Significance was calculated using the Student's t-test: * p < 0.05.

FIG. 26. shows a model for AR- and -SZNCR-mediated, EGR1 -dependent but p53- independent regulation of p21. In the absence of either SLNCR (top panel) or AR (bottom panel), EGR1 binds to its cognate DNA binding site in the CDKN1A promoter and increases expression. Once SLNCR and AR levels exceed the required threshold, SLNCR recruits AR to EGR1 -bound chromatin to repress gene expression.

For any figure showing a bar histogram, curve, or other data associated with a legend, the bars, curve, or other data presented from left to right for each indication correspond directly and in order to the boxes from top to bottom of the legend. Detailed Description of the present invention

The present invention is based, at least in part, on the discovery that the N-terminal, regulatory domain of AR interacts with single-stranded, pyrimidine rich RNA in a sequence-specific manner, and that single-stranded nucleic acids designed to sterically block the interaction of AR and such RNA without significantly altering the expression of AR and/or RNA (e.g., 2'-deoxy-2'-fluoro-D-arabinonuleic acid (2'-FANA)-modified single-stranded nucleic acids that bind AR or SLNCR to modulate SLNCR-mediated melanoma invasion). I. Definitions

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

Unless otherwise specified herein, the terms "antibody" and "antibodies" broadly encompass naturally-occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

The term "antibody" as used herein also includes an "antigen-binding portion" of an antibody (or simply "antibody portion"). The term "antigen-binding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 : 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab , Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also

encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov, S.M., et al. (1994) Mo/. Immunol. 31 : 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof {e.g., humanized, chimeric, etc.). Antibodies may also be fully human. The terms "monoclonal antibodies" and "monoclonal antibody composition", as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term "polyclonal antibodies" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it

immunoreacts.

The term "antisense" nucleic acid polypeptide comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA polypeptide, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid polypeptide can hydrogen bond to a sense nucleic acid polypeptide.

The term "body fluid" refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, peritoneal fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In a preferred embodiment, body fluids are restricted to blood-related fluids, including whole blood, serum, plasma, and the like.

The terms "cancer" or "tumor" or "hyperproliferative disorder" refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer is generally associated with uncontrolled cell growth, invasion of such cells to adjacent tissues, and the spread of such cells to other organs of the body by vascular and lymphatic menas. Cancer invasion occurs when cancer cells intrude on and cross the normal boundaries of adjacent tissue, which can be measured by assaying cancer cell migration, enzymatic destruction of basement membranes by cancer cells, and the like. In some embodiments, a particular stage of cancer is relevant and such stages can include the time period before and/or after angiogenesis, cellular invasion, and/or metastasis. Cancer cells are often in the form of a solid tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,

leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,

craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,

promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the method of the present invention is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small- cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, brenner, or undifferentiated. In some embodiments, the present invention is used in the treatment, diagnosis, and/or prognosis melanoma and its subtypes.

The term "classifying" includes "to associate" or "to categorize" a sample with a disease state. In certain instances, "classifying" is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods and systems of classifying use of a so-called training set of samples having known disease states. Once established, the training data set serves as a basis, model, or template against which the features of an unknown sample are compared, in order to classify the unknown disease state of the sample. In certain instances, classifying the sample is akin to diagnosing the disease state of the sample. In certain other instances, classifying the sample is akin to differentiating the disease state of the sample from another disease state.

The term "coding region" refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term "noncoding region" refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).

The term "complementary" refers to the broad concept of sequence

complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

The term "control" refers to any reference standard suitable to provide a comparison to the expression products in the test sample. In one embodiment, the control comprises obtaining a "control sample" from which expression product levels are detected and compared to the expression product levels from the test sample. Such a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository. In another preferred embodiment, the control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment. It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention. In one embodiment, the control may comprise normal or non-cancerous cell/tissue sample. In another preferred embodiment, the control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In the former case, the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level. In another preferred embodiment, the control may comprise normal cells, cells from patients treated with combination chemotherapy and cells from patients having benign cancer. In another embodiment, the control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population. Such a population may comprise normal subjects, cancer patients who have not undergone any treatment (i.e., treatment naive), cancer patients undergoing therapy, or patients having benign cancer. In another preferred embodiment, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control. In particularly preferred embodiments, the control comprises a control sample which is the same lineage and/or type as the test sample. In another embodiment, the control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In one embodiment a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome. In another preferred embodiment, a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome. As demonstrated by the data below, the methods of the present invention are not limited to use of a specific cut-point in comparing the level of expression product in the test sample to the control.

As used herein, the term "costimulate" with reference to activated immune cells includes the ability of a costimulatory molecule to provide a second, non-activating receptor mediated signal (a "costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion, e.g., in a T cell that has received a T cell-receptor-mediated signal. Immune cells that have received a cell-receptor mediated signal, e.g., via an activating receptor are referred to herein as "activated immune cells."

The term "diagnosing cancer" includes the use of the methods, systems, and code of the present invention to determine the presence or absence of a cancer or subtype thereof in an individual. The term also includes methods, systems, and code for assessing the level of disease activity in an individual. Diagnosis can be performed directly by the agent providing therapeutic treatment. Alternatively, a person providing therapeutic agent can request the diagnostic assay to be performed. The diagnostician and/or the therapeutic interventionist can interpret the diagnostic assay results to determine a therapeutic strategy. Similarly, such alternative processes can apply to other assays, such as prognostic assays.

A molecule is "fixed" or "affixed" to a substrate if it is covalently or non-covalently associated with the substrate such the substrate can be rinsed with a fluid (e.g., standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate. The term "gene expression data" or "gene expression level" as used herein refers to information regarding the relative or absolute level of expression of a gene or set of genes in a cell or group of cells. The level of expression of a gene may be determined based on the level of RNA, such as mRNA, encoded by the gene. Alternatively, the level of expression may be determined based on the level of a polypeptide or fragment thereof encoded by the gene. Gene expression data may be acquired for an individual cell, or for a group of cells such as a tumor or biopsy sample. Gene expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in conjunction with a microarray or chip reading device. Such gene expression data can be manipulated to generate gene expression signatures.

The term "gene expression signature" or "signature" as used herein refers to a group of coordinately expressed genes. The genes making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response. The genes can reflect biological aspects of the tumors in which they are expressed, such as the cell of origin of the cancer, the nature of the non-malignant cells in the biopsy, and the oncogenic mechanisms responsible for the cancer.

The term "homologous" or "homology" as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-

TATGGC-3' share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

The term "host cell" is intended to refer to a cell into which a nucleic acid of the present invention, such as a recombinant expression vector of the present invention, has been introduced. The terms "host cell" and "recombinant host cell" are used

interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The term "humanized antibody," as used herein, is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell, for example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.

The term "humanized antibody", as used herein, also includes antibodies in which

CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

As used herein, the term "immune cell" refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, the term "immune response" includes T cell mediated and/or B cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.

The term "immunotherapeutic agent" can include any molecule, peptide, antibody or other agent which can stimulate a host immune system to generate an immune response to a tumor or cancer in the subject. Various immunotherapeutic agents are useful in the compositions and methods described herein. Numerous anti-cancer agents in the immunotherapeutic agent class are well-known in the art and include, without limitation, antibodies that block or inhibit the function of PD-1, PD-L1, PD-L2, CTLA4, and the like. As used herein, the term "inhibit" includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. For example, cancer is "inhibited" if at least one symptom of the cancer, such as hyperproliferative growth, is alleviated, terminated, slowed, or prevented. As used herein, cancer is also "inhibited" if recurrence or metastasis the cancer is reduced, slowed, delayed, or prevented.

As used herein, the term "inhibitory signal" refers to a signal transmitted via an inhibitory receptor (e.g., CTLA-4 or PD-1) for a polypeptide on an immune cell. Such a signal antagonizes a signal via an activating receptor (e.g., via a TCR, CD3, BCR, or Fc polypeptide) and can result in, e.g., inhibition of second messenger generation; an inhibition of proliferation; an inhibition of effector function in the immune cell, e.g., reduced phagocytosis, reduced antibody production, reduced cellular cytotoxicity, the failure of the immune cell to produce mediators, (such as cytokines (e.g., IL-2) and/or mediators of allergic responses); or the development of anergy.

As used herein, the term "interaction," when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with one another. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules. The activity may be a direct activity of one or both of the molecules. Alternatively, one or both molecules in the interaction may be prevented from binding their ligand, and thus be held inactive with respect to ligand binding activity (e.g., binding its ligand and triggering or inhibiting an immune response). To inhibit such an interaction results in the disruption of the activity of one or more molecules involved in the interaction. To enhance such an interaction is to prolong or increase the likelihood of said physical contact, and prolong or increase the likelihood of said activity.

An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

As used herein, an "isolated protein" refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations, in which compositions of the present invention are separated from cellular components of the cells from which they are isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of having less than about 30%, 20%, 10%, or 5% (by dry weight) of cellular material. When an antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

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 present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.

The term "melanoma" generally refers to cancers derived from melanocytes.

Although melanocytes are predominantly located in skin, they are also found in other parts of the body, including the eye and bowel. Although cutaneous melanoma is most common, melanoma can originate from any melanocyte in the body. Though melanoma is less than five percent of the skin cancers, it is the seventh most common malignancy in the U.S. and is responsible for most of the skin cancer related deaths. The incidence has increased dramatically in the last several decades due to altered sun exposure habits of the population. Several hereditary risk factors are also known. Other important risk factors are the number of pigment nevi, the number dysplastic nevi, and skin type. An increased risk is coupled to many nevi, both benign and dysplastic, and fair skin. Familial history of malignant melanomas is a risk factor, and approximately 8-12% of malignant melanoma cases are familial. Additional details are well-known, such as described in US Pat. Pubis. 2012- 0269764 and 2013-0237445.

Malignant melanomas are clinically recognized based on the ABCD(E) system, where A stands for asymmetry, B for border irregularity, C for color variation, D for diameter >5 mm, and E for evolving. Further, an excision biopsy can be performed in order to corroborate a diagnosis using microscopic evaluation. Infiltrative malignant melanoma is traditionally divided into four principal histopathological subgroups: superficial spreading melanoma (SSM), nodular malignant melanoma (NMM), lentigo maligna melanoma (LMM), and acral lentiginous melanoma (ALM). Other rare types also exists, such as desmoplastic malignant melanoma. A substantial subset of malignant melanomas appear to arise from melanocytic nevi and features of dysplastic nevi are often found in the vicinity of infiltrative melanomas. Melanoma is thought to arise through stages of progression from normal melanocytes or nevus cells through a dysplastic nevus stage and further to an in situ stage before becoming invasive. Some of the subtypes evolve through different phases of tumor progression, which are called radial growth phase (RGP) and vertical growth phase (VGP).

Malignant melanomas are staged according to the American Joint Committee on Cancer (AJCC) TNM-classification system, where Clark level is considered in 'reclassification. The T stage describes the local extent of the primary tumor, i.e., how far the tumor has invaded and imposed growth into surrounding tissues, whereas the N stage and M stage describe how the tumor has developed metastases, with the N stage describing spread of tumor to lymph nodes and the M stage describing growth of tumor in other distant organs. Early stages include: TO-1, NO, MO, representing localized tumors with negative lymph nodes. More advanced stages include: T2-4, NO, MO, localized tumors with more widespread growth and Tl-4, Nl-3, MO, tumors that have metastasized to lymph nodes and Tl-4, Nl-3, Ml, tumors with a metastasis detected in a distant organ.

Stages I and II represent no metastatic disease and for stage I (Tla/b-2a,N0,M0) prognosis is very good. The 5-year survival for stage I disease is 90-95%, for stage II (T2b- 4-b,N0,M0) the corresponding survival rate ranges from 80 to 45%. Stages III (Tla-4- b,Nla-3,M0) and IV (T(aII),N(aII),Mla-c) represent spread disease, and for these stages 5- year survival rates range from 70 to 24%, and from 19 to 7%, respectively. "Clark's level" is a measure of the layers of skin involved in a melanoma and is a melanoma prognostic factor. For example, level I involves the epidermis. Level II involves the epidermis and upper dermis. Level III involves the epidermis, upper dermis, and lower dermis. Level IV involves the epidermis, upper dermis, lower dermis, and subcutis. When the primary tumor has a thickness of >1 mm, ulceration, or Clark level IV- V, sentinel node biopsy (SNB) is typically performed. SNB is performed by identifying the first draining lymph node/s {i.e., the SN) from the tumour. This is normally done by injection of radiolabelled colloid particles in the area around the tumour, followed by injection of Vital Blue dye. Rather than dissection of all regional lymph nodes, which was the earlier standard procedure, only the sentinel nodes are generally removed and carefully examined. Following complete lymph node dissection is only performed in confirmed positive cases.

In addition to staging and diagnosis, factors like T-stage, Clark level, SNB status, Breslow's depth, ulceration, and the like can be used as endpoints and/or surrogates for analyses according to the present invention. For example, patients who are diagnosed at an advanced stage with metastases generally have a poor prognosis. For patients diagnosed with a localized disease, the thickness of the tumor measured in mm (Breslow) and ulceration can be endpoints for prognosis. Breslow's depth is determined by using an ocular micrometer at a right angle to the skin. The depth from the granular layer of the epidermis to the deepest point of invasion to which tumor cells have invaded the skin is directly measured. Clark level is important for thin lesions (<1 mm). Other prognostic factors include age, anatomic site of the primary tumor and gender. The sentinel node (SN) status can also be a prognostic factor, especially since the 5-year survival of SN-negative patients has been shown to be as high as 90%. Similarly, overall survival (OS) can be used as a standard primary endpoint. OS takes in to account time to death, irrespective of cause, e.g. if the death is due to cancer or not. Loss to follow-up is censored and regional recurrence, distant metastases, second primary malignant melanomas and second other primary cancers are ignored. Other surrogate endpoints for survival can be used, as described further herein, such as disease-free survival (DFS), which includes time to any event related to the same cancer, i.e. all cancer recurrences and deaths from the same cancer are events.

In addition to endpoints, certain diagnostic and prognostic markers can be analyzed in conjunction with the methods described herein. For example, lactate dehydrogenase (LDH) can be measured as a marker for disease progression. Patients with distant metastases and elevated LDH levels belong to stage IV Mlc. Another serum biomarker of interest is S100B. High S100B levels are associated with disease progression, and a decrease in the S100B level is an indicator of treatment response. Melanoma-inhibiting activity (MIA) is yet another serum biomarker that has been evaluated regarding its prognostic value. Studies have shown that elevated MIA levels are rare in stage I and II disease, whereas in stage III or IV, elevation in MIA levels can be seen in 60-100% of cases. Additional useful biomarkers include RGS 1 (associated with reduced relapse-free survival (RFS)), osteopontin (associated with both reduced RFS and disease-specific survival (DSS), and predictive of SLN metastases), HER3 (associated with reduced survival), and NCOA3 (associated with poor RFS and DSS, and predictive of SLN metastases). In addition, HMB-45, Ki-67 (MIB 1), MITF and MART -1 /Mel an- A or combinations of any described marker may be used for staining (Ivan & Prieto, 2010, Future Oncol. 6(7), 1163-1175; Linos et al, 2011, Biomarkers Med. 5(3) 333-360). In a literature review Rothberg et al. report that melanoma cell adhesion molecule

(MCAM)/MUC18, matrix metalloproteinase-2, Ki-67, proliferating cell nuclear antigen (PCNA) and pl6/INK4A are predictive of either all-cause mortality or melanoma specific mortality (Rothberg et al, 2009 J. Nat. Cane. Inst. 101(7) 452-474).

Currently, the typical primary treatment of malignant melanoma is radical surgery. Even though survival rates are high after excision of the primary tumour, melanomas tend to metastasize relatively early, and for patients with metastatic melanoma the prognosis is poor, with a 5-year survival rate of less than 10%. Radical removal of distant metastases with surgery can be an option and systemic chemotherapy can be applied, but response rates are normally low (in most cases less than 20%), and most treatment regiments fail to prolong overall survival. The first FDA-approved chemotherapeutic agent for treatment of metastatic melanoma was dacarbazine (DTIC), which can give response rates of

approximately 20%, but where less than 5% may be complete responses. Temozolamid is an analog of DTIC that has the advantage of oral administration, and which have been shown to give a similar response as DTIC. Other chemotherapeutic agents, for example different nitrosureas, cisplatin, carboplatin, and vinca alkaloids, have been used, but without any increase in response rates. Since chemotherapy is an inefficient treatment method, immunotherapy agents have also been proposed. Most studied are interferon-alpha and interleukin-2. As single agents they have not been shown to give a better response than conventional treatment, but in combination with chemotherapeutic agents higher response rates have been reported. For patients with resected stage IIB or III melanoma, some studies have shown that adjuvant interferon alfa has led to longer disease free survival. For first- or second-line stage III and IV melanoma systemic treatments include: carboplatin, cisplatin, dacarbazine, interferon alfa, high-dose interleukin-2, paclitaxel, temozolomide, vinblastine or combinations thereof (NCCN Guidelines, ME-D, MS-9-13). Recently, the FDA approved Zelboraf™ (vemurafenib, also known as INN, PLX4032, RG7204 or R05185426) for unresectable or metastatic melanoma with the BRAF V600E mutation (Bollag et al. (2010) Nature 467:596-599 and Chapman et al. (2011) New Eng. J. Med. 364:2507-2516). Another recently approved drug for unresectable or metastatic melanoma is Yervoy®(ipilimumab) an antibody which binds to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) (Hodi et al. (2010) New Eng. J. Med. 363 :711-723). Others recently reported that patients with KIT receptor activating mutations or over-expression responded to Gleevac® (imatinib mesylate) (Carvajal et al. (2011) JAMA 305:2327-2334). In addition, radiation treatment may be given as an adjuvant after removal of lymphatic metastases, but malignant melanomas are relatively radioresistant. Radiation treatment might also be used as palliative treatment. Melanoma oncologists have also noted that BRAF mutations are common in both primary and metastatic melanomas and that these mutations are reported to be present in 50-70% of all melanomas. This has led to an interest in B-raf inhibitors, such as sorafenib, as therapeutic agents.

The term "modulate" includes up-regulation and down-regulation, e.g., enhancing or inhibiting a response.

The "normal" or "control" level of expression of AR or IncRNA is the level of expression of the AR or IncRNA in cells of a subject, e.g., a human patient, not afflicted with an AR- and/or IncRNA-mediated disease. An "over-expression" or "significantly higher level of expression" of AR or IncRNA refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6. 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expression activity or level of the AR or IncRNA in a control sample {e.g., sample from a healthy subject not having the AR- and/or IncRNA-mediated disease) and preferably, the average expression level of AR or IncRNA in several control samples. A "significantly lower level of expression" of AR or IncRNA refers to an expression level in a test sample that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6. 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the expression level of the AR or IncRNA in a control sample {e.g., sample from a healthy subject not having the AR- and/or IncRNA-mediated disease) and preferably, the average expression level of the AR or IncRNA in several control samples.

The term "nuclear receptor target drugs" refers to a agents that inhibit the expression and/or activity of nuclear receptors involved in regulating gene expression of gene sets. Nuclear receptors target drugs are well-known in the art and include, without limitation, luteinizing hormone-releasing hormone (LHRH) analogs, androgen receptor inhibitors, anti-androgens, hormone blocking drugs, nuclear receptor agonists, nuclear receptor antagonists, selective receptor modulators, selective androgen receptor modulators (SARMs), selective estrogen receptor modulators (SERMs), selective progesterone receptor modulators (SPRMs), selective glucocorticoid receptor agonists (SEGRAs), and selective glucocorticoid receptor modulators (SEGRMs). Specific agents are also well-known in the art and include, without limitation, leuprolide (Lupron®, Eligard®), goserelin (Zoladex®), triptorelin (Trelstar®), histrelin (Vantas®), degarelix (Firmagon®), bicalutamide

(Casode®), enzalutamide (Xtandi®), flutamide (Eulexin®), nilutamide (Nilandron®), ketoconazole (Nizoral®), abiraterone (Zytiga®), dexamethasone, megestrol acetate

(Megace®), medroxyprogesterone acetate (MP A), ethisterone, norethindrone acetate, norethisterone, norethynodrel, ethynodiol diacetate, norethindrone, norgestimate, norgestrel, levonorgestrel, medroxyprogesterone acetate, desogestrel, etonogestrel, drospirenone, norelgestromin, desogestrel, etonogestrel, gestodene, dienogest,

drospirenone, elcometrine, nomegestrol acetate, trimegestone, tanaproget, BMS948, mifepristone, 4-hydroxytamoxifen, CF PAl, Cyproterone acetate (Androcur®, Cyprostat®, Siterone®), chlormadinone acetate (Clordion®, Gestafortin®, Lormin®, Non-Ovlon®, Normenon®, Verton®), 17-hydroxyprogesterone (17-OHP), THC, clotrimazole, PK11195 [l-(2-chlorophenyl)-N-methyl-N-(l-methylpropyl)-3-isoquinoli necarboxamide], meclizine, androstanol, CITCO [6-(4-chlorophenyl)imidazo [2,1-£][1,3] thiazole-5-carbaldehyde O- (3,4-dichlorobenzyl) oxime], zearalenone (ZEN), T0901317, S07662, enobosarm, BMS- 564,929, LGD-4033, AC-262,356, JNJ-28330835, LGD-2226, LGD-3303, S-40503, S-23, clomifene, femarelle, ormeloxifene, raloxifene, tamoxifen, toremifene, lasofoxifene, ospemifene, afimoxifene, arzoxifene, bazedoxifene, gulvestrant (Faslodex®, ICI- 182780), CDB-4124, asoprisnil, proellex, mapracorat (BOL-303242-X, ZK 245186), fosdagrocorat (PF-04171327), ZK 216348, and 55D1E1. Additional exemplary agents include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of nuclear receptor targets, or fragments thereof.

The term "pre-malignant lesions" as described herein refers to a lesion that, while not cancerous, has potential for becoming cancerous. It also includes the term "pre- malignant disorders" or "potentially malignant disorders." In particular this refers to a benign, morphologically and/or histologically altered tissue that has a greater than normal risk of malignant transformation, and a disease or a patient's habit that does not necessarily alter the clinical appearance of local tissue but is associated with a greater than normal risk of precancerous lesion or cancer development in that tissue (leukoplakia, erythroplakia, erytroleukoplakia lichen planus (lichenoid reaction) and any lesion or an area which histological examination showed atypia of cells or dysplasia.

The term "probe" refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

The term "prognosis" includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis cancer in an individual. For example, the prognosis can be surgery, development of a clinical subtype of melanoma, development of one or more clinical factors, development of intestinal cancer, or recovery from the disease. In some embodiments, the term "good prognosis" indicates that the expected or likely outcome after treatment of melanoma is good. The term "poor prognosis" indicates that the expected or likely outcome after treatment of melanoma is not good.

The term "resistance" refers to an acquired or natural resistance of a cancer sample or a mammal to a cancer therapy ( i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more. The reduction in response can be measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal who is known to have no resistance to the therapeutic treatment. A typical acquired resistance to chemotherapy is called "multidrug resistance." The multidrug resistance can be mediated by P-glycoprotein or can be mediated by other mechanisms, or it can occur when a mammal is infected with a multi-drug-resistant microorganism or a combination of microorganisms. The determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician, for example, can be measured by cell proliferative assays and cell death assays as described herein as "sensitizing." In some embodiments, the term "reverses resistance" means that the use of a second agent in combination with a primary cancer therapy (e.g., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e.g., p<0.05) when compared to tumor volume of untreated tumor in the circumstance where the primary cancer therapy (e.g., chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically.

The term "sensitize" means to alter cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancer with a cancer therapy (e.g., chemotherapeutic or radiation therapy. In some embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the cancer therapy (e.g., chemotherapy or radiation therapy). An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94: 161-173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69: 615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993 : 415-432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivity or resistance may also be measured in animal by measuring the tumor size reduction over a period of time, for example, 6 month for human and 4-6 weeks for mouse. A composition or a method sensitizes response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.

The term "SLNCR," unless otherwise specified, refers to the known SLNCR

(SLNCR1) IncRNA, as well as its isoforms, such as SLNCR2 (SLNCR4A) and SLNCR3 (SLNCR4B), and biologically active fragments thereof. SLNCR1 , also known as

LINC00673 in the art, is a 2,257 nucleotide sequence associated with Ref Seq Gene ID NR 036488.1 and Entrez Gene ID 100499467. It is located immediately downstream of another IncRNA known as LINC0051 1 on human chromosome 17. SLNCR2 (SLNCR4A) and SLNCR3 (SLNCR4B) are isoforms described herein. SLNCR and its isoforms act as a scaffold to bring together one or more transcription factors and associated co-activators and/or co-repressors for translocation to the nucleus and activation and/or repression of gene expression. In cancer cells, such as melanoma cells, SLNCR and its isoforms mediate one or more of the following functions: 1) expression or activity of MMP9; 2)

downregulation of naturally-occurring SLNCR isoforms; 3) modulation of the expression of one or more genes listed in Figures 7, 14, 16, 17, 19, and 31 ; 4) the expression of

PLA2G4C, CT45A6, EGR2, RP1 1 -820L6.1, EGR1, ATF3, VCX3A, SPCS2, FABP5, MAGEA2B, RPL41P1, RPS17, HNRNPAIPIO, TXNIP, RPL21P75, EIF3CL, RPL7, CT45A3, GTF2IP1, CDK7, HIST1H1C, CT45A1, BTG2, RPS27, RP1 1 -3P17.3, FDCSP, CITED4, IL34, and PD-L1 ; 5) cellular proliferation; 6) cell death; 7) cellular migration; 8) genomic replication; 9) angiogenesis induction; 10) cellular invasion; 1 1) cancer metastasis; 12) binding to one or more protein transcription factors (TF) selected from the group consisting of SRC-l/NCOA-1 (e.g., REF SEQ: NP_671766.1, NP_671756.1, and

NP_003734.3); PXR/NR1I2 (e.g., REF SEQ: NP_003880.3, NP_071285.1, and

NP_148934.1); PAX5 (e.g., REF SEQ: NP_001267476.1, NP_001267477.1,

NP_001267480.1, NP_001267482.1, NP_001267483.1, NP_001267484.1,

NP_001267485.1, NP_001267479.1, NP_001267478.1, NP_057953.1, and

NP OO 1267481.1); EGR-1 (e.g., REF SEQ: NP_001955.1); AR (e.g., REF SEQ:

NP_000035); E2F-1 (e.g., REF SEQ: NP_005216.1); CAR/NR1I3 (e.g., REF SEQ:

NP_001070950.1, NP_001070949.1, NP_001070949.1, NP_001070947.1,

NP_001070945.1, NP_001070946.1, NP_001070944.1, NP_001070942.1,

NP_001070941.1, NP_001070940.1, and NP_001070939.1); PBX1 (e.g., REF SEQ:

NP_001 191892.1, NP_001 191890.1, and NP_002576.1); ATF2 (e.g., REF SEQ:

NP_001243021.1, NP_001243019.1, NP_001871.2, NP_001243023.1, NP_001243022.1, and NP_001243020.1); C/EBP (e.g., REFSEQ: NP_001272758, NP_001272807,

NP_001239225, NP_005186, NP_005751, and NP_001796); BRN-3/POU4F 1 (e.g.,

REFSEQ: NP_006228.3); HNF4 (e.g., REFSEQ: NP_000448 and NP_004124); NF-kB (e.g., REFSEQ: NP_001 158884, NP_001 138610, NP_001070962, NP_006500, and NP_001278675); AP2 (e.g., REFSEQ: NP_001027451, NP_003212.2, NP_001025177.1, NP_001273.1, NP_003213.1, NP_758438.2, and NP_848643.2); OCT4/POU5F 1 (e.g., REFSEQ: NP_001272916.1, NP_001272915.1, NP_001 167002.1, NP_976034.4,

NP_002692.2), SP1(REFSEQ: NP_001238754.1, NP_003100.1, and NP_612482.2);

STAT 5 (e.g., REFSEQ: NP 001275649.1, NP_001275648.1, NP_001275647.1,

NP_003143.2, and NP_036580.2); p53 (e.g., REFSEQ: NP_001 1 19584.1, NP_000537.3, NP_001263626.1, NP_001263690.1, NP_001263689.1, NP_001 1 19590.1,

NP_001 1 19587.1, NP_001 1 19586.1, NP_001 1 19585.1, NP_001263628.1,

NP_001263627.1 , NP_001263625.1 , NP_001263624.1 , NP_001 1 19589.1 , and

NP_001 1 19588.1); TFIID (e.g., REFSEQ: NP OOl 165556.1, NP OO 1273003.1,

NP_003175.1, NP_1 14129.1, NP_003176.1, NP_001280654.1, NP_008882.1,

NP_001 177344.1, NP_005633.1, NP_612639.1, NP_001015892.1, NP_057059.1, NP_006275.1, NP_001257417.1, NP_001 128690.1, NP_005636.1, and NP_003478.1); SLIRP (e.g., REFSEQ: NP_1 12487.1, NP_001254792.1, NP_001254793.1); STAT3 (e.g., REFSEQ: NP_003141.2, NP_644805.1, NP_998827.1); REST (e.g., REFSEQ:

NP_005603.3, NP_001 180437.1, including isoforms of REST, such as REST4 (e.g.,

REFSEQ: AEJ31941.1 and UniProt: L0B3Z2, A0A087X1C2, L0B 1 S6, and

A0A087X1C2)); and DAX1 (e.g., REFSEQ: NP_000466.2), optionally wherein the SLNCR- TF complex can translocate to the nucleus; 13) regulation of immune response and/or immune evasion; and 14) modulation of one or more genes listed in Tables S5 and S6 affected by SLNCR overexpression. It has been determined herein that certain SLNCR structural features common or different among the SLNCR isoforms are related to SLNCR function. For example, a highly conserved approximately 301 nucleotide sequence common to the SLNCR 1 -3 isoforms (referred to as "SLNCR cons" herein) is sufficient for cancer cell invasion. Similarly, a portion or all of an approximately 1 1 1 nucleotide sequence common to the SLNCR 1-3 isoforms (referred to as "SLNCR delta cons" herein) is required for cancer cell invasion. The representative SLNCR sequences, as well as annotations of structural domains and biologically active fragments associated with SLNCR function are shown in WO 2017/007941. The term "synergistic effect" refers to the combined effect of two or more anticancer agents or chemotherapy drugs can be greater than the sum of the separate effects of the anticancer agents or chemotherapy drugs alone.

The term "subject" refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a condition of interest (e.g., cancer). The term "subject" is interchangeable with "patient." In some embodiments, a subject does not have any cancer other than melanoma. In other embodiments, the subject has melanoma but does not have one or more other cancers of interest. For example, in some embodiments, a subject does not have renal cell carcinoma, head or neck cancer, and/or lung cancer.

The language "substantially free of chemical precursors or other chemicals" includes preparations of antibody, polypeptide, peptide or fusion protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of antibody, polypeptide, peptide or fusion protein having less than about 30% (by dry weight) of chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, still more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, and most preferably less than about 5% chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals.

The term "substantially pure cell population" refers to a population of cells having a specified cell marker characteristic and differentiation potential that is at least about 50%, preferably at least about 75-80%, more preferably at least about 85-90%, and most preferably at least about 95% of the cells making up the total cell population. Thus, a "substantially pure cell population" refers to a population of cells that contain fewer than about 50%), preferably fewer than about 20-25%, more preferably fewer than about 10-15%, and most preferably fewer than about 5% of cells that do not display a specified marker characteristic and differentiation potential under designated assay conditions.

As used herein, the term "survival" includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence-free survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., an mRNA, hnRNA, cDNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a marker of the present invention and normal post-transcriptional processing (e.g., splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript. In some embodiments, transcribed polynucleotides are "long non-coding RNAs" or "lcRNAs" that are defined as transcribed polynucleotides that do not naturally encode a translated protein. lcRNAs are generally sequences longer than about 100 nucleotides and can be as long as up to tens of kilobases, although the length definition is a matter of convenience for distinguishing traditionally small nucleic acids like microRNAs, siRNAs, and piwi-associated RNAs. lcRNAs may be located separate from protein coding genes (long intergenic ncRNAs or lincRNAs), or reside near or within

protein coding genes (Guttman et al. (2009) Nature 458:223-227; Katayama et al. (2005) Science 309: 1564-1566; Kim et al. (2010) Nature 465: 182-187; De Santa et al. (2010) PLoSBiol. 8:el000384).

As used herein, the term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

As used herein, the term "unresponsiveness" includes refractivity of immune cells to stimulation, e.g., stimulation via an activating receptor or a cytokine. Unresponsiveness can occur, e.g., because of exposure to immunosuppressants or exposure to high doses of antigen. As used herein, the term "anergy" or "tolerance" includes refractivity to activating receptor-mediated stimulation. Such refractivity is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by lack of cytokine production, e.g., IL-2. T cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, reexposure of the cells to the same antigen (even if reexposure occurs in the presence of a costimulatory polypeptide) results in failure to produce cytokines and, thus, failure to proliferate. Anergic T cells can, however, proliferate if cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by a multimer of the API sequence that can be found within the enhancer (Kang et al. (1992) Science 257: 1134).

As used herein, the term "high level of secondary structure" refers to a strong hairpin structure which is predicted to form within a given single-stranded nucleic acid sequence, as predicted by RNAStructure (Reuter et al. (2010) BMC Bioinformatics 11 : 129) or similar RNA secondary structure prediction software programs. In some embodiments, the single-stranded nucleic acid sequence has less than or equal to 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or any range in between inclusive (e.g., 5-15%), of the nucleotides base pair with each other. In other embodiments, the single-stranded nucleic acid sequence has a sequence within which the nucleotides form fewer than or equal to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base pairs, or any range in between inclusive (e.g., 5-15 base pairs), with each other.

"AR- and/or IncRNA-mediated disease" refers to disease or condition associated with an aberrant expression or activity of AR and/or an IncRNA. The AR- and/or lncRNA- mediated diseases include, but are not limited to, cancer (e.g., melanoma, lung

adenocarcinoma, lung squamous cell carcinoma, cervical cancer, ovarian cancer, uterine cancer, pancreatic cancer, colorectal cancer, lower grade glioma, glioblastoma multiforme, breast cancer, endometrial cancer, prostate cancer, testicular cancer, thyroid cancer, osteosarcoma, esophageal cancer, liver cancer or bladder cancer), inflammatory disease (e.g., acne vulgaris, asthma, or rheumatoid arthritis), metabolic diseases, spontaneous abortion, alopecia, Alzheimer disease, androgen-insensitivity syndrome, heart disease (e.g., artiosclerosis, coronary artery disease, or cardiomyopathy), azoospermia, cholelithiasis, cognition disorders, cryptorchidism, depressive disorder, diabetes mellitus, diabetic retinopathy, sex development disorders, bone fractures, hyperglycemia, hypertension, hypospadias, hypertrophy, hyperandrogenism, infertility, klinefelter syndrome, liver cirrhosis, liver disease, lupus, migraine disorder, Menkes Kinky Hair Syndrome, muscular atrophy, nerve degeneration, obesity, oligospermia, osteoarthritis, osteoporosis, Paget Disease, Polycystic Ovary Syndrome, Primary Ovarian Insuffiency, Schizophrenia, spinal fractures, thrombocytosis, thyroid diseases, Tourette syndrome, Turner syndrome, and autoimmune disease (e.g., lupus, type I diabetes, or multiple sclerosis).

There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE

Alanine (Ala, A) GCA, GCC, GCG, GCT

Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT

Aspartic acid (Asp, GAC, GAT

Cysteine (Cys, C) TGC, TGT

Glutamic acid (Glu, GAA, GAG Glutamine (Gin, Q) CAA, CAG

Glycine (Gly, G) GGA, GGC, GGG, GGT

Histidine (His, H) CAC, CAT

Isoleucine (lie, I) ATA, ATC, ATT

Leucine (Leu, L) CTA, CTC, CTG, CTT,

Lysine (Lys, K) AAA, AAG

Methionine (Met, M) ATG

Phenylalanine (Phe, F) TTC, TTT

Proline (Pro, P) CCA, CCC, CCG, CCT

Serine (Ser, S) AGC, AGT, TCA, TCC,

Threonine (Thr, T) ACA, ACC, ACG, ACT

Tryptophan (Trp, W) TGG

Tyrosine (Tyr, Y) TAC, TAT

Valine (Val, V) GTA, GTC, GTG, GTT

Termination signal (end) TAA, TAG, TGA

An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNA coding for a fusion protein or polypeptide of the present invention (or any portion thereof) can be used to derive the fusion protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for a fusion protein or polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and/or disclosure herein of a nucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.

Similarly, description and/or disclosure of a fusion protein or polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

Finally, exemplary single-stranded nucleic acid sequences as well as the nucleic acid and protein sequences of the N-terminal region of AR encompassed within the scope of compositions-of-matter and methods of the present invention are shown below.

Table 1: single-stranded oligos tested for androgen receptor binding

WT-41 : CUGGAGGUAUUUUUCCCUCUCCACCCUGGUCUUCUCCUGUA (SEQ ID NO: 1)

WT-28: UUCCCUCUCCACCCUGGUCUUCUCCUGU (SEQ ID NO: 2)

WT-20: UAUUUUUCCCUCUCCACCCU (SEQ ID NO: 3)

MUT L: AUCGCUCUCAACCCUGGUCUUCUCCUGU (SEQ ID NO: 4)

MUT R: UUCCCUCUCCACCCUGGUAGUCUCAGGU (SEQ ID NO: 5)

MUT FL: AUCGCUCUCAACCCUGGUAGUCUCAGGU (SEQ ID NO: 6)

MUT PT: UCCUUUCCUCACCCUGGUCUUCUUCCGU (SEQ ID NO: 7)

MUT I: UUCCCUCUCCAGCAUGGUCUUCUCCUGU (SEQ ID NO: 8)

WT-17: UAUUUUUCCCUCUCCAC (SEQ ID NO: 9)

MUT-20: UAUUUUUCCCUCUCCACCCU (SEQ ID NO: 10)

Stem-loop: UGGAGGUAUUUUUCCCUCUCCAC (SEQ ID NO: 11)

REP-UC : UCUCUCUCUCUCUCUCUCUC (SEQ ID NO : 12)

REP-UUUC: UUCUUUCUUUCUUUCUUUCU (SEQ ID NO: 13)

REP-CCCU: CCCUCCCUCCCUCCCUCCCU (SEQ ID NO: 14)

UCUCCA: CCCUCUCCAUCUCCACUC (SEQ ID NO: 15)

UCUCCU: CCCUCUCCUUCUCCUCUC (SEQ ID NO: 16)

WT-11 : UUCCCUCUCCA (SEQ ID NO: 17)

WT-10: CUUCUCCUGU (SEQ ID NO: 18)

HOT AIR: UUCCAGCCUCCAGGCCCUGCCUUCUGCCUGC (SEQ ID NO: 19)

SRA1 : UCCCCGCAUCAGAGACUUCUCCUGG (SEQ ID NO: 20)

PCGEM1 : UUACAAGACACAGGCCUACUCCUAG (SEQ ID NO: 21) HOXA11-ASl : CCUUUUGUUUUUCCCUCUCCAGG (SEQ ID NO: 22)

Table 2: FANA-modified oligos that block the interaction of AR and SLNCR1

MIMIC 1 : UUCCCUCTCCACCCUGGTCTUCUCCUGU (SEQ ID NO: 23)

MIMIC 2: CCUCUCCACCCTGGUCUUCUC (SEQ ID NO: 24)

ANTISENSE 1 : ACAGGAGAAGACCAGGGUGGAGAGGGAA (SEQ ID NO: 25) ANTISENSE 2: GGAGAAGACCAGGGUGGAGAG (SEQ ID NO: 26)

Table 3:

N-terminal region of AR Amino Acid sequence (SEQ ID NO: 27)

MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEAASAAPPGASLLL LQQQQQQQQQQQQQQQQQQQQQETSPRQQQQQQGEDGSPQAHRRGPTGYLVLD EEQQPSQPQSALECHPERGCVPEPGAAVAASKGLPQQLPAPPDEDDSAAPSTLSLLG PTFPGLSSCSADLKDILSEASTMQLLQQQQQEAVSEGSSSGRAREASGAPTSSKDNY LGGTSTISDNAKELCKAVS VSMGLGVEALEHLSPGEQLRGDCMYAPLLGVPPAVR PTPCAPLAECKGSLLDDSAGKSTEDTAEYSPFKGGYTKGLEGESLGCSGSAAAGSS GTLELP S TL SL YK S GALDE A A A YQ SRD YYNFPL AL AGPPPPPPPPHPH ARIKLENPL DYGSAWAAAAAQCRYGDLASLHGAGAAGPGSGSPSAAASSSWHTLFTAEEGQLY GPCGGGGGGGGGGGGGGGGGGGGGGGGEAGAVAPYGYTRPPQGLAGQESDFTA PDVWYPGGMVSRVPYPSPTCVKSEMGPWMDSYSGPYGDMRLETARDHVLPIDYY FPPQ

N-terminal region of AR cDNA sequence (SEQ ID NO: 28)

ATGGAAGTGCAGTTAGGGCTGGGAAGGGTCTACCCTCGGCCGCCGTCCAAGAC CTACCGAGGAGCTTTCCAGAATCTGTTCCAGAGCGTGCGCGAAGTGATCCAGA

ACCCGGGCCCCAGGCACCCAGAGGCCGCGAGCGCAGCACCTCCCGGCGCCAGT

TTGCTGCTGCTGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCA

GCAGCAGCAGCAGCAGCAGCAGCAGCAAGAGACTAGCCCCAGGCAGCAGCAG

CAGCAGCAGGGTGAGGATGGTTCTCCCCAAGCCCATCGTAGAGGCCCCACAGG CTACCTGGTCCTGGATGAGGAACAGCAACCTTCACAGCCGCAGTCGGCCCTGG

AGTGCCACCCCGAGAGAGGTTGCGTCCCAGAGCCTGGAGCCGCCGTGGCCGCC

AGCAAGGGGCTGCCGCAGCAGCTGCCAGCACCTCCGGACGAGGATGACTCAGC

TGCCCCATCCACGTTGTCCCTGCTGGGCCCCACTTTCCCCGGCTTAAGCAGCTG

CTCCGCTGACCTTAAAGACATCCTGAGCGAGGCCAGCACCATGCAACTCCTTCA GCAACAGCAGCAGGAAGCAGTATCCGAAGGCAGCAGCAGCGGGAGAGCGAGG GAGGCCTCGGGGGCTCCCACTTCCTCCAAGGACAATTACTTAGGGGGCACTTCG ACCATTTCTGACAACGCCAAGGAGTTGTGTAAGGCAGTGTCGGTGTCCATGGGC CTGGGTGTGGAGGCGTTGGAGCATCTGAGTCCAGGGGAACAGCTTCGGGGGGA TTGCATGTACGCCCCACTTTTGGGAGTTCCACCCGCTGTGCGTCCCACTCCTTGT GCCCCATTGGCCGAATGCAAAGGTTCTCTGCTAGACGACAGCGCAGGCAAGAG CACTGAAGATACTGCTGAGTATTCCCCTTTCAAGGGAGGTTACACCAAAGGGCT AGAAGGCGAGAGCCTAGGCTGCTCTGGCAGCGCTGCAGCAGGGAGCTCCGGGA CACTTGAACTGCCGTCTACCCTGTCTCTCTACAAGTCCGGAGCACTGGACGAGG C AGCTGCGT ACC AGAGTCGCGACT ACT AC AACTTTCC ACTGGCTCTGGCCGGAC CGCCGCCCCCTCCGCCGCCTCCCCATCCCCACGCTCGCATCAAGCTGGAGAACC CGCTGGACTACGGCAGCGCCTGGGCGGCTGCGGCGGCGCAGTGCCGCTATGGG GACCTGGCGAGCCTGCATGGCGCGGGTGCAGCGGGACCCGGTTCTGGGTCACC CTCAGCCGCCGCTTCCTCATCCTGGCACACTCTCTTCACAGCCGAAGAAGGCCA GTTGTATGGACCGTGTGGTGGTGGTGGGGGTGGTGGCGGCGGCGGCGGCGGCG GCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGAGGCGGGAGCTGTAGCCCC CTACGGCTACACTCGGCCCCCTCAGGGGCTGGCGGGCCAGGAAAGCGACTTCA CCGCACCTGATGTGTGGTACCCTGGCGGCATGGTGAGCAGAGTGCCCTATCCCA GTCCCACTTGTGTCAAAAGCGAAATGGGCCCCTGGATGGATAGCTACTCCGGA CCTTACGGGGACATGCGTTTGGAGACTGCCAGGGACCATGTTTTGCCCATTGAC TATTACTTTCCACCCCAGAA

II. Nucleic Acids that block AR-lncRNA interaction. Related Agents, and Compositions Novel agents and compositions of the present invention are provided herein. Such agents and compositions can bind to AR or IncRNA, and block the interaction between AR and IncRNA. In some embodiments, such agents and compositions do not significantly reduce the expression level of AR or IncRNA. Such agents and compositions can also be used for the prevention and treatment of cancers, such as melanoma, as well as conditions in which AR or IncRNA is associated or aberrantly expressed (e.g., inflammatory disease and autoimmune disease). Exemplary agents include single-stranded nucleic acids that either (i) reverse complement to IncRNA' s AR binding seqeunce, which bind to IncRNA to generate double stranded RNA incapable of AR binding, or (ii) mimics of the IncRNA AR binding sequence, which bind directly to AR to preclude IncRNA binding. In some embodiments, the single-stranded nucleic acid is a RNA molecule. In some other embodiments, the single-stranded nucleic acid is a RNA/DNA chimera,

a. Single-Stranded Nucleic Acids

In some embodiments, the single-stranded nucleic acid binds to AR to preclude IncRNA binding. The single-stranded nucleic acid may localize in both cytoplasm and nucleus, or at least partially to the nucleus. The single-stranded nucleic acid can be at least 11 nucleotides in length, and are less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, or 12 nucleotides in length. In a preferred embodiment, the single-stranded nucleic acid is 17 nucleotides in length. The single-stranded nucleic acid may be longer than 17 nucleotides, but may not comprise a high level of secondary structure. The single- stranded nucleic acid comprises at least one UCUCCU or UCUCCA motif, e.g., the single-stranded nucleic acid may contain one, two, three, or more such motifs. If two or more such motifs are present in the single- stranded nucleic acids, they may not be consecutive. An internal sequence with optimal length between any two of such motifs may be required. In certain embodiments, the internal seqeunce is 9 nucleotides in length. In some embodiments, the single-stranded nucleic acid is at least 20 nucleotides in length, and contain at least 65%, preferably >75% (e.g, >80%, >85%, >90%, or >95%), pyrimidine (U/T and C) content within the about 17- 20 nucleotide region containing the UCUCCU or UCUCCA motif.

In some other embodiments, the single-stranded nucleic acid binds to IncRNA to generate double-stranded RNA incapable of AR-binding. Such single-stranded nucleic acid may contain at least one AGGAGA or UGGAGA motif, for example, it may contain one, two, three or more of such motifs. If the IncRNA contains consecutive UCUCCU or UCUCCA motifs, the single-stranded nucleic acid may bind to both motifs simultaneously, with the appropriate internal sequence to facilitate specific and high affinity interaction.

In some embodiments, IncRNA includes SLNCR, HOXA1 l-AS-203, SRA1, PCGEM1, and HOTAIR. In certain embodiments, the IncRNA is SLNCR, and the single- stranded nucleic acid binds to SLNCR to generate double-stranded RNA incapable of AR- binding. Such single-stranded nucleic acid may exhibit at least partial nuclear localization. It may be at least 20 nucleotides long, and are less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, or 21 nucleotides in length. In a preferred embodiment, the single-stranded nucleic acid is 28 nucleotides in length. The single-stranded nucleic acid may be longer than 28 nucleotides, but may not comprise a high level of secondary structure. In some embodiments, the single-stranded nucleic acid may contain two intact AGGAGA or UGGAGA motifs (e.g., an AGGAGA motif followed by a UGGAGA motif). In some other embodiments, the first A in the AGGAGA motif, or the last A in the UGGAGA motif, may be lacking. The two AGGAGA or UGGAGA motifs are linked by an internal seqeunce. In certain embodiment, the internal sequence is 9 nucleotides in length, and may have more than 75% homogloy (e.g., more than 80%, 85%, 90%, 95%, 99% homology) to the sequence AGACCAGGG. In certain preferred embodiment, the internal sequence has 100% homology to the sequence

AGACCAGGG. In some embodiments, the single-stranded nucleic acid is at least 30 nucleotides long, and may contain at least 65% purine (G and A), preferably >75% (e.g., >80%, >85%, >90%, >95%, >99%), within the about 30 nucleotides surrounding the AGGAGA or UGGAGA motifs.

Also provided herein are compositions comprising one or more nucleic acids comprising or capable of expressing at least 1, 2, 3, 4, 5, 10, 20 or more singled-strande nucleic acids that block the AR-lncRNA interaction. In some embodiments, a composition may comprise a library of nucleic acids comprising or capable of expressing said singled- strande nucleic acids, or pools of said singled-stranded nucleic acids. A pool of nucleic acids may comprise about 2-5, 5-10, 10-20, 10-30 or more nucleic acids comprising or capable of expressing said singled-stranded nucleic acids.

In one embodiment, binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.

b. Chemical Modifications, Production, and Delivery of Nucleic Acids

Singled-stranded nucleic acids of the methods and compositions presented herein can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of cellular nucleic acids (e.g., small RNAs, mRNA, lncRNA, and/or genomic DNA). Alternatively, the expression plasmid can produce RNA which encodes mRNA, miRNA, pre-miRNA, pri-miRNA, miRNA*, piwiRNA, anti-miRNA, or a miRNA binding site, or a variant thereof. For example, selection of plasmids suitable for expressing the miRNAs, methods for inserting nucleic acid sequences into the plasmid, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng et al. (2002), Molecular Cell 9: 1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat.

Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference.

Alternatively, singled-stranded nucleic acids are oligonucleotide probes that are generated ex vivo and which, when introduced into the cell, results in hybridization with cellular nucleic acids {e.g., IncRNA) or binding to AR. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as singled-stranded nucleic acids are phosphoramidate,

phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

Singled-stranded nucleic acids can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof. Singled-stranded nucleic acids can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc., and may include other appended groups such as peptides {e.g., for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.

W088/09810, published December 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134, published April 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988), Pharm. Res. 5:539-549). To this end, singled-stranded nucleic acids may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross- linking agent, transport agent, hybridization-triggered cleavage agent, etc.

Singled-stranded nucleic acids may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-

(carboxyhydroxytiethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1 -methyl guanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3 -methyl cytosine, 5 -methyl cytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-

3- N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Singled-stranded nucleic acids may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In certain embodiments, a compound comprises a singled-stranded nucleic acid conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting nucleic acid. In certain such embodiments, the moiety is a cholesterol moiety (e.g., antagomirs) or a lipid moiety or liposome conjugate. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In certain embodiments, a conjugate group is attached directly to the singled-stranded nucleic acid. In certain embodiments, a conjugate group is attached to the singled-stranded nucleic acid by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol,

unsaturations (e.g., double or triple bonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidom ethyl) cyclohexane-l-carboxylate (SMCC), 6- aminohexanoic acid (AHEX or AHA), substituted C1 -C 10 alkyl, substituted or

unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C 10 alkynyl. In certain such embodiments, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises the singled-stranded nucleic acid having one or more stabilizing groups that are attached to one or both termini of the singled-stranded nucleic acid to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the singled-stranded nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'- terminus (3 '-cap), or can be present on both termini. Cap structures include, for example, inverted deoxy abasic caps. Suitable cap structures include a 4',5'-methylene nucleotide, a l-(beta-D- erythrofuranosyl) nucleotide, a 4'-thio nucleotide, a carbocyclic nucleotide, a 1,5- anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threo-pentofuranosyl nucleotide, an acyclic 3',4'-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide, an acyclic 3,5- dihydroxypentyl nucleotide, a 3 '-3 '-inverted nucleotide moiety, a 3 '-3 '-inverted abasic moiety, a 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted abasic moiety, a 1,4-butanediol phosphate, a 3'-phosphoramidate, a hexyl phosphate, an aminohexyl phosphate, a 3'- phosphate, a 3'-phosphorothioate, a phosphorodithioate, a bridging methylphosphonate moiety, and a non-bridging methylphosphonate moiety 5'-amino-alkyl phosphate, a 1,3- diamino-2-propyl phosphate, 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2- aminododecyl phosphate, a hydroxypropyl phosphate, a 5'-5'-inverted nucleotide moiety, a 5'-5'-inverted abasic moiety, a 5'-phosphoramidate, a 5'-phosphorothioate, a 5'-amino, a bridging and/or non-bridging 5'-phosphoramidate, a phosphorothioate, and a 5'-mercapto moiety.

It is to be understood that additional well-known nucleic acid architecture or chemistry can be applied. Different modifications can be placed at different positions to prevent the singled-stranded nucleic acid from activating RNase H and/or being capable of recruiting the RNAi machinery. In another embodiment, they may be placed such as to allow RNase H activation and/or recruitment of the RNAi machinery. The modifications can be non-natural bases, e.g. universal bases. It may be modifications on the backbone sugar or phosphate, e.g., 2'-0-modifications including LNA or phosphorothioate linkages. As used herein, it makes no difference whether the modifications are present on the nucleotide before incorporation into the oligonucleotide or whether the oligonucleotide is modified after synthesis.

Preferred modifications are those that increase the affinity of the singled-stranded nucleic acid for complementary sequences of IncRNAs, i.e. increases the tm (melting temperature) of the singled-stranded nucleic acid base paired to a complementary sequence. Such modifications include 2'-0-flouro, 2'-0-methyl, 2'-0-methoxyethyl. The use of LNA (locked nucleic acid) units, phosphoramidate, PNA (peptide nucleic acid) units or IN A

(intercalating nucleic acid) units is preferred. For shorter singled-stranded nucleic acids, it is preferred that a higher percentage of affinity increasing modifications are present. If the singled-stranded nucleic acid is less than 12 or 10 units long, it may be composed entirely of LNA units. A wide range of other non-natural units may also be build into the singled- stranded nucleic acid, e.g., morpholino, 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA) and arabinonucleic acid (ANA). In a preferred embodiment, the fraction of units modified at either the base or sugar relatively to the units not modified at either the base or sugar is selected from the group consisting of less than less than 99%, 95%, less than 90%, less than 85%) or less than 75%, less than 70%, less than 65%, less than 60%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%), less than 10%, and less than 5%, less than 1%, more than 99%, more than 95%, more than 90%, more than 85% or more than 75%, more than 70%, more than 65%, more than 60%, more than 50%, more than 45%, more than 40%, more than 35%, more than

30%, more than 25%, more than 20%, more than 15%, more than 10%, and more than 5% and more than 1%.

Singled-stranded nucleic acids can also contain a neutral peptide-like backbone. Such molecules are termed peptide nucleic acid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93 : 14670 and in Eglom et al. (1993) Nature 365:566. One advantage of PNA oligomers is their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of the DNA. In yet another embodiment, singled-stranded nucleic acids comprise at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In a further embodiment, singled-stranded nucleic acids are a-anomeric

oligonucleotides. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al. (1987) Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al. (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

Singled-stranded nucleic acids of the methods and compositions presented herein may be synthesized by standard methods known in the art, e.g., by use of an automated

DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988) Nucl. Acids Res. 16:3209, methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. For example, an isolated miRNA can be chemically synthesized or recombinantly produced using methods known in the art. In some instances, miRNA are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), Cruachem (Glasgow, UK), and Exiqon (Vedbaek, Denmark).

Singled-stranded nucleic acids can be delivered to cells in vivo. A number of methods have been developed for delivering singled-stranded nucleic acids to cells; e.g., singled-stranded nucleic acids can be injected directly into the tissue site, or modified singled-stranded nucleic acids, designed to target the desired cells {e.g., singled-stranded nucleic acids linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically. Alternatively, singled-strand nucleic acids can be FANA-modified and gymnotically delivered to desired cells {e.g., short-term melanoma cell cultures).

Singled-stranded nucleic acids of the methods and compositions presented herein may be prepared by any method known in the art for the synthesis DNA and RNA molecules. These include techniques for chemically synthesizing

oligodeoxyribonucleotides and oligoribonucleotides well-known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. One of skill in the art will readily understand that polypeptides, small nucleic acids, and antisense

oligonucleotides can be further linked to another peptide or polypeptide {e.g., a

heterologous peptide), e.g., that serves as a means of protein detection. Non-limiting examples of label peptide or polypeptide moieties useful for detection in the invention include, without limitation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; epitope tags, such as FLAG, MYC, HA, or HIS tags; fluorophores such as green fluorescent protein; dyes; radioisotopes; digoxygenin; biotin; antibodies; polymers; as well as others known in the art, for example, in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999).

c. Combinatory Therapy

Singled-stranded nucleic acids can be incorporated into pharmaceutical

compositions and administered to a subject in vivo. The compositions may contain a single such molecule or agent or any combination of agents described herein. Based on the genetic pathway analyses described herein, it is believed that such combinations of agents is especially effective in diagnosing, prognosing, preventing, and treating melanoma. Thus, "single active agents" described herein can be combined with other pharmacologically active compounds ("second active agents") known in the art according to the methods and compositions provided herein. It is believed that certain combinations work synergistically in the treatment of particular types of melanoma. Second active agents can be large molecules {e.g., proteins) or small molecules {e.g., synthetic inorganic, organometallic, or organic molecules).

Examples of large molecule active agents include, but are not limited to,

hematopoietic growth factors, cytokines, and monoclonal and polyclonal antibodies.

Typical large molecule active agents are biological molecules, such as naturally occurring or artificially made proteins. Proteins that are particularly useful in this invention include proteins that stimulate the survival and/or proliferation of hematopoietic precursor cells and immunologically active poietic cells in vitro or in vivo. Others stimulate the division and differentiation of committed erythroid progenitors in cells in vitro or in vivo. Particular proteins include, but are not limited to: interleukins, such as IL-2 (including recombinant IL-II ("rIL2") and canarypox IL-2), IL-10, IL-12, and IL-18; interferons, such as interferon alfa-2a, interferon alfa-2b, interferon alpha-nl, interferon alpha-n3, interferon beta-la, and interferon gamma-lb; GM-CF and GM-CSF; and EPO.

Particular proteins that can be used in the methods and compositions provided herein include, but are not limited to: filgrastim, which is sold in the United States under the trade name Neupogen® (Amgen, Thousand Oaks, Calif); sargramostim, which is sold in the United States under the trade name Leukine® (Immunex, Seattle, Wash.); and recombinant EPO, which is sold in the United States under the trade name Epogen® (Amgen, Thousand Oaks, Calif.). Recombinant and mutated forms of GM-CSF can be prepared as described in U.S. Pat. Nos. 5,391,485; 5,393,870; and 5,229,496; all of which are incorporated herein by reference. Recombinant and mutated forms of G-CSF can be prepared as described in U.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755; all of which are incorporated herein by reference.

When antibodies are used, the therapy is called immunotherapy. Antibodies that can be used in combination with the methods described herein include monoclonal and polyclonal antibodies. Examples of antibodies include, but are not limited to, ipilimumab (Yervoy®), trastuzumab (Herceptin®), rituximab (Rituxan®), bevacizumab (Avastin®), pertuzumab (Omnitarg®), tositumomab (Bexxar®), edrecolomab (Panorex®), and G250. Compounds of the present invention can also be combined with, or used in combination with, anti-TNF-a antibodies. Large molecule active agents may be administered in the form of anti-cancer vaccines. For example, vaccines that secrete, or cause the secretion of, cytokines such as IL-2, G-CSF, and GM-CSF can be used in the methods, pharmaceutical compositions, and kits provided herein. See, e.g., Emens, L. A., et al., Curr. Opinion Mol. Ther. 3(l):77-84 (2001).

Second active agents that are small molecules can also be used to in combination as provided herein. Examples of small molecule second active agents include, but are not limited to, anti-cancer agents, antibiotics, immunosuppressive agents, and steroids.

In some embodiments, well-known "combination chemotherapy" regimens can be used. In one embodiment, the combination chemotherapy comprises a combination of two or more of cyclophosphamide, hydroxydaunorubicin (also known as doxorubicin or adriamycin), oncovorin (vincristine), and prednisone. In another preferred embodiment, the combination chemotherapy comprises a combination of cyclophsophamide, oncovorin, prednisone, and one or more chemotherapeutics selected from the group consisting of anthracycline, hydroxydaunorubicin, epirubicin, and motixantrone.

Examples of other anti -cancer agents include, but are not limited to: acivicin;

aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine;

ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor); chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;

dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate;

fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate;

liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;

masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;

metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid;

nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;

sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa;

tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate;

trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;

amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;

andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein- 1 ; antiandrogen, prostatic carcinoma; antiestrogen;

antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin

B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;

bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; capecitabine; carboxamide-amino- triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;

carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;

chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cyclosporin A; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin

B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-;

dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;

doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate;

exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;

flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;

formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;

idramantone; ilmofosine; ilomastat; imatinib (e.g., Gleevec®), imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon;

leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan;

lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;

miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide;

mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim;

Erbitux, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;

naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin;

neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen (Genasense®); 06-benzylguanine; octreotide; okicenone;

oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;

paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol;

panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol;

phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine

hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; nbozymes; RII retinamide; rohitukine; romurtide; roquinimex; rubiginone B 1 ; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D;

spiromustine; splenopentin; spongistatin 1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin;

swainsonine; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; teniposide;

tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin;

thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;

triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Specific second active agents include, but are not limited to, chlorambucil, fludarabine, dexamethasone (Decadron®), hydrocortisone, methylprednisolone, cilostamide, doxorubicin (Doxil®), forskolin, rituximab, cyclosporin A, cisplatin, vincristine, PDE7 inhibitors such as BRL-50481 and IR-202, dual PDE4/7 inhibitors such as IR-284, cilostazol, meribendan, milrinone, vesnarionone, enoximone and pimobendan, Syk inhibitors such as fostamatinib disodium (R406/R788), R343, R-l 12 and Excellair® (ZaBeCor Pharmaceuticals, Bala Cynwyd, Pa.).

Moreover, singled-stranded nucleic acids in combination with nuclear receptor inhibitors are described herein,

d. Vectors and Host Cells

In some embodiments, vectors and/or host cells are further provided. In one aspect of the present invention pertains to the use of vectors, preferably expression vectors, containing a nucleic acid encoding RNAs that block AR-lncRNA interaction. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. In one embodiment, adenoviral vectors comprising a nucleic acid encoding RNAs that block AR-lncRNA interaction are used.

The recombinant expression vectors of the present invention comprise a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of RNA desired, etc. The recombinant expression vectors of the present invention can be designed for expression of the desired nucleic acid in prokaryotic or eukaryotic cells. For example, a single-stranded nucleic acid can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Examples of suitable yeast expression vectors include pYepSecl (Baldari, et al., (\9%l) EMBO J. 6:229-234), pMFa (Kurjan and

Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Examples of suitable baculovirus expression vectors useful for insect cell hosts include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3 :2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39). Examples of suitable mammalian expression vectors include pCDM8 (Seed, B. (19S7) Nature 329:840) and pMT2PC (Kaufman et al. (\9 l) EMBO J. 6: 187-195).

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type {e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters such as in melanoma cancer cells are well-known in the art (see, for example, Pleshkan et al. (2011) Acta Nat. 3 : 13-21).

Another aspect of the present invention pertains to host cells into which a recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, single-stranded nucleic acids can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Fao hepatoma cells, primary hepatocytes, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or

electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.

A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well-known in the art. III. Methods of Selecting Agents and Compositions

Another aspect of the present invention relates to methods of selecting agents (e.g., single-stranded nucleic acids) which bind to AR or IncRNA, and block AR-lncRNA interaction. Such methods can use screening assays, including cell-based and non-cell based assays.

In one embodiment, the invention relates to assays for screening candidate or test compounds which bind to AR or IncRNA, and block AR-lncRNA interaction. Such compounds include, without limitation, single-stranded nucleic acids.

In one embodiment, an assay is a cell-based assay, comprising contacting a cell with a test single-stranded nucleic acid and determining the ability of the test single-stranded nucleic acid to block the interaction between AR (either full-lentgh or N-terminal region of AR) and IncRNA as measured by direct binding or by measuring a parameter of cancer.

For example, in a direct binding assay, the IncRNA, AR protein, or a fragment(s) thereof (e.g., N-terminal region of AR), can be coupled with a radioisotope or enzymatic label such that binding of the IncRNA or a fragment(s) thereof to AR protein or a fragment(s) thereof (e.g., N-terminal region of AR) can be determined by detecting the labeled molecule in a complex. For example, the IncRNA, AR protein, or a fragment(s) thereof, can be labeled with ¹²⁵ 1, ³⁵ S, ¹⁴ C, or ¾, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, AR protein or a fragment(s) thereof (e.g., N-terminal region of AR) can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. AR protein, or a fragment(s) thereof (e.g., N-terminal region of AR) can also be detected directly with a specific anti-AR antibody. LncRNA or a fragment(s) thereof can be biotinylated, and the labed RNA probes can then be detected with streptavidin-HRP and chemiluminescent.

It is also within the scope of this invention to determine the ability of a single- stranded nucleic acid to block the interactions beween a IncRNA or a fragment thereof, and AR protein or a fragment(s) thereof (e.g., N-terminal region of AR), without the labeling of any of the interactants (e.g., using a microphysiometer as described in McConnell, H. M. et al. (1992) Science 257: 1906-1912). As used herein, a "microphysiometer" (e.g.,

Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between compound and receptor.

In a preferred embodiment, determining the ability of a single-stranded nucleic acid to block the interaction betweem a IncRNA or a fragment thereof, and AR protein or a fragment(s) thereof (e.g., N-terminal region of AR), can be accomplished by determining the activity of one or more members of the set of interacting molecules. For example, the activity of AR or IncRNA can be determined by detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker), or detecting a cellular response regulated by AR or IncRNA, (e.g., modulations of biological pathways identified herein, such as cell proliferation, cell invasion, and/or transcription of AR- and/or lncRNA-regulated genes).

In yet another embodiment, an assay of the present invention is a cell-free assay in which a IncRNA or a fragment(s) thereof, and AR protein or a fragment(s) thereof (e.g., N- terminal region of AR protein), are contacted with a test single- stranded nucleic acid, and the ability of the test single-stranded nucleic acid to block the interaction between the IncRNA or a fragment(s) thereof, and AR protein or a fragment(s) thereof (e.g., N-terminal region of AR protein), is determined. Blocking of the interaction between the IncRNA or a fragment(s) thereof, and AR protein or a fragment(s) thereof (e.g., N-terminal region of AR protein), can be determined either directly or indirectly as described above. Determining the ability of the single-stranded nucleic acid to block the interaction between the IncRNA or a fragment(s) thereof, and AR protein or a fragment(s) thereof (e.g. , N-terminal region of AR protein) can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63 :2338- 2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological polypeptides. AR protein or a fragment thereof (e.g., N-termial of AR protein) can be immobilized on a BIAcore chip, and IncRNA or a fragment thereof can be tested for binding to the immobilized AR protein or fragment thereof. An example of using the BIA technology is described by Fitz et al. (1997) Oncogene 15:613.

The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of AR proteins. In the case of cell-free assays in which a membrane-bound form of AR protein is used it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the AR protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether) _n , 3-[(3-cholamidopropyl)dimethylamminio]-l-propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l-propane sulfonate (CHAPSO), or N- dodecyl=N,N-dimethyl-3-ammonio-l -propane sulfonate.

In one or more embodiments of the above described assay methods, it may be desirable to immobilize either the IncRNA or a fragment(s) thereof, or AR protein or a fragment(s) thereof, to facilitate separation of complexed from uncomplexed forms of the reactants, as well as to accommodate automation of the assay. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase-base fusion proteins, can be adsorbed onto glutathione Sepharose® beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with IncRNA in the presence or absence of the test single-stranded nucleic acid, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity determined using standard techniques.

In an alternative embodiment, determining the ability of the test single-stranded nucleic acid to modulate the interaction between IncRNA or a fragment thereof, and AR protein or a fragment thereof (e.g., the N-terminal region of AR), can be accomplished by determining the ability of the test single-stranded nucleic acid to modulate the activity of a gene, e.g., nucleic acid, or gene product, e.g., polypeptide, that functions downstream of the interaction. For example, cellular proliferation or invasion can be determined by monitoring cell number count, cellular movement, matrigel assays, induction of proliferation- and/or invasion-related gene expression, and the like, as described further herein.

In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell- based or a cell-free assay, and the ability of the agent to modulate the interaction between IncRNA and AR protein or a fragment thereof can be confirmed in vivo, e.g., in an animal such as an animal model for cellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

V. Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) an AR- and/or IncRNA-medidated disease.

1. Prophylactic Methods

In one aspect, the present invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of AR and/or a IncRNA, by administering to the subject an agent (e.g, a single-stranded nucleic acid) which binds to AR or the IncRNA, and blocks AR-lncRNA interaction. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the AR and/or IncRNA expression or activity aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.

2. Therapeutic Methods

Another aspect of the present invention pertains to methods for treating a subject affliated with a disease or condition associated with an aberrant expression or activity of AR and/or a IncRNA, by administering to the subject an agent (e.g, a single-stranded nucleic acid) which binds to AR or the IncRNA, and blocks AR-lncRNA interaction. The disease or condition associated with an aberrant expression or activity of AR and/or a IncRNA include cancer, such as melanoma, as well as inflammatory and autoimmune diseases. Accordingly, the interaction between IncRNA or a fragment thereof and AR protein or a fragment(s) thereof can be modulated in order to modulate cancer growth and/or metastasis, as well as the immune response.

Modulatory methods of the present invention involve contacting a cell with a single- stranded nucleic acid of the present invention, that binds to AR or a IncRNA and block the interaction between AR and the IncRNA within the cell. An agent that blocks the interaction between AR and a IncRNA can be an agent as described herein.

These modulatory methods can be performed in vitro {e.g., by contacting the cell with the agent) or, alternatively, by contacting an agent with cells in vivo {e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a condition or disorder that would benefit from blocking the interaction between AR and a IncRNA, e.g., a disorder characterized by unwanted, insufficient, or aberrant expression or activity of AR and/or IncRNA. In one embodiment, the method involves administering an agent {e.g., an agent identified by a screening assay described herein), or combination of agents that blocks the interaction between AR and a IncRNA.

In addition, these modulatory agents can also be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, and/or radiotherapy. The preceding treatment methods can be administered in conjunction with other forms of conventional therapy {e.g., standard-of-care treatments for cancer well-known to the skilled artisan), either consecutively with, pre- or post-conventional therapy. For example, these modulatory agents can be administered with a therapeutically effective dose of chemotherapeutic agent. In another embodiment, these modulatory agents are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapeutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular melanoma, being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.

IV. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of a single-stranded nucleic acid that binds to AR or IncRNA, and blocks the interaction between AR and the IncRNA, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4)

intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.

The phrase "therapeutically-effective amount" as used herein means that amount of a single-stranded nucleic acid that binds to AR or IncRNA, and blocks the interaction between AR and the IncRNA, which is effective for producing some desired therapeutic effect, e.g., cancer treatment, at a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable" is employed herein to refer to those agents, materials, compositions, and/or 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, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and

polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more

pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the

pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well-known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the

gastrointestinal tract, optionally, in a delayed manner. Examples of embedding

compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active agent may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more therapeutic agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent. Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a single-stranded nucleic acid that binds to AR or IncRNA and blocks the interaction between AR and the IncRNA, include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to a therapeutic agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a single-stranded nucleic acid that binds to AR or IncRNA and blocks the interaction between AR and the IncRNA, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The single-stranded nucleic acid that binds to AR or IncRNA and blocks the interaction between AR and the IncRNA, can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. Transdermal patches have the added advantage of providing controlled delivery of a therapeutic agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more therapeutic agents in combination with one or more

pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of

microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.

Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of a single- stranded nucleic acid that binds to AR or IncRNA and blocks the interaction between AR and the IncRNA, in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

When the agents of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

The nucleic acid molecules of the present invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. V. Administration of Agents

The prevention, and/or treatment modulating agents of the present invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the nucleic acid to be administered in which any toxic effects are outweighed by the therapeutic effects of the nucleic acid. The term "subject" is intended to include living organisms, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Administration of an agent as described herein can be in any pharmacological form including a therapeutically active amount of an agent alone or in combination with a pharmaceutically acceptable carrier.

Administration of a therapeutically active amount of the therapeutic composition of the present invention is defined as an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, a therapeutically active amount of a single-stranded nucleic acid may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the nucleic acid to elicit a desired response in the individual. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

It will also be appreciated that the effective dosage of the therapeutic composition of the present invention used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result from the results of diagnostic assays. In addition, the therapeutic composition of the present invention can also be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, and/or radiotherapy. A therapeutic composition of the present invention can also be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post-conventional therapy. For example, the therapeutic composition of the present invention can be administered with a therapeutically effective dose of chemotherapeutic agent. In another embodiment, the therapeutic composition of the present invention can be administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physicians' Desk Reference (PDR) discloses dosages of chemotherapeutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer, e.g., melanoma, being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician. In another embodiment, the therapeutic composition of the present invention can be administered in conjunction with one or more nuclear receptor targeting drugs.

The agents of the present invention described herein can be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral

administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. For example, for administration of agents, by other than parenteral administration, it may be desirable to coat the agent with, or co-administer the agent with, a material to prevent its inactivation.

An agent can be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non- ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

The agent may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions of agents suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the composition will preferably be sterile and must be fluid to the extent that easy

syringeability exists. It will preferably be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating an agent of the pbresent invention (e.g., a single-stranded nucleic acid) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the agent plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When the agent is suitably protected, as described above, the agent can be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form", as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention are dictated by, and directly dependent on, (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

In addition, the agents of the present invention described herein can be administered using nanoparticle-based composition and delivery methods well-known to the skilled artisan. For example, nanoparticle-based delivery for improved nucleic acid (e.g., small RNAs) therapeutics are well-known in the art (Expert Opinion on Biological Therapy 7: 1811-1822).

Exemplification

This invention is further illustrated by the following examples, which should not be construed as limiting. Example 1: Materials and Methods for Examples 2-6 and 14-17

Reagents

Biotinylated and unmodified RNA probes were purchased from Integrated DNA Technologies. Full-length, recombinant AR protein containing an N-terminus DDDK tag was purchased from Abeam (ab82609). N-terminal recombinant AR protein containing an N-terminal 6x HIS tag was purchased from RayBiotech (RB-14-0003P). Steric-blocking 2'-FANA modified oligos were gifts from AUM LifeTech. The plasmid expressing full- length SLNCR1 was previously described (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Site-directed mutagenesis was used to obtain all SLNCR1 mutant plasmids described herein, using oligo sequences listed in Table 4.

RNA electrophoretic mobility shift assays

REMSAs were performed using Thermo Fisher Scientific LightShift

Chemiluminescent RNA EMS A (REMSA) Kit, according to manufacturer's instructions. Briefly, 20 μΐ binding reactions were assembled in low-adhesion tubes in IX binding buffer (lOmM HEPES pH 7.3, 20 mM KC1, 1 mM MgCl ₂ , 1 mM DTT), with 2 μg of yeast tRNA, the indicated amount of either recombinant full-length or N-terminal recombinant protein,

0.5 nM final concentration of the biotinylated RNA probe, and 10 μΜ of unlabeled competitor RNA (if applicable). Reactions were incubated at room temperature for 20 minutes, at which time 5 μΐ of loading dye was added, and 20 μΐ was electrophoresed on

Bio-Rad's 5% Mini-PROTEAN ^® TBE Gel, 10 well, 30 μΐ (Cat number #4565013). RNA and protein/RNA complexes were transferred to GE Healthcare Amersham Hybond -N+ Membrane in 0.5x TBE at 400 mA for 30 minutes in 0.5X TBE on Bio-Rad's Trans Blot Turbo Transfer System. Detection was performed as described, using Bio-Rad's

ChemiDoc™ XRS+ System. MEME analysis (at World Wide Web address of

www.meme-suite.org) was performed using the normal motif discovery mode, searching for any number of motif repetitions using 0-order background model of sequences within the input sequences WT-41, WT-28, WT-20, WT-17, MUT R, MUT L, MUT PT, MUT I, HOXA11-AS1.

Plasmid construction

The plasmid expressing full-length SLNCR1 was previously described (Schmidt, K., et a/., Cell Rep, 2016. 15(9): p. 2025-37). Site-directed mutagenesis was used to obtain all SLNCR1 mutant plasmids described here, using oligo sequences listed in Table 4.

Table 4. Oligos used in this study

* SDM = site-directed mutagenesis primers. Mutations from wild-type SLNCR1 are highlighted in bolded font. The SLNCRl ^{m MUT 1 md 2} expressing plasmids was generated through PCR amplification of SLNCRl^ ^{MUT 2} using the indicated plasmids. Cell Culture

A375 cells were purchased from ATCC. WM1976 cells were from the collection of the Wistar Institute (Philadelphia, PA). Cells were were cultured as adherent cells in DMEM (Dulbecco's modified eagle medium, Invitrogen) without glutamine supplemented with 10% fetal bovine serum (FBS). Lipofectamine® 2000 (Life Technologies) was used for all plasmid transfections.

Surface Plasmon Resonance SPR experiments were performed on a Biacore 3000. Full length AR protein (Abeam) was dialyzed against 10 mM NaOAc pH 5 and subsequently coupled to a CM5 chip (GE) using EDC/NHS coupling. The system was equilibrated to running buffer (lOmM HEPES pH 7.3, 20 mM KC1, 1 mM MgCl ₂ , 1 mM DTT) and RNA (IDT) was subsequently injected (30 uL/min flow rate, one minute injection followed by 5-15 minutes dissociation).

Denatured control RNA SHAPE probing

WM1976 cells were grown in 6-well plates (2 wells per experiment) to -80% confluency. Media was aspirated, cells were washed once in PBS, and total RNA was extracted using 1 mL TRIzol reagent (Thermo Fisher) per well. Total RNA (15 μΐ.) was combined with 5 iL of lOx denaturing buffer [500 mM HEPES pH 8, 40 mM EDTA] and 25 μΙ_, of formamide, and the mixture was incubated at 95 °C for 1 minute. 5 μΙ_, of lOx SHAPE reagent [250 mM 5-nitroisatoic anhydride (5NIA)] was added to the denatured RNA and incubation at 95 °C continued for 1 min. Denatured modified RNA was purified using a 1.8x ratio of Agencourt RNA Clean XP beads (Beckman Coulter) and eluted in 88 μΐ, of nucl ease-free water.

In-cell SHAPE probing

WM1976 cells were grown in 6-well plates (4 wells per experiment) to -80% confluency. Cells were washed once in PBS before adding to each well 900 μΙ_, of PBS. To two wells, 100 μL· of lOx SHAPE reagent [250 mM 5-nitroisatoic anhydride (5NIA)] was added while 100 μΙ_, of neat DMSO were added to each of the remaining two wells. Plates were mixed concurrently with reagent addition and then incubated at 37 °C for 10 minutes. Media was aspirated, cells were washed once in PBS, and total RNA was extracted using 1 mL TRIzol reagent (Thermo Fisher) per well. Resulting pellets were resuspended in 88 μΙ_, of water per 2 wells.

In-cell DMS probing

WM1976 cells were grown in 6-well plates (4 wells per experiment) to -80% confluency. Cells were washed once in PBS before adding to each well 900 μΙ_, of DMS Folding Buffer [300 mM Bicine pH 8, 100 mM NaCl, 5 mM MgCl ₂ ]. To two wells, 100 μΙ_, of lOx DMS reagent [1 :5 dimethyl sulfate (DMS): ethanol] was added while 100 μΙ_, of neat ethanol were added to each of the remaining two wells. Plates were mixed concurrently with reagent addition and then incubated at 37 °C for 6 minutes. DMS was quenched with 200 μΙ_, of beta-mercaptoethanol and cells were placed on ice for 5 minutes. Excess buffer was removed, cells were washed once in PBS, and total RNA was extracted using 1 mL TRIzol reagent (Thermo Fisher) per well. Resulting pellets were resuspended in 88 μΐ. of water per two wells.

Cell-Free RNA extraction and protein digestion

WM1976 cells were grown in two 10 cm dishes to -80% confluency. Both plates were washed once in PBS before scraping and lysis in 2.5 mL of ice-cold cytoplasmic lysis buffer [40 mM Tris pH 8, 175 mM NaCl, 6 mM MgCh, 1 mM CaCl ₂ , 256 mM Sucrose, 0.5% Triton-X 100, 0.5 Units^L RNasin (Promega), 0.45 Units/μΐ DNase I (Roche)]. Cells were lysed for 5 minutes on ice with intermittent mixing. Cell nuclei were pelleted at 3000 x g for 5 minutes at 4 °C (nuclear fraction), and the resulting supernatant (cytoplasmic fraction) was transferred to a new tube. A volume of 2.5 mL of proteinase K buffer [40 mM Tris pH 8, 200 mM NaCl, 1.5% Sodium Dodecyl Sulfate, and 0.5 mg/mL Proteinase K] was added to the nuclear pellets. The supernatant (cytoplasmic fraction) volume was increased to 5 mL to include a final concentration of 200 mM NaCl, 1.5% Sodium Dodecyl Sulfate, and 0.5 mg/mL Proteinase K (Thermo Fisher). Proteins were digested for 45 minutes at 23 °C with intermittent mixing.

RNA separation and bu ffer exchange

Nucleic acids were extracted twice with 1 volume of Phenol Chloroform Isoamyl Alcohol (25:24: 1) that was pre-equilibrated with either l . lx SHAPE Folding Buffer [110 mM HEPES pH 8, 110 mM NaCl, 5.5 mM MgCl ₂ ] or l . lx DMS Folding Buffer [330 mM Bicine pH 8, 110 mM NaCl, 5.5 mM MgCl ₂ ]. Excess Phenol was removed through two subsequent 1 volume chloroform extractions. The final aqueous layer was buffer exchanged into 1. lx SHAPE Folding Buffer or 1. lx DMS Folding Buffer using PD-10 Desalting Columns (GE Healthcare Life Sciences). The resulting RNA solution (3.5 mL nuclear/7 mL cytoplasmic) was incubated at 37 °C for 20 minutes before being split into two equal volumes for control and probe treatment samples.

5NIA SHAPE probins of extracted RNA

A l/10 ^th volume of lOx SHAPE reagent [250 mM 5-nitroisatoic anhydride (5NIA)] in DMSO was added to one sample of RNA while neat DMSO was added to the other. Samples were incubated at 37 °C for 10 minutes.

DMS probing of extracted RNA

A 1/10 volume of lOx DMS Solution [1 :5 Dimethyl Sulfate (DMS):Ethanol] was added to one sample of RNA while neat ethanol was added to the other. Samples were incubated at 37 °C for 6 minutes. DMS was quenched with 1/5 volume of beta- mercaptoethanol and immediately placed on ice for 5 minutes.

Precipitation of probe-reacted RNA

Cell-free RNA was precipitated with 1/10 volume of 2 M NH ₄ OAc and 1 volume of isopropanol. RNA was pelleted at 10,000 x g for 10 minutes at 4 °C. After 1 wash in 75% ethanol, the resulting pellet was dried and then resuspended in 88 μΙ_, of water.

DNase Treatment of cell-derived RNAs

To 88 μΐ, of in-cell or cell-free RNA samples, 10 iL of lOx TURBO DNase buffer and 4 Units of TURBO DNase (Thermo Fisher) were added and the mixture was incubated at 37 °C for 1 hour. Following this incubation, 2 more Units of TURBO DNase were added and incubation continued at 37 °C for another 1 hour. RNA was purified using a 1.8x ratio of Agencourt RNAClean XP magnetic beads (Beckman Coulter) and eluted into 20-30 μΙ_, of nuclease-free water.

SLNCR MaP

From each sample of RNA from WM1976 in-cell and cell-free probing experiments,

1-3 μg were subjected to mutational profiling (MaP) reverse transcription (Siegfried, N.A., et al, Nature Methods, 2014. 1 1 : p. 959-965; Smola, M.J., et al, Nat Protoc, 2015. 10: p. 1643-1669) using a primer specific to SLNCR. The cDNA generated was buffer exchanged over Illustra microspin G-50 columns (GE Healthcare). Output cDNA (5 μΕ) was used as a template for 50 μΙ_, PCR reactions (Q5 Hot-start polymerase, NEB) with primers made to amplify nt 403-780 of SLNCR1 and add adapter sequences [lx Q5 reaction buffer, 250 nM each primer, 200 μΜ dNTPs, 3% DMSO, 0.02 Units^L Q5 Hot-start polymerase]. PCR proceeded in a touchdown format: 98 °C for 2 minutes, 20 cycles of [98 °C for 10 s , 68 °C (decreasing by 1 °C each cycle until reaching 63 °C) for 30 s , 72 °C 30 s], and 72 °C for 2 min. Step 1 PCR products were purified using a 0.8x ratio of Agencourt AMPure XP beads (Beckman Coulter) and eluted in 20 μΙ_, of nuclease-free water. Purified PCR products (3 ng) were used as a template in 50 μΙ_, of PCR meant to add multiplex indices and remaining sequence necessary for Illumina sequencing [lxQ5 reaction buffer, 500 nM each index primer, 200 μΜ dNTPs, 3% DMSO, 0.02 Units^L Q5 Hot-start polymerase]. Step 2 PCR proceeded as follows: 98 °C for 2 minutes, 10 cycles of [98 °C for 10 s , 66 °C for 30 s , 72 °C 20 s], and 72 °C for 2 min. Step 2 PCR products were purified using a 0.8x ratio of Agencourt AMPure XP beads (Beckman Coulter) and eluted in 20 μΙ_, of nuclease-free water. SLNCR Conserved Region RT primer: 5'-GGATCAGTCCTTCCCATCC

SLNCR step 1 amp F: 5'- GACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNACCTCCGCGAGTCTGG

SLNCR step 1 amp R: 5'- CCCTACACGACGCTCTTCCGATCTNNNNNGGATCAGTCCTTCCCATCC

Truseq LT step 2 with index F: 5'-CAAGCAGAAGACGGCATACGAG-(index) ₈ - GTGACTGGAGTTCAGAC

Truseq LT Universal Adapter R: 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCC GATCT

Ul snRNA MaP

The procedure for Ul snRNA MaP RT was similar to that of SLNCR MaP with minor modifications: (1) Ul-specific primers were used for RT and step 1 PCR, (2) During step 1 PCR, the touchdown protocol began with an annealing temperature of 72 °C and only decreased to 64 °C, and (3) Step 1 PCR included 500 nM of each primer, no DMSO, and only 25 μΙ_, of total volume.

Ul RT primer: 5 '-C AGGGGAAAGCGCGAA

Ul step 1 F: 5'-

GACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNATACTTACCTGGCA

Ul step 1 R: 5'-

CCCTACACGACGCTCTTCCGATCTNNNNNCAGGGGAAAGCGCGAA

Sequencing and profile generation

Purified SLNCR amplicons were pooled and sequenced on an Illumina Miseq instrument, outputting 2 x 300 paired end datasets. For Ul libraries, 2x 150 paired end datasets were generated instead. Reverse transcription of SLNCR yielded three products, corresponding to 3 alternatively spliced forms of SLNCR (SLNCR1: 2257 nucleotides, SLNCR2: 2522 nucleotides, newly observed isoform SLNCR4: 2160 nucleotides). Data from these isoforms were separated before alignment by fragment lengths after merging paired reads. SLNCR1 -specific merged reads were then aligned to the

SLNCR1 /UNC00673 sequence (NCBI Reference NR_036488.1) using the ShapeMapper2 software (Busan, S., and K M. Weeks, RNA, 2018. 24(2): p. 143-148; Smola, M.J., et al, Nat Protoc, 2015. 10: p. 1643-1669) with the command shapemapper—target [TARGET]. fa -modified -U [MERGED-TREATED-READS] fastq -untreated -U [MERGED- UNTREATED-READS]. fastq -denatured -U [MERGED-DENATURED-RE ADS] fastq. When aligning DMS reads, the denatured control was excluded. Ul reads were similarly aligned to the Ul snRNA sequence (NCBI Reference NR_004430.2).

Discovery of in-cell SHAPE protections with deltaSHAPE

In-cell SHAPE and DMS protection sites were identified through comparison of in- cell and cell-free reactivities using the deltaSHAPE program with default settings (Smola, M.J., et al, Biochemistry, 2015. 54: p. 6867-6875; Smola, M.J., et al, Proc. Nat. Acad, of Sci. of the U. S. A., 2016. 113 : p. 10322-10327). In-cell data were scaled such that the 95 ^th percentile reactivity matched the average of cell-free 95 ^th percentile reactivities. Significant nucleotide protections were identified in both nuclear and cytoplasmic fractions with the criteria of being centered in window of 5 nucleotides that contains 3 nucleotides with positive smoothed deltaSHAPE values (cell -free minus in-cell), positive Z-factors, and Z- scores above 1. Additional regions with smoothed deltaSHAPE values above 1 and Z- factors above 0.5 were also considered to account for skewing of Z-score by highly reactive sites.

RNA structure modeling with SuperFold

Base-pairing probabilities, Shannon entropy, and arcplot base-pairing projections were generated by the SuperFold analysis pipeline (Smola, M.J., et al, Nat Protoc, 2015. 10: p. 1643-1669). For FIG. 4F-4H, SLNCRl ^403'780 data were scaled to the Ul

normalization factor created by the boxplot approach (Hajdin, C.E., et al, Proc. Nat. Acad, of Sci. of the U. S. A., 2013. 110: p. 5498-5503), where reactivities above the larger of the 90 ^th percentile and 1.5 x the interquartile range are first removed as outliers and the top 10% of remaining reactivities are averaged (the normalization factor). The original SLNCRl data were divided by this Ul normalization factor to obtain Ul -scaled reactivity profiles. Low SHAPE, low entropy regions were identified by two methods: [1] as nucleotides where the 50 nucleotide window-median SHAPE and entropy values fall below 0.4 and 0.03, respectively (FIG. 4F-4H), and [2] nucleotides where the 50 nucleotide window-median SHAPE and entropy values fall below the global medians (FIG. 4M-40). Regions separated by 5 or fewer nucleotides were combined and only regions spanning at least 30 nucleotides were retained before expanding to include overlapping base-pair arcs. RNA secondary structure representations of SLNCRl were generated from ShapeMapper and SuperFold outputs using VARNA (Darty, K., et al, Bioinformatics, 2009. 25: p. 1974- 1975). Invasion Assays

A375 cells were seeded at 25 x 10 ⁴ cells in a 6-well plate and transfected with 2,500ng of the indicated plasmid 24 hours later. For assays using FANA-modified oligos, media was changed 7-8 hours post-transfection to media containing 1 μΜ final

concentration of the indicated oligo. Approximately 28 hours post-transfection, 2.5 x 10 ⁴ cells in serum-free media were plated in either BD BioCoat™ matrigel inserts or uncoated control inserts (Corning), placed into DMEM with 30% FBS (fetal bovine serum), and incubated for 16 hours. Cells that did not migrate or invade were removed using a cotton tipped swap, chambers were rinsed twice with PBS, and stained using Fisher HealthCare™ PROTOCOL™ Hema 3™ Fixative and Solutions. Cells were imaged on 20x magnification in 8 fields of view for 3 independent replicates. All statistical analyses were performed using GraphPad Prism version 7.01 for Windows (GraphPad Software, La Jolla California USA).

RNA extraction and quanti fication

RNA was isolated using Qiagen RNeasy® Mini Kit and treated with DNase. cDNA was generated using Bio-Rad's iScript™ cDNA synthesis kit and quantified as previously described (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). For tissue specific expression analysis, cDNA was generated from Human Total RNA Master Panel II (Clontech). AR mRNA expression was taken from the Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NFMH, and NINDS (Consortium, G.T., Science, 2015. 348(6235): p. 648-60). The GTEx data used for the analyses described in this manuscript were obtained from The Human Protein Atlas available at World Wide Web address of www.proteinatlas.org (Uhlen, M., et al., Science, 2015. 347(6220): p. 1260419).

Example 2: The N-terminal region of AR binds to single-stranded RNA in a sequence-specific manner

Previously, RNA immunoprecipitation assays were used to identify an

approximately 100 nucleotide (nt) region of SLNCRl required for interaction with AR (nts

568-673) (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). To further characterize the interaction of AR with SLNCRl, RNA electrophoretic mobility shift assays (REMSA) were used. Increasing concentration of the full-length AR protein significantly retards the migration of a 41 nt single-stranded biotinylated RNA probe corresponding to wild-type SLNCR1 nucleotides 597-637 (WT-41), indicating that AR binds this RNA sequence, and that that this RNA sequence is sufficient for AR binding (FIG. 1 A-1B). At higher AR concentrations (>200 nM), a second shift was observed, possibly resulting from AR protein dimerization or binding of multiple proteins on a single RNA probe.

AR is comprised of discreet protein domains, including a C-terminal ligand binding domain, a flexible hinge region, a DNA-binding domain, and an NTD required for interactions with many protein and RNA co-factors, including the IncRNAs SRA1 and PCGEM1 (FIG. 1A) (Lanz, R.B., et al, Cell, 1999. 97: p. 17-27; Yang, L., et al, Nature, 2013. 500(7464): p. 598-602). The IncRNA PCGEM1 binds to the N-terminal regulatory region of AR (Yang, L., et al, Nature, 2013. 500(7464): p. 598-602). To test if the

SLNCR1 sequence WT-41 also binds to the N-terminal region of AR, REMSA using a truncated version of AR (amino acids 2-566), spanning the N-terminal region (FIG. 2A) were repeated. Indeed, the truncated version of AR (amino acids 2-566) spanning AR NTD also binds and slows migration of WT-41, indicating that SLNCR1 binds to and interacts with the N-terminus of AR (FIG. 1 A and FIG. 1C). Even at high concentrations, the N- terminal AR does not result in a secondary downward shift of the RNA probe as is observed with full-length AR, possible due to the inability of the truncated protein to dimerize. Thus, the N-terminal protein was used for the majority of the subsequent experiments.

RNA-binding proteins may bind RNA in a sequence- and/or structure-specific manner. Since the 5' end of WT-41 is highly predicted to form a stable stem-loop structure, while the 3' end is likely unstructured, whether AR binds to shorter sequence SLNCR1 fragments WT-28 and WT-20 was tested (FIG. 1 A). These shorter UC-rich sequences are unlikely to form any stable secondary structures. N-terminal AR significantly reduces migration of both WT-28 and WT-20, indicating that AR binds to single-stranded pyrimidine-rich RNAs (FIG. 1C). Thus, REMSAs revealed that AR NTD also binds shorter UC-rich SLNCR1 sequences WT-28 and WT-20 (FIG. 1C). Addition of unlabeled probes corresponding to WT-41 successfully outcompetes binding of labeled WT-41, WT- 28, or WT-20 to either full-length or N-terminal AR, confirming that that the observed mobility shifts result from specific RNA binding (FIG. 2B, FIG. 2C, and FIG. 4C).

Combined, these data show that the AR NTD is sufficient for binding to short sequences of pyrimidine-rich RNA.

To investigate if AR binds RNA in a sequence specific-manner, REMSA was used to test the effects of various mutations of WT-28 on AR binding. Mutations in either the left, 5' end of WT-28 (MUT L, containing 3 base changes), or the right, 3' end of the RNA (MUT R, containing 4 base changes), do not abolish AR-binding (FIG. 3 A and FIG. 3B). A slightly altered migration pattern of MUT L was noted, however, indicating modified AR binding compared to WT-28 or MUT R. Interestingly, combining MUT L and MUT R mutations (MUT full-length, or MUT FL) completely abolishes AR binding. This result indicates that either (i) AR binds to pyrimidine-rich RNA in a non-sequence specific manner, and combining MUT L and MUT R (consisting of pyrimidine - to - purine mutations) into a single sequence drops the total pyrimidine percentage below the threshold required for AR binding, or (ii) WT-28 contains two AR binding motifs, only one of which is mutated in MUT L and MUT R. Inconsistent with pyrimidine-rich sequence binding, AR does not bind 20 nts probes consisting of UC, UUUC or CCCU repeats (REP-UC, REP- UUUC, or REP-CCCU, respectively, FIG. 4A and FIG. 4B). Consistent with sequence- specific binding of two AR-binding motifs, a UCUCCU/A motif is present on both the 5' and 3' end of WT-28 (FIG. 3 A). Thus, a series of experiments was designed to test if AR specifically recognized the UCUCCU/A motif. First, a probe containing 7 pyrimidine mutations (U to C or C to U) across the total length of WT-28 was generated. In addition, this probe was designed so that while the endogenous motifs were mutated, a single, non- endogenous motif (UCUCCU) just upstream but partially overlapping with motif 1 (MUT shifted motif, MUT PT) was generated. MUT PT's migration pattern upon AR binding is similar to MUT L, which contains only one UCUCCU motif, indicateing that AR binding to both probes is comparably altered compared to WT-28. Second, mutation of the sequence internal to these two motifs (MUT internal, MUT I) does not alter AR binding (FIG. 3 A and 3B). Third, probes corresponding to the SLNCR1 sequence shifted slightly upstream of WT-28, and importantly containing only motif UCUCCA motif (WT-17 and WT-20), are bound by AR (FIG. 3C and 3E). Finally, mutating 2 nts of this motif in the absence of any additional motifs, abolished AR binding (WT-20 versus MUT-20, FIG. 3C and 3E), thus confirming that AR binding requires at least one intact UCUCCU/A motif. It was determined that AR does not bind 18 nt probes containing two repeats of these motifs (either UCUCCU or UCUCCA, FIG. 4A), indicateing that AR is unable to bind to two consecutive motifs. Consistent with previous data, introduction of non-endogenous nucleotides at the 3' end of WT-17 to induce a stable stem -loop structure abolished binding (FIGS. 3C, 3D and 3E), confirming specific binding to single-stranded RNAs. Collectively, these data reveal that AR binds to single-stranded, pyrimidine-rich RNA containing at least one UCUCCU/A motif.

RNA-binding proteins may bind RNA in a sequence- and/or structure-specific manner. A series of mutational analyses was used to probe the potential sequence requirements of the AR/RNA interaction, identifying a set of wild-type and mutated RNAs that retain the ability to bind AR NTD (FIG. 3A-3E and 3G). Motif enrichment analysis (multiple EM for motif elicitation, MEME) of this set of tested RNAs identified a significantly enriched (E-value = 1.3 x 10 ^"13 ) 9 nt motif CYUYUCCWS (Y = pyrimidine [C or U], W = weak [A or U] and S = strong [C or G]) required for AR-RNA binding, indicating that AR NTD binds to RNA in a sequence-specific manner (FIG. 3G) (Bailey, T.L., et al., Nucleic Acids Research, 2009. 37: p. W202-208). SLNCR1 contains 2 of these motifs (nts 612-620 and nts 627-635, motif 1 and motif 2), located in close proximity to each other. Both motifs are contained within WT-41 and WT-28 (FIG. 2 A and 3G).

Several lines of evidence indicate that only one motif is required for interaction with AR. First, WT-20 contains only one motif and is still bound by AR NTD (FIG. 1C).

Second, mutations in either motif 1 (MUT L [left], containing 3 base changes), or motif 2 of WT-28 (MUT R [right], containing 4 base changes) do not abolish AR-binding when the other motif is present (FIG. 3B and 3C). The altered migration pattern of MUT L, however, indicates that AR engages each motif in a unique way. Third, combining MUT L and MUT R mutations (MUT full-length, or MUT FL) completely abolished AR binding (FIG. 3 A- 3B). Fourth, and perhaps most importantly, mutating 2 nts of the single motif in WT-20 abolished AR binding (WT-20 versus MUT-20, FIG. 3B and 3D). Fifth, truncation of WT- 20 to a 17 nt probe ending at the 3' end of motif 1 (WT-17) retains AR binding (FIG. 3D). Together, these data show that a single CYUYUCCWS motif is sufficient for specific recognition by AR NTD.

Considering that the CYUYUCCWS motif identified above is a short pyrimidine- rich sequence, it was determined if pyrimidine-rich sequences that do not contain this motif also confer AR binding. AR does not bind 20 nt probes consisting of UC, UUUC or CCCU repeats (REP-UC, REP -UUUC, or REP-CCCU, respectively) or to 18 nt probes containing two sequential repeats of a UCUCCA motif (FIG. 4A-4B). Further, SLNCR1 contains a near consensus motif approximately 100 nts upstream (nts 547-555, CUUCUCCAU) of motifs 1 and 2 that falls outside of the SLNCR1 ^{56 '631} region previously identified to be required for AR (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). REMSA confirms that AR NTD does not bind a 15-nt probe corresponding to this upstream motif, indicating that a U in position 9 is not accepted. Mutations between motifs 1 and 2 that do not alter either CYUYUCCWS motif do not affect AR binding (FIG. 3 A-3B). A probe containing 7 pyrimidine transitions (U to C or C to U) across WT-28 (MUT -pyrimidine transitions, or MUT PT) that disrupt both of the original motifs 1 and 2 but introduce a new

CYUYUCCWS consensus motif (CCUUUCCUC) retains AR binding (FIG. 3A-3B).

These data further indicate that AR specifically binds the CYUYUCCWS motif.

To test whether RNA structure interferes with AR binding, AR-RNA binding was compared in the presence or absence of secondary structure (FIG. 1 A and 3D-3F).

Introduction of non-endogenous nucleotides at the 5' end of WT-17 that induce a stable stem-loop structure encompassing 6 nts of motif 1 abolished AR NTD binding (FIG. 3D- 3E). Surface plasmon resonance (SPR) using full-length protein confirms that AR has a ~3 fold higher affinity for WT-20 (KD = 251 nM), which contains an unstructured motif 1 alone, than WT-41 (KD = 741 nM), which includes motif 1 in a stem-loop and an unstructured motif 2 (FIG. 3F). Together, these data reveal that AR NTD binds pyrimidine- rich RNA containing at least one loosely-structured CYUYUCCWS motif.

In agreement with previous work indicating that SLNCR1- and AR-mediated phenotypes occur in the absence of androgens (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37), data presented herein indicate that an AR monomer can bind to SLNCR1 in the absence of protein dimerization. Specifically, (1) AR binds RNA independently of androgens in vitro (FIG. IB); (2) AR NTD, which is incapable of forming head-to-tail dimers, binds RNA in vitro (FIG. 3B); and (3) only one AR binding motif is required for AR/RNA interaction (FIG. 3 A-3G). Thus, it was considered if AR has differential affinity to motif 1 versus motif 2. Although WT-41 contains both AR binding motifs, only motif 2 is likely accessible for AR binding, as motif 1 forms a highly-stable stem-loop structure occluded from AR binding (FIG. 1 A). Considering that AR displayed a ~3-fold higher affinity for WT-20 (containing only motif 1 : CCUCUCCAC) compared to WT-41 (where only motif 2 is accessible: CUUCUCCUG), AR has a higher affinity for motif 1 compared to motif 2 (FIG. 3F).

Example 3: SLNCRVs AR binding motif is required for SLNCR1- and AR-mediated melanoma invasion

To test if the in vitro REMSA results recapitulate AK-SLNCR1 interactions in the cell, whether mutations in the AR binding motif identified above affect known AR- ,SZNCR7-mediated phenotypic changes was investigated. Ectopic expression of SLNCRl significantly increases the invasiveness, as quantified by matrigel invasion assays, of A375 melanoma cells in an AR-dependent manner through transcriptional upregulation of the matrix metalloproteinase MMP9 [3]. Moreover, ectopic expression of a highly conserved, -300 nt sequence of SLNCRl containing the AR-binding sequence is sufficient to induce increased melanoma invasion. Thus, A375 invasion was quantified after expression of wild-type, full-length SLNCRl, SLNCRl ^com , or SLNCRl harboring site-directed mutations in the AR binding motif 1, motif 2, or both motif 1 and 2 (SLNCR^™ ⁷ \ SLNCRl^™ ⁷² , or SLNCRl^^ ^{7 1 and 2} , respectively). Consistent with our in vitro data, mutation of either motif 1 or motif 2 slightly negated SLNCRL s ability to increase melanoma invasion, while mutation of both completely abolished SLNCRl -mediated invasion (FIGs. 5 A and 5B). Importantly, and in agreement with results from invasion assays, although all SLNCRl mutants are expressed at similar levels, MMP9 levels are significantly increased only with expression of either full-length or the conserved sequence of SLNCRl (FIG. 5C).

Example 4: Sequence based identification of novel AR-interacting IncRNA

HOXA11-AS-203

Previously, a region of sequence similarity between multiple IncRNAs predicted or shown to bind to AR was identified, including HOTAIR, SRAI, and PCGEMI, that overlaps with the WT-28 SLNCRl sequence (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Of these IncRNAs, only SRAI contains an intact potential AR-binding motif (motif 2- UCUCCU). Indeed, AR does result in a slight, but reproducible, shift of the SRAI probe that is successfully competed away by unlabeled WT-41 probe (FIG. 6A and 7A), indicating that SRAI directly binds AR, though further work is required to confirm this interaction. In contrast, probes corresponding to the conserved region of HOTAIR or PCGEMI, which do not contain a UCUCCU/A motif, do not bind N-terminal AR in the REMSA assay (FIGs. 6A and 6B). The conditions used in the assay do not account for additional protein or RNA factors, nor do they enable mammalian-specific post- translational modifications of AR, that may be required for mediating these interactions in vivo. Indeed, the AR-PCGEM1 interaction requires both protein- and AR-modifications, as discussed later. These results further confirm that, in a minimal in vitro system, AR requires the presence of at least one UCUCCU/A motif for RNA binding. The ability to predict IncRNA binding partners, and more generally function, from sequence analysis alone remains largely uninvestigated. A region of sequence similarity between multiple IncRNAs predicted or shown to bind to AR, including HOTAIR, SRA1, and PCGEM1, that overlaps with the WT-28 SLNCR1 sequence was previously identified (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Of these IncRNAs, only SRAJ contains an intact potential AR-binding motif (CUUCUCCUG). Indeed, in REMSA, addition of AR results in a slight, but reproducible, shift of the SRA1 probe that is successfully competed away by unlabeled WT-41 probe (FIG. 6 A and 7 A), indicating that SRA1 directly binds AR.

In contrast, probes corresponding to the conserved region of HOTAIR or PCGEMI, which do not contain a CYUYUCCWS motif, do not bind AR NTD in REMSA assays (FIG. 6A-6B). The conditions used in REMSA assays do not account for additional protein or RNA factors, nor do they enable mammalian-specific post-translational modifications of AR, that may be required for mediating these interactions in vivo. Indeed, the AR- PCGEM1 interaction requires both protein- and AR-modifications, as discussed later.

These results further confirm that, in a minimal in vitro system, AR requires the presence of at least one CYUYUCCWS motif for RNA binding. Thus, nucleotide BLAST search (available on the World Wide Web at

blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) was used to identify additional non-coding RNAs containing either a containing a consensus or near-consensus AR binding motif, corresponding to the sequences of WT-28 or WT-20. A 1,169 nt transcript expressed divergently from the HOXA11 gene, IncRNA HOXA1 l-AS-203 (Ensembl transcript ENST00000520395, gene ENSG00000240990), contains a region of high similarity to WT-20 (FIG. 6C and 6D). A probe corresponding to 23 nt of the

HOXA1 l-AS-203 sequence is significantly shifted upon incubation with the N-terminal

AR, indicating that the N-terminal region of AR directly binds to this sequence. Moreover, RT-qPCR profiling of a panel of human tissues reveals that this IncRNA isoform is expressed in AR-expressing tissues, including the prostate, uterus, and testis (FIG. 7B), indicating that this interaction may occur in vivo. HOXA1 l-AS-203 and AR are co- expressed in several human tissues, including the prostate, uterus, and testis, indicating that this interaction occurs in vivo (FIG. 7B). These data demonstrate that the AR-binding RNA motif identified herein may be predictive of the ability of a IncRNA to bind to AR; these data indicate that perturbing AR binding to IncRNAs can block AR-dependent functions of IncRNAs in multiple tissues. These data demonstrate that the presence of the AR-binding RNA motif identified here may be predictive of a IncRNA's ability to bind to AR.

Example 5: Inhibiting the AK-SLNCR1 interaction using 2'-FANA-modified oligonucleotides negates SZJVCRi-mediated melanoma invasion

To develop candidate novel therapeutics inhibiting the SLNCRl- and AR-mediated invasion, and to assess the therapeutic potential of inhibiting the SLNCR1-AR interaction short (21-28 nucleotide) oligos were designed that are either (i) reverse complement or antisense to SLNCRL s AR binding sequence, which bind to SLNCRl to generate double stranded RNA incapable of AR binding (ANTISENSE 1 and ANTISENSE 2), or (ii) mimics of the SLNCRl AR binding sequence, which bind directly to AR to preclude or inhibit SLNCR binding (MEVIIC 1 and MIMIC 2, FIG. 9A). These oligonucleotides are composed of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2 -FANA) and unmodified DNA nucleotides specifically arranged to enhance intracellular stability of the oligonucleotide without eliciting RNase H mediated cleavage and therefore downregulation of SLNCRl (FIG. 10A) (Ferrari, N., et al, Ann N Y Acad Sci, 2006. 1082: p. 91-102). Importantly, these probes are designed to block both CYUYUCCWS motifs present in SLNCRl. Both SLNCRl- and AR-binding FANA modified oligonucleotides are capable of sterically blocking the AR- SLNCRl interaction in vitro (FIG. 9B). Addition of ANTISENSE oligonucleotides upwardly shifts the biotinylated probe, confirming the formation of dsRNA. Gymnotic delivery {i.e. delivery of naked oligos independent of any agents) of 2'- deoxy-2'-fluoro-D-arabinonucleic acid (2 -FANA) modified oligos to A375 melanoma cells (FIG. 8) significantly reduces SLNCRl -mediated melanoma invasion, without affecting SLNCRl expression (FIGs. 9C and 10A). Consistently, while ectopic expression of SLNCRl significantly increases MMP9 mRNA as previously shown, addition of the oligo blockers attenuates this increase (FIG. 10B). In the presence of MFMIC 2, a reproducible trend, though not significant, for increased MMP9 levels was observed, indicating that this oligo does not block the SLNCR1-AR interaction as well in cells. MFMIC 1 and

ANTISENSE 1 negate SLNCRl -mediated MMP9 upregulation more efficiently than MFMIC 2 and ANTISENSE 2, which are truncated by 7 nts. Collectively, these data demonstrate that single-stranded AR- or SLNCRl -targeting oligonucleotides can be used to block the interaction of AR and SLNCRl, attenuating ,SZNCR7-mediated melanoma invasion. Example 6: Minimal RNA sequence length required for AR binding

To determine the minimal RNA sequence length required for AR N-terminal domain binding, REMSA was used to check for AR binding to shorter biotinylated probes corresponding to wild-type SLNCR. AR binds to 11 nucleotide long sequence containing a UCUCCA motif (WT-11, FIG. 11). AR does not bind to a 10 nucleotide sequence containing an UCUCCU sequence (WT-10). Thus, AR requires at least 11 nucleotides for efficient RNA binding.

Example 7: Materials and Methods for Examples 8-12 and 18-19

Cell Culture

MSTCs (WM1976 (p53 wild-type) and WM858 (BRAF ^V600E , p53 ^MUT )) are from the Wistar Institute collection, A375 (BRAF ^V600E , p53 wild-type, CDKN2A ^E61* ) and SK-MEL- 28 (BRAF ^V600E , EGFR ^P753S , P53 ^L145R ) cells were purchased from ATCC. Unless otherwise indicated, cells were grown in DMEM (Invitrogen) without glutamine supplemented with 10% fetal bovine serum (FBS). All siRNAs were transfected using RNAiMAX® (Thermo Fisher). AR and SLNCR targeting siRNAs were used at 10 nM final concentration. EGR1 targeting siRNA was used at 20 nM. Hormone-deprived cells were cultured in phenol-red free DMEM without glutamine (Invitrogen) with 5% charcoal -stripped FBS (Sigma- Aldrich). For knockdown and flutamide proliferation assays, cells were seeded at 0.4 x 10 ⁴ cells/well in a 96-well plate. For assays using FANA-modified oligos (AUM

Technologies), cells were seeded at 0.3 x 10 ⁴ cells/well in a 96-well plate. Cells were treated with FANA-oligos, flutamide, or transfected with the indicated siRNAs 24 hours post seeding, and proliferation was measured using a 1 : 10 dilution of WST-1 proliferation reagent (Roche) at the indicated time points. Cells were incubated for one hour at 37°C, and absorbance at 450 nM was measured. For cell cycle analyses, cells were harvested and washed 72 hours post-transfection, fixed in cold 70% ethanol for 2 hours, and incubated in LifeTech PI/RNaseA solution for 30 minutes at 37 degrees. For analysis of apoptosis, cells were seeded at 30 x 10 ⁴ cells/well in 6-well plate, harvested 72 hours post-transfection, and stained using Biolegends Pacific Blue™ Annexin V Apoptosis Kit with 7-AAD. Cells were analyzed on a Fortessa X-20 and populations were identified and quantified using

FlowJo software. For luciferase assays, A375 cells were seeded in 96-well plates at 0.75 x 10 ⁴ cells per well, transfected 24 hours later with the indicated siRNAs. Fifty micrograms of either wild-type or mutated CDKN1A reporter plasmid and 50 μg of a pCMV-renilla luciferase control vector were transfected Lipofectamine® 2000 (Life Technologies) 24 hours post-transfection of siRNAs. Luciferase activity was measured another 24 hours later using Promega Dual-Glo® Luciferase Assay system. The CDKN1A reporter plasmid was generated by replacing the MMTV promoter in pGL4.36 vector (Promega) using Gibson Cloning (Gibson, D.G., et al, Nature Methods, 2009. 6: p. 343-345). Sequences for all siRNAs and oligos used in this study can be found in Table 17. For transcription factor activation arrays, WM1976 cells were seeded in 6-well tissue culture treated dishes, transfected 24 hours later with either scramble or -SLNCR (1) siRNA, and were harvested and fractionated using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit, according to the manufacturer's instructions, 3 days after transfection. Ten

micrograms of nuclear lysate was used directly as input into the Signosis TF Activation Profiling Plate Array I. For AR ChlP-seq, A375 cells were cultured in phenol -red-free DMEM without glutamine (Invitrogen), supplemented with 5% charcoal-stripped FBS, and transfected with the indicated plasmid 24 hr post-seeding. For EGRl ChlP-seq, A375 cells were cultured in DMEM without glutamine (Invitrogen), supplemented with 10% FBS, and grown to -80% confluency. Cells were crosslinked in 1% formaldehyde for 15 min 48 hr post-transfection, and the reaction was quenched by addition of 0.125 M glycine. ChlP-seq was performed by Active Motif.

Table 5: siRNA sequences

siEGRl target sequence: CAGGACAATTGAAATTTGCTA

siSLNCR (1) target sequence: TTAGGTCAAATAGGATCTAAA

siSLNCR (2): AAAGACGTTTACACCGAGAAA

siAR (1): CAGGAATTCCTGTGCATGAAA

siAR (2): C AC GGGA AGTTT AGAGAGC T A

siAR (3): CTGCTACTCTTCAGCATTATT

RNA immunoprecipitation and chromatin immunoprecipitation and sequencing.

AR and EGRl RIP assays were performed as previously described with minor modifications (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). For EGRl RIP, IgG or a-EGRl antibody was added to A375 cell lysate at a final concentration of 0.5 μg and rotated at 4°C for 2 hours. Lysate was then incubated with Protein A Dynabeads® (Life Technologies) (25 μΐ slurry) for 1 hour at 4°C and samples were processed as described. Fold enrichment of SLNCR was calculated as the fold enrichment in the IgG or EGRl IP compared to input control after normalization to the indicated mRNA transcript (18S, GAPDH, ACTIN).

For AR ChlP-seq, A375 cells were cultured in phenol-red-free DMEM without glutamine (Invitrogen), supplemented with 5% charcoal -stripped FBS, and transfected with the indicated plasmid 24 hour post-seeding. For EGR1 ChlP-seq, A375 cells were cultured in DMEM without glutamine (Invitrogen), supplemented with 10% FBS, and grown to -80% confluency. Cells were crosslinked in 1% formaldehyde for 15 min 48 hour post- transfection, and the reaction was quenched by addition of 0.125 M glycine. ChlP-seq was performed by Active Motif using Santa Cruz AR (H-280), or Cell Signaling EGR1 (44D5). After chromatin isolation and fractionation, 75-nt reads were generated by Illumina sequencing (using NextSeq 500), and were mapped to human reference genome (hgl9) using the BWA algorithm with default settings. The 3' ends of aligned reads were extended in silico using Active Motif software to a length of 150-200 bp. Fragment density was determined based on the number of reads corresponding to 32-nucleotide genomic bins. Peak calling, to identify intervals with local enrichment in reads, was performed using

MACS (Zhang, Y., et al, Genome Biol, 2008. 9: p. R137). MACS default cutoff p-value is le ^"7 for narrow peaks and le ^"1 for broad peaks. Peak filtering was performed by removing false ChlP-Seq peaks as defined within the ENCODE blacklist. Active regions were defined by the start coordinate of the most upstream interval and the downstream coordinate of the most downstream interval. Active genes are defined as any active region present within 10,000 bps upstream or downstream of an annotated gene.

RNA-sequencing and analysis

Total RNA was isolated from WM1976 transfected with either scramble, si-SLNCR (1) or si-SLNCR (2) siRNAs, in duplicate, using Trizol. Sequencing cDNA libraries were prepared from lug of total RNA using the Illumina TruSeq RNA sample preparation kit (v2). Libraries were pooled and sequenced on the Illumina HiSeq 2500 platform.

Normalized read counts (FPKM) were generated in Cufflinks v2.1.1 (available on the World Wide Web at cole-trapnell-lab.github.io/cufflinks/) by mapping onto the hgl9 build of the human transcriptome (available on the World Wide Web at

support.illumina.com/sequencing/sequencing_software/igeno me.html). Raw FASTQ sequence was mapped using Bowtie (Langmead, B., et al., Genome Biol, 2009. 10(3): p. R25), and differentially expressed genes were identified using CuffDiff (available on the World Wide Web at cufflinks.cbcb.umd.edu/), comparing duplicate scramble controls against duplicate conditions of both SLNCR-specific knockdowns. Values represented in the heatmaps were generated by CuffDiff comparison of duplicate scramble controls versus duplicates of only one siRNA duplicate. GeneOntology Enrichment Analysis was performed in MetaCore (Thompson Reuters), against a control background set of genes expressed in skin cells.

Gene expression of AR- and ^NCR-target genes was accessed using cBioPortal (Cerami, E., et al, Cancer Discov, 2012. 2: p. 401-404; Gao, J., et al, Sci Signal, 2013. 6: p. pll). All statistics (including T-tests, ANOVAs, and correlations) were calculated using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla California USA (on the World Wide Web at www.graphpad.com). Bonferroni correction for multiple hypothesis testing was performed by defining the significance threshold as the critical p=0.05 divided by the total number of comparisons. BAM files from RNA-seq and ChlP- seq were visualized using the Integrated Genome Viewer (on the World Wide Web at broadinstitute.org/igv/) (Robinson, J.T., et al, Nat Biotechnol, 2011. 29(l):24-6;

Thorvaldsdottir, H., Robinson, J.T., and J.P. Mesirov, Brief Bioinform, 2013. 14(2): 178- 92).

Apoptosis assay

Cells were seeded at 30 x 10 ⁴ cells/well in 6-well plate. Transfected with ΙΟηΜ final or indicated siRNAs using RNAiMAX 24 post-seeding. 72 hours post-transfection, cells were prepped for FACs using Biolegends Pacific Blue™ Annexin V Apoptosis Kit with 7-AAD.

TF activation array

WM1976 cells were seeded in 6-well tissue culture treated dishes, transfected 24 hours later with either scramble or si-SLNCR (1) siRNAs, and 48 hours post transfections cells were harvested and fractionated using NE-PER fractionation kit. Approximately 10 μg of the nuclear fraction for input directly in Signosis' Transcription Factor Activation Array I. Alternatively, cells transfected with siRNA were harvested and fractionated using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit, according to the manufacturer's instructions, 3 days after transfection. Ten micrograms of nuclear lysate was used directly as input into the Signosis TF Activation Profiling Plate Array I.

Cell cycle analysis Fixed in cold 70% ethanol for 2 hours. Incubate in LifeTech PI/RNaseA solution for 30 minutes at 37 degrees. Ran on FACs machine Fortessa X-20, analyzed using FlowJo software.

Protein extraction and analysis

Unless otherwise indicated, lysate was prepared using M-PER Mammalian Protein

Extraction Reagent (Thermo Scientific), according to manufacturer's instructions. Samples were separated on BioRad Any kD™ Mini -PROTEAN® TGX™ Precast Protein Gels and transferred to LF-PVDF using the mixed MW protocol on the BioRad Transblot Turbo. The following antibodies were used: Santa Cruz p53 (DO-1) sc-126 at 1 :200, AR (M-20) sc-816 at 1 :200, Cell Signaling P21 Wafl/Cipl (12D1) at 1 : 1000, Cell Signaling Egrl (44D5) at 1 : 1000, Cell Signaling S6 Ribosomal Protein (5G10) at 1 : 1000, and Cell Signaling GAPDH (14C10) at 1 :5000. Santa Cruz AR (H-280) was used for AR ChJP- PCR.

72 hours post-transfection of the indicated cells with 10 nM of the indicated siRNAs, lysate was prepared using M-PER Mammalian Protein Extraction Reagent

(Thermo Scientific), according to manufacturer's instructions. Samples were separated on BioRad Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels and transferred to LF- PVDF using the mixed MW protocol on the BioRad Transblot Turbo. The following antibodies were used: Santa Cruz p53 (DO-1) sc-126 at 1 :200, AR (M-20) sc-816 at 1 :200, Cell Signaling P21 Wafl/Cipl (12D1) at 1 : 1000, Cell Signaling Egrl (44D5) at 1 : 1000, Cell Signaling GAPDH (14C10) at 1 :5000.

RNA electrophoretic mobility shift assays

REMSAs were performed using Thermo Fisher Scientific LightShift

Chemiluminescent RNA EMS A (REMSA) Kit, according to manufacturer's instructions. Briefly, 20 μΐ binding reactions were assembled in low-adhesion tubes in IX binding buffer (lOmM HEPES pH 7.3, 20 mM KCl, 1 mM MgC12, 1 mM DTT), with 2 μg of yeast tRNA, the indicated amount of recombinant EGR1 corresponding to amino acids 282-433 (Aviva Systems Biology, catalogue number OPCD02876), 0.5 nM final concentration of the biotinylated SLNCRl, and 10 μΜ of unlabeled SLNCRl where indicated. Reactions were incubated at room temperature for 20 minutes, 5 μΐ of loading dye was added, and 20 μΐ was electrophoresed on Bio-Rad's 5% Mini-PROTEAN® TBE Gel, 10 well, 30 μΐ. RNA and protein/RNA complexes were transferred to GE Healthcare Amersham Hybond -N+ Membrane in 0.5x TBE at 400 mA for 30 minutes in 0.5X TBE on Bio-Rad's Trans Blot Turbo Transfer System. Detection was performed according to LightShift REMSA kit, using Bio-Rad's ChemiDoc™ XRS+ System.

TCGA informatics and statistical analyses

GeneOntology Enrichment Analysis was performed in MetaCore (Thompson Reuters), against a control background set of genes expressed in skin cells. Gene expression of AR- and SZNCR-target genes, p21 protein levels, and p53 mutational status were accessed using cBioPortal (Cerami, E., et al, Cancer Discov, 2012. 2: p. 401-404; Gao, J., et al, Sci Signal, 2013. 6: p. pll). SLNCR RNA expression values were derived from normalized read coverage across the SLNCR genomic range. Raw RNA-Seq data for cutaneous melanoma (SKCM) was downloaded from the NCI Genomic Data Commons (GDC). The RNA-Seq data was in the format of BAM files representing an alignment, using the STAR aligner, of raw reads to hg38. Human gene models were downloaded from RefSeq on November 3rd, 2017, and the SLNCR ranges were defined from the range of LINC00673. For each patient, the total number of reads aligning the SLNCR genomic region was obtained by parsing the output of samtools flagstat and SLNCR-specific counts were normalized by the total number of reads aligning to hg38. The expression values for non-SLNCR genes were obtained by downloading a results table from Xenabrowser (on the World Wide Web at https://toil.xenahubs.net/download/tcga_rsem_isoform_tpm.gz) . TPM expression values were computed from a reanalysis of the TCGA dataset under the TOIL framework using RSEM. Hierarchical multiple regression analysis was performed using R v3.2.2, where the model estimates and p-values, based on the t-statistic, were calculated using the 'lm' function. SLNCR and EGR1 mRNA expression values were log2

transformed to account for non-normal distributions. Expression of AR, p53 and p21 protein was accessed from Level 4 cross-batch normalized data in The Cancer Proteome Atlas (Li, J., et al, Nature Methods, 2013. 10: p. 1046-1047). All continuous variables were converted to z-scores in order to improve interpretability of the model output. To determine the set of parameters present in the final model, standard reduction techniques were used, including iterative removal of the least significant parameter along with evaluation of the Akaike's information criterion (AIC) and ANOVA comparisons of model fit. P53-deficient melanomas were defined as (i) primary melanomas or metastases of known melanoma origin, (ii) patients with no prior treatment, and (iii) harboring nonfunctional p53 mutations, as defined by the TP53 database (p53.fr) (Leroy, B., et al, Hum Mutat, 2014. 35: p. 756-765). Patients containing R248W or Y220C gain of function p53 mutations were excluded based on reported regulation of p21 (Di Fiore, R., et al, Bone, 2014. 60: p. 198-212; Song, H., Hollstein, M., and Y. Xu, Nature Cell Biol, 2007. 9: p. 573-580; Xu, J., et al., Cell Death Dis, 2014. 5: p. el 108). Bonferroni correction for multiple hypothesis testing was performed by defining the significance threshold as the critical P-value (0.05) divided by the total number of comparisons. BAM files from RNA- seq and ChlP-seq were visualized using the Integrated Genome Viewer (on the World Wide Web at broadinstitute.org/igv/) (Robinson, J.T., et al, Nat Biotechnol, 2011. 29(l):24-6; Thorvaldsdottir, H., Robinson, J.T., and J.P. Mesirov, Brief Bioinform, 2013. 14(2): 178- 92).

Statistics

T-tests, ANOVAs, and correlations were calculated using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla California USA (on the World Wide Web at www.graphpad.com). In proliferation assays error bars represent the mean ± SD of 3 technical replicates. Significance was calculated using the two-way analysis of variance (ANOVA), with the Dunnett test for multiple comparison testing. For binding enrichments, statistical significance was calculated using a either a Binomial test (two-sided) by comparing the observed versus expected probabilities under independence or using a Fisher Exact test of the co-bound targets, using GraphPad Prism software. In cell cycle assays, cell populations were analyzed using FlowJo software, and significance was calculated using GraphPad Prism software. Bars represent the average percent of total cells in the indicated stage of the cell cycle, and error bars represent SD from 3 independent replicates. RT- qPCR data is represented as the fold change compared to scramble control, normalized to GAPDH. Error bars represent standard deviations calculated from 3 reactions. Protein levels were quantified using ImageJ, and are presented as a fold change normalized to GAPDH levels. Bars represent mean ± SD from 3 independent biological replicates.

Significance for RNA and protein quantification was calculated using the Student's t-test. Significance was calculated using a two-tailed Student's t-test. Transcription Factor Activation Array is represented by relative luminescence mean ± SD from 2 independent biological replicates.

Example 8: AR and SLNCR exhibit sex-specific differences in human melanomas

Previous work revealed that SLNCR directly binds AR, recruiting it to specific genomic locations to modulate AR transcriptional activity (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). Since AR has been implicated as a potential oncogene in melanoma, and SLNCR (but not AR) expression is associated with worse overall melanoma survival (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37), it indicates that SLNCR imparts oncogenic functions to AR. If true, this functional relationship may explain why (i) AR expression is not associated with melanoma survival, and (ii) canonical AR

functionality alone is unable to account for the observed melanoma gender bias. To first determine if SLNCR demonstrates sex-specific expression or effects, SLNCR expression was interrogated from 150 sequenced melanomas from The Cancer Genome Atlas (TCGA). An analysis variance model (ANOVA) reveals a significant gender-specific effect of SLNCR expression related to stage of melanoma (p-value = 0.0384, FIG. 12A).

Specifically, SLNCR expression is higher in females prior to local dissemination (Stages I and II). In agreement with these results, SLNCR expression is significantly higher in females prior to lymph node metastasis (NX/NO versus N1/N2/N3, FIG. 12B). Notably, however, expression in males, or in total melanoma patients, is not significantly altered upon metastasis, possibly a consequence of lower expression in pre-metastatic males compared to females (FIGs. 12 A, 12B and 13 A). Taken together, these results reveal that SLNCR expression is gender- and stage-specific.

Male melanomas express significantly higher levels of AR than female melanomas (p-value = 0.046, FIG. 13B). Thus, if SLNCR mediates AR's oncogenic functions, SLNCR expression would likely be associated with gender-specific melanoma survival.

Intriguingly, while high SLNCR expression (RPKM > 14.1) is associated with worse melanoma survival total melanoma patients (log-rank p-value = 0.043, (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37)) and females (log rank p-value = 0.048, FIG. 1C), this association is not as significant in males (log-rank p-value = 0.17). Moreover, males exhibited a slight trend of worse survival than females at low levels of SLNCR (p-value = 0.18, FIG. 12B). Said another way, males appear to be hyper-sensitized to low levels of SLNCR compared to females. It is believed that \ow-SLNCR male melanomas express higher levels of AR than \ow-SLNCR female melanomas, rendering the low levels of SLNCR capable of inducing oncogenic effects of AR. Consistent with the observations, male \ow-SLNCR melanomas express significantly higher levels of AR (p = 0.02, two- tailed) than \ow-SLNCR female melanomas. Among hig -SLNCR melanomas, where no gender-specific effects are observed, similar levels of AR are expressed in males and females. Collectively, these data indicate that there is a functional relationship between SLNCR and AR with possible implications for the melanoma gender bias, warranting further investigation.

Example 9: The IncRNA SLNCR and ligand-independent AR increase melanoma cell proliferation

While previous work focused on the most prevalent SLNCR isoform, SLNCR1, the role of all SLNCR isoforms in AR functionality and melanoma etiology were fully characterized here. To account for possible overlapping functions between the multiple SLNCR isoforms, siRNAs were designed to knockdown of all SLNCR isoforms, resulting in -60-80% knockdown of SLNCR in two patient-derived melanoma short-term cultures (MSTCs), WM1976 and WM858 (FIG. 13A). Importantly, MSTCs have undergone minimal passages outside of the patient and provide an accurate genetic model of melanoma (Lin, W.M., et al., Cancer Res, 2008. 68(3): p. 664-73). Using RNA-sequencing (RNA-seq), melanoma cells were transcriptionally profiled before and after siRNA- mediated knockdown of SLNCR in WM1976, the MSTC which expresses higher levels of SLNCR (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Following siRNA- mediated depletion of SLNCR, 222 genes are significantly dysregulated (p-value < 0.01, FIG. 12E, Table 6A and Table 6B). Specifically, 131 genes are significantly upregulated compared to a scramble siRNA control, while knockdown of SLNCR decreases expression of 91 genes. Gene Ontology (GO) Enrichment analysis these differentially expressed (DE) genes (MetaCore) identifies a significant enrichment of genes involved in multiple cancer- relevant processes, including cell adhesion/motility, apoptosis, differentiation, response to stress and proliferation (FIG. 12F). Indeed, roles for SLNCR1 were previously identified in melanoma invasion and motility (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). The possible roles for SLNCR in melanoma proliferation were particularly interesteing, as SLNCR has been implicated in proliferation of lung cancer and other cancers. Consistent with a role in regulation of cell proliferation, knockdown of SLNCR significantly decreases proliferation of both MSTCs compared to a scramble siRNA control (p-value < 0.0001, FIG. 12G). Proliferation of the melanoma cell line A375 is also significantly reduced upon knockdown of SLNCR, albeit to a lesser extent than observed in the MSTCs, possibly due to (i) lower endogenous levels of SLNCR in these cells (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37), (ii) lower efficiency of SLNCR depletion (FIG. 13C) and/or (iii) the faster proliferative rate of A375 cells, which shortens the observable duration of knockdown. Importantly, knockdown of SLNCR does not significantly alter the number of apoptotic cells in either MSTC, or in A375 melanoma cells (FIG. 13D). Collectively, these experiments indicate that SLNCR increases melanoma proliferation.

GO Enrichment analysis of the 222 DEGs upon SLNCR knockdown identifies significant enrichment of genes involved in multiple cancer-relevant processes, including cell adhesion/motility, apoptosis, differentiation, response to stress and proliferation (FIG. 12F). Possible roles for SLNCR in melanoma proliferation is of particular interest, as SLNCR has been implicated in proliferation of pancreatic, gastric, and non-small-cell lung cancers (Huang, M., et al, Mol Ther, 2017. 25: p. 1014-1026; Lu, W., et al, Molecular Cancer, 2017. 16: p. 118; Shi, X., et al, Oncotarget, 2016. 7: p. 25558-25575; Zheng, J., et al, Nature Genetics, 2016. 48: 747-757). Consistent with enrichment of cell proliferation genes identified with GO analysis (-22%, or 49 out of 222 DEGs, p = 2.54 x 10 ^"48 , FIG. 12F), interrogation of The Cancer Genome Atlas (TCGA) data reveals that SLNCR expression is significantly correlated with the mitotic growth rate of primary melanomas (Spearman r = 0.20, p = 0.0083, FIG. 13F), with slow-growing melanomas expressing significantly lower levels of SLNCR (mitotic growth rate <1 versus >1 mitosis/mm ² , p = 0.014, FIG. 12H). SLNCR knockdown significantly decreased proliferation of both MSTCs compared to a scramble siRNA control (p < 0.0001, FIG. 12G). Proliferation of the melanoma cell line A375 is slightly reduced upon SLNCR knockdown (p<0.0001) likely due to lower endogenous levels of SLNCR which limits the fold depletion and range of knockdown-related phenotypes (FIG. 12G) (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Importantly, knockdown of SLNCR did not alter the percentage of apoptotic cells in either MSTC, or in A375 melanoma cells (FIG. 13D). Collectively, these experiments indicate that SLNCR increases melanoma proliferation. Because depleting SLNCRl alone did not affect cell growth, SLNCR isoforms likely share an overlapping role in regulation of melanoma proliferation, with SLNCR2 and SLNCR3 able to functionally compensate in the absence of SLNCRl (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025- 37).

Previous work revealed that SLNCR directly binds AR, recruiting it to specific genomic locations to modulate AR transcriptional activity (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Consistent with previous studies implicating AR in melanoma cell growth and proliferation (Morvillo, V., et al, Melanoma Research, 2002. 12: p. 529- 538; Morvillo, V., et al, Pigment Cell Res, 1995. 8: p. 135-141), the anti-androgen flutamide significantly decreases melanoma cell proliferation (FIG. 15A, p<0.0001, FIG. 15 A). While this indicates an androgen-dependent role for AR in melanoma proliferation, SLNCR and AR interact even in the absence of canonical ligand-induced AR activation (i.e., in hormone-deprived cells, Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). While standard cell culture conditions use fetal bovine serum which contains exogenous hormones, it is unknown if these standard cell culture conditions accurately reflect the natural hormone state of the melanoma tumor microenvironment. To test if AR regulates melanoma proliferation in the absence of androgen, cell proliferation of hormone-deprived cells (cells grown in phenol-red free media supplemented with charcoal stripped media) was quantified before and after depletion of AR. Two AR-targeting siRNAs result in -60- 90% knockdown oiAR in all 3 hormone-deprived melanoma cells (FIG. 15B and 15C). Knockdown of AR significantly decreases proliferation of hormone-deprived melanoma cells (p<0.0001, FIG. 14A), confirming that AR regulates melanoma proliferation in an androgen-independent manner (FIG. 14A). To confirm that SLNCR and AR cooperatively regulate melanoma cell growth and proliferation, short single-stranded RNA oligos were designed to sterically block the interaction of SLNCR and AR. These oligos block the interaction either by direct binding to AR, mimicking the sequence region of SLNCR responsible for mediating this interaction (MEVIIC 1 or MEVIIC 2) and dominantly repress AR-binding SLNCR, or are the reverse complement to the SLNCR sequence required for AR binding, generating dsRNA that is unable to bind AR (ANTISENSE 1 or ANTISENSE 2). The antisense oligonucleotides are specifically designed to bind to SLNCR without eliciting RNase H-mediated degradation of SLNCR. Gymnotic delivery (i.e. delivery without the use of transfection reagents) of either AR- or _^ SZNCR-binding 2'-deoxy-2'- fluoro-D-arabinonucleic acid (2'-FANA) modified oligonucleotides significantly decreases melanoma proliferation (FIG. 14B) without decreasing SLNCR expression (FIG. 14B and 15D). Moreover, these oligos contain FANA modifications, enabling reagent-free gymnotic uptake into the cells. Steric blocking oligonucleotides occasionally upregulate SLNCR expression 2-3 fold, possibly resulting from a feedback loop regulating SLNCR expression that is initiated upon inhibition of SLNCR function. Decreased cell proliferation upon inhibition of the SLNCR-AR interaction, despite increased SLNCR expression, further indicates that SLNCR and AR cooperatively regulate melanoma proliferation. Thus, these steric blocking oligos confirm that SLNCR and AR cooperatively regulate melanoma cell growth and represent a possible novel melanoma therapeutic. Example 10: SLNCR and AR cooperatively regulate expression of a subset of melanoma genes

To gain mechanistic insights into the molecular pathways regulating AR- and ^NCR-mediated cell proliferation, AR chromatin immunoprecipitation and massively parallel sequencing (ChlP-seq) was used to identify genomic loci bound by ligand-free AR (i.e., hormone-deprived cells). Because AR expression in the MSTCs is below the level detectable by western blot and are therefore technically challenging, AR ChlP-seq was performed from A375 melanoma cells, which express detectable levels of AR (FIG. 15B). To identify loci bound specifically by androgen-independent AR, ChlP-seq was performed from hormone-deprived cells. Moreover, to confirm the sensitivity of ChlP-seq to detect potentially subtle IncRNA-induced changes in AR function, AR binding sites were identified in cells transfected with either an empty or ,SZNCR/-expressing vector, previously shown to affect AR occupancy at least 1 target gene (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). Because SLNCR1 regulates AR occupancy at at least one genomic region (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37), cells were transfected with either an empty or ,SZNCR/-expressing vector, representing either endogenous SLNCR levels or ,SZNCR/-overexpression conditions. AR ChlP-seq identified a total of 9,974 AR binding regions (referred to as 'active regions', 5,717 for the empty vector and 8,239 for the >SZNCR/-expressing vector) in hormone-deprived A375 melanoma cells (Table 7). AR ChlP-seq identified a total of 5,717 AR binding regions in hormone- deprived A375 melanoma cells with only endogenous SLNCR1 levels (listed under l_A375_pNL7_minus_lncRNA) (Table 7). Consistent with IncRNA-regulated AR function, SLNCR1 expression increased the number of AR active regions to 8,239, identifying a total of 4,257 unique AR binding sites (listed under

2_A375_pNL97_plus_lncRNA: : 1 Peak Value on the table 7) (FIG. 16 A). The majority of the binding events in both the vector and ,SZNCR-expressing cells occurred within 10,000 bases pairs of annotated genes (as defined by NCBI, 78.66% and 79.13%, respectively, as shown in Tables 8 and 14), hereafter referred to as 'active genes,' indicating that AR regulates gene expression even in the absence of canonical androgen signaling. Table 7 contains active regions identified for both the vector control (pNL7 minus IncRNA), as well as the SLNCR expression condition (pNL97 plus IncRNA), and all the statistics is shown in Table 8. Moreover, SLNCR1 overexpression significantly increased tag density at transcriptional start sites (FIG. 16A). Several previously identified AR-target genes, including C15orf40, POLR2A, and WDR70 (Wang, B.D., et al, Prostate Cancer, 2013. 2013 : p. 763569; Massie, C.E., et al, EMBO Rep, 2007. 8(9): p. 871-8), display strong (average peak intensities -60-110) and clear AR ChlP-seq peaks, consistent with direct AR binding and confirming the accuracy of the AR ChlP-seq analysis. Moreover, AR ChlP- seq analysis also identified known ligand-independent AR targets, including R6A1 (Average peak -40) MIPEP (average peak -175), and WWOX (average peak -40) (Lin, B., et al., PLoS One, 2009. 4(8): p. e6589; Massie, C.E., et al., EMBO Rep, 2007. 8(9): p. 871- 8; Wang, B.D., et al., Prostate Cancer, 2013. 2013 : p. 763569).

Several lines of evidence indicate that SLNCRl regulates AR chromatin occupancy.

SLNCRl overexpression (i) increased the number of AR active regions (from 5,717 to 8,239 active regions, with 4,257 unique sites), without increasing expression or altering localization of AR (FIG. 16A) (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37), (ii) increased tag density at transcriptional start sites (FIG. 16 A), (iii) increased AR occupancy at 101 of 112 sites with altered AR binding as identified by differential binding analysis

(using model-based analysis of ChlP-seq, MACS; Tables 9A and 9B); and (iv) dysregulated 9 (9.2%) of the possible 98 associated genes (included in our RNA-seq analysis) exhibiting differential AR binding (p<0.05) (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Collectively, these data indicate that SLNCRl recruits AR to particular genomic loci.

Because AR binds multiple SLNCRl- and -SZNCR-regulated genes even in the absence of ectopically expressed SLNCRl (Table 8), all identified AR-bound genes were considered in subsequent analyses.

To identify candidate AR- and -SZNCR-regulated genes, genes that are both AR- bound (active region within 10,000 bp of gene annotation) as determined by AR ChlP-seq (Tables 8 and 9), and -SZNCR-regulated, as determined by RNA-seq (Tables 6A and 6B) were searched. Interestingly, consistent with a functional relationship between SLNCR and AR, almost half of -SZNCR-regulated genes are directly bound by AR. For example, 25.3% of genes (9, 139 out of 36,074 NCBI-defined genes) were bound by AR but 43.2% of SJNCR-regulated genes (96/222) were bound by AR (Binomial Test p<0.0001,FIG. 16B and Table 10), indicating that AR directly regulates a large number of SLNCR

transcriptional targets. Additionally, 45.5% of genes (50/110) dysregulated by SLNCRl overexpression were bound by AR (Binomial Test p=0.0001) and 43.8% (53/121) of genes dysregulated by SLNCRl knockdown were bound by AR (Binomial Test p=0.0003), further confirming that AR-binding is enriched on SLNCR- and SLNCR1 -regulated genes (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). Consistent with SLNCR and AR

cooperatively regulating melanoma cell proliferation, AR-binding is enriched among the SJNCR-regulated proliferative genes (-37%, 18/49, FIG. 17C). Interestingly, 53 AR-bound genes are significantly upregulated upon SLNCR knockdown, while 43 genes are significantly downregulated, indicating that the directional regulation of cooperative ARJSLNCR function depends on genomic context.

Many of the AR-bound, -SZNCR-regulated genes are known or believed to play important roles in melanoma etiology, including the Gro oncogene and chemokine ligand CXCL2 (log2 fold change -2.0), the JUN oncogene (log2 fold change 0.9), the STAT3 transcription factor (Log2 fold change -0.9), the interleukin IL24 (log2 fold change 2.5), and the melanoma cell adhesion molecule MCAM (log2 fold change 1.2). To validate that the analysis faithfully identified AR- and -SZNCR-regulated genes, RT-qPCR was used to quantify levels of JUN, CXCL2, and STAT3 before and after siRNA-mediated knockdown of either SLNCR or AR. RT-qPCR of several AR-bound, SJNCR-regulated genes (JUN, CXCL2, and STAT3) before and after siRNA-mediated knockdown of either SLNCR or AR confirms that SLNCR and AR regulate expression of these target genes in both WM1976 and A375 cells (FIG. 17A). In agreement with RNA-seq, knockdown of SLNCR

significantly increases levels of JUN (1.5 - 2.5 fold), while significantly decreasing levels of CXCL2 and STAT3 (0.75 - 0.5 fold) in both WM1976 and A375 cells. Knockdown of AR recapitulates increased JMVlevels (1.5 - 2.5 fold), while decreased STAT3 levels are observed in A375 cells only (0.5 fold), possibly due to lower levels of AR (and therefore a weakened phenotype) in WM1976 cells. Interestingly, contrary to decreased levels upon SLNCR knockdown, AR knockdown significantly increases levels of CXCL2 (1.25 - 1.75 fold), revealing that SLNCR and AR may regulate expression of target genes in an opposing manner. Integrative analysis of SLNCR RNA-seq and AR ChlP-seq dataset reveals that AR-binding is enriched on ffiNCR-regulated genes, and indicate that AR and SLNCR similarly regulate expression of many of these target genes both in vitro and in vivo.

To confirm SLNCR- regulated expression of several of these candidate target genes in vivo, expression correlations from The Cancer Genome Atlas (TCGA) was interrogated. As expected, SLNCR expression is positively correlated with expression of STAT3

(Spearman correlation coefficients of r = 0.22, p-value < 0.01). SLNCR expression is also correlated with TMEM59 and FAM46A expression (Spearman correlation coefficients r = 0.49, p-value < 0.0001 and r = 0.29, p-value < 0.001, respectively), genes which are significantly decreased upon SLNCR depletion (Log2 fold change -0.79 and -0.7, respectively), while SLNCR expression is negatively correlated with mRNA levels of ZYX (Spearman correlation coefficient of -0.43), a gene which is significantly upregulated with SLNCR knockdown (Log2 fold change 0.8). Analysis of TCGA expression data reveals that AR is significantly correlated with expression of over half of -SZNCR-regulated genes (148/222), 66 of which are also bound by AR based on our ChlP-seq analysis (Table 6D). Correcting for multiple hypothesis testing (Bonferroni correction) maintained significance of AR correlation with 92 _^ STJVCR-regulated genes, 43 of which are bound by AR. There is a significant concordance between target gene correlations with SLNCR and AR expression (Spearman r = 0.2, p = 0.003 overall), further confirming that SLNCR and AR cooperatively regulate expression of many of these target genes, in vivo. Collectively, integration of SLNCR RNA-seq and AR ChlP-seq analyses identifies many _^ STJVCR-regulated, AR-bound genes, many of which are implicated in melanoma growth and progression.

Example 11: SLNCR and AR cooperatively inhibit expression of the cyclin-dependent kinase inhibitor p21 in a p53-independent manner

The mechanism of AR- and -SZNCR-mediated regulation of one representative gene was examined. Because SLNCR and AR cooperatively increase melanoma cell

proliferation, AR-bound and -SZNCR-regulated genes implicated in melanoma cell growth were searched for. Out of the 222 ffiNCR-regulated genes, 49 (-22%) are implicated in cell proliferation. Moreover, 18 of these 49 proliferative genes (-37%) are directly bound by ligand-independent AR, indicateing that AR and SLNCR increase cell proliferation through regulation of multiple genes (FIG. 17C). Of these, the -SZNCR-mediated regulation of CKDN1A, the gene encoding the tumor suppressive cyclin dependent kinase (CDK) inhibitor 1A (p21 ^Cipl/Wafl ), was particularly interesting, for several reasons: (i) p21 is an important regulator of cell cycle progression and anti -proliferative pathways, inducing Gl or G2 cell cycle arrest (Gire, V., and V. Dulic, Cell Cycle, 2015. 14: p. 297-304; Giuliano, S., et al., Pigment Cell Melanoma Res, 2011. 24: p. 295-308; Yanagi, T., et al., The Journal of Biological Chemistry, 2003. 278: p. 39906-39911), (ii) its expression is commonly dysregulated in multiple tumors , including melanoma (Abbas, T., and A. Dutta, Nat Rev Cancer, 2009. 9: p. 400-414; Jiang, H., et al., Oncogene, 1995. 10: p. 1855-1864; Vidal, M.J., et al., Melanoma Research, 1995. 5: p. 243-250), (iii) while expression is tightly correlation to wildtype p53 activity, p53 -independent regulation remains unknown and (iv) it is known to be transcriptionally regulated by other non-coding RNAs (Dimitrova, N., et al., Molecular Cell, 2014. 54: p. 777-790; Leveille, N., et al., Nature Communications, 2015. 6: p. 6520; Morris, K.V., et al., PLoS Genetics, 2008. 4: p. el000258). Additionally, (v) p21 inhibits melanoma proliferation, as depletion of p21 increases proliferation of A375 cells (FIG. 18G and 18H, p<0.0001) (Yanagi, T., et al., The Journal of Biological

Chemistry, 2003. 278: p. 39906-39911). In agreement with the RNA-seq analysis, knockdown of SLNCR in WM1976, as well as A375 cells, results in a significant upregulation of CDKN1A mRNA or p21 mRNA (-1.5-2.5 fold increase, FIG. 18A). To test if AR also inhibits expression of CDKN1A, mRNA levels were quantified following knockdown of AR and observed a similar increase in CDKN1A (i.e., p21 mRNA) levels 1.3-2.5 fold increase, FIG. 18B). Furthermore, increased p21 mRNA levels upon knockdown of either AR or SLNCR correspond to an increase in p21 protein levels (-1.4-3 fold increase, FIG. 18C and 18D). Thus, SLNCR and AR transcriptionally repress p21 expression.

Expression of p21 may be regulated by the related tumor suppressor protein p53, or through less well-characterized p53-independent mechanisms. Neither p53 mRNA (TP53) nor protein are significantly altered upon knockdown of AR or SLNCR in WM1976 or A375 (p53 ^WT ) cells, indicating that p21 is regulated in a p53 independent manner (FIG. 19A - FIG. 19C). Moreover, knockdown of SLNCR or AR significantly upregulates p21 mRNA and protein levels in the p53 mutant (p53 ^L145R , inactive) primary malignant melanoma cell line SK-MEL-28 (-1.5-4 fold increase in both mRNA and protein, FIGs. 18E and F), confirming p53-independent regulation. Thus, the data indicates that AR and SLNCR directly inhibit expression of p21 in a p53-independent manner.

In addition to cell proliferation, p21 regulates many additional cellular processes.

Since SLNCR regulates p21 expression, whether SLNCR knockdown mimics p21 -induced melanoma phenotypes was investigated herein. First, p21 inhibits CDK activity, leading to Gl or G2 cell cycle arrest. Consistently, knockdown of SLNCR induces G2 cell cycle arrest in WM858 (p53 ^MUT ) cells and WM1976 (p53 ^WT ) cells, observed as a significant increase in the percent G2/M cells while the percent of Gl/GOdecreased (FIG. 20A) (Gire, V., and V. Dulic, Cell Cycle, 2015. 14: p. 297-304; Giuliano, S., et al., Pigment Cell Melanoma Res, 2011. 24: p. 295-308; Yanagi, T., et al., The Journal of Biological Chemistry, 2003. 278: p. 39906-39911). Knockdown of SLNCR does not significantly alter A375 cell cycle progression (FIG. 19A), likely due to lower overall levels of SLNCR in these cells.

Knockdown of AR does not affect cell cycle progression in any of these cells (FIG. 2 IB), possibly due to pleotropic effects of the transcription factor. These data demonstrate that SLNCR depletion phenocopies p21 induction of G2/M melanoma cell cycle arrest.

In addition to inducing cell cycle arrest, p21 also regulates activity of multiple transcription factors (Abbas, T., and A. Dutta, Nat Rev Cancer, 2009. 9: p. 400-414). Thus, nuclear transcription factor activity in WM1976 cells was quantified before and after depletion of SLNCR. Therefore, nuclear transcription factor binding to specific DNA motifs in WM1976 cells before and after depletion of SLNCR was quantified (FIG. 20B and 20D). SLNCR knockdown reduced DNA binding of two transcription factors bound to and regulated by SLNCR1 (AR and Brn3a) by 60%, as measured by transcription factor activation array. Knockdown of SLNCR significantly reduces AR and BRN3a activity (-60%), transcription factors bound and regulated by SLNCR1. SLNCR knockdown also decreased DNA binding by other candidate ^NCR-interacting proteins, including EGR-1 (70%), E2F-1 (30%), ATF2 (70%), and the ATF2-containing Activator Protein 1 (API) transcription factor heterodimer (-60%) (Schmidt, K., et al, Cell Rep, 2016. 15(9): p. 2025-37). Decreased DNA binding by AR occurred independently of altered AR

expression or localization (FIG. 19D) (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025- 37). SLNCR knockdown also alters activity and DNA binding of known p21 targets, decreasing activity of the estrogen receptor (ER) and C/EBP (both -40% reduced activity) (Fritah, A., et al, Mol Cell Biol, 2005. 25(6): p. 2419-30; Harris, T.E., et al, J Biol Chem, 2001. 276(31): p. 29200-9), and significantly increasing SMAD activity (-2.7 fold) and DNA binding by SMAD (-270%) (Dai, M., et al, Breast Cancer Res, 2012. 14(5): p.

R127). Moreover, E2F-1 is both a candidate SLNCR interactor (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37) and known to be directly inhibited by p21 (Dimri, G.P., et al, Mol Cell Biol, 1996. 16(6): p. 2987-97; Teplyuk, N.M., et al, Oncotarget, 2015. 6(6): p. 3770-83; Isaeva, A R. and V.I. Mitev, J Dermatol Sci, 2009. 55(2): p. 134-6; Jung, Y.S., Y. Qian, and X. Chen, Cell Signal, 2010. 22(7): p. 1003-12) and is consistently

downregulated upon knockdown of SLNCR (30%). This data confirms that SLNCR both directly (through protein-RNA interactions) and indirectly (through p21 -mediated regulation) affects and regulates activity of multiple transcription factors. Collectively, SLNCR knockdown phenocopies p21 -mediated cell cycle arrest and transcription factor regulation, indicating that SLNCR knockdown induces a biologically-relevant upregulation of p21.

Because SLNCR knockdown dysregulated the activity of multiple transcription factors (FIG. 20B), it is next investigated if altered transcription factor activity might explain SLNCR-regulated transcriptional networks, and transcriptional effects of SLNCR not directly attributed to AR binding. Importantly, since AR is a known mediator of SLNCR-regulation, the analysis was limited to the 126 _^ STJVCR-regulated genes not bound by AR (FIG. 16B). Indeed, when limiting the analysis to the 126 _^ STJVCR-regulated genes not bound by AR (FIG. 16B), a significant enrichment of -SZNCR-regulated transcription factor networks was observed, as identified by the transcription factor activation array (FIG. 20B), including ER, AR, C/EBP, EGR1 and E2F1 (FIG. 20C). Interestingly, SLNCR knockdown also significantly alters expression of many genes in the CREBl and STAT3 networks, i.e., ST AT3 -regulated genes, a transcription factor whose expression is regulated by both SLNCR and AR (Table 10 and FIG. 17A). STAT3 was previously identified as being AR-bound and -SZNCR-regulated (Table 10), indicating that SLNCR directly regulates STAT3 expression. Enrichment of genes in the CREBl network may be artifact resulting from a high degree of overlap between transcription factor targets. However, depletion of SLNCR does not appear to affect STAT3 activity (FIG. 20D), warranting further

investigation into the nature of STAT3 regulation. Collectively, these studies indicate that, in addition to cooperative transcriptional regulation of AR-bound genes, SLNCR regulates expression of additional non-AR bound genes through modulation of transcription factor activity, possibly through inhibition of p21. Moreover, SLNCR knockdown mimics known p21 -induced phenotypes, including cell cycle arrest and altered transcription factor activity. Thus, SLNCR-mediated regulation of p21 expression results in biologically-meaningful phenotypic changes.

Example 12: SLNCR binds EGR1 and recruits AR to EGR-bound loci

To investigate how SLNCR and AR mechanistically regulate target gene expression,

AR ChlP-seq data was interrogated to identify either known or novel motifs present and enriched in AR-bound genes (AR ChlP-seq datasets). MEME and TOMTOM analysis

(Bailey, T.L., et al., Nucleic Acids Research, 2009. 37: p. W202-208) of AR ChlP-seq peaks identifies an enrichment of a conserved motif similar to the DNA binding motif of

RE 1 -Silencing Transcription Factor (REST, or Neuron-Restrictive Silencer Factor, NRSF), a transcriptional repressor known to mediate AR activity in prostate cancer ((p=le ^"191 , FIG. 23A) (Svensson, C, et al., Nucleic Acids Res, 2014. 42(2): p. 999-1015). However, the REST motif was not enriched in AR binding sites among ,SZNCR-regulated genes. Instead, the DNA binding site of the EGRl transcription factor was enriched (p=2.24e ^"05 , FIG. 22A). Limiting the analysis to only AR ChlP-seq peaks found near or within SLNCR- regulated genes identifies a unique conserved motif highly similar to the EGRl DNA binding motif (p-value 2.24e-05, FIG. 22A), indicating that AR binds to -SZNCR-regulated genes through a distinct mechanism, perhaps in cooperation with EGRl .

Both previous (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37) results and results described herein (FIG. 20A-20C) indicate that EGRl and SLNCR directly and/or functionally interact. In further support of a direct physical interaction, incubation of biotinylated, full length SLNCRl with A375 melanoma cell lysate followed by streptavidin pulldown significantly enriches EGRl and AR (FIG. 22B), as well as the RNA binding ribosomal protein Rps6 (FIG. 22A). This interaction was independently validated using RNA immunoprecipitation (RIP) assays, which enriched SLNCR (-4-10 fold) in RNAs immunoprecipitating with EGRl (FIG. 22F). These data confirm that endogenous levels of SLNCR and EGRl interact in A375 cells.

To distinguish between direct interaction of SLNCR and EGRl versus an indirect interaction mediated by secondarily-associated macromolecules, RNA electrophoretic mobility shift assays (REMSA) was performed. As shown by REMSA, recombinant EGRl protein (corresponding to amino acids 282-433) significantly alters the mobility of full length, in vitro transcribed biotinylated SLNCRl in a protein concentration dependent manner (FIG. 22B-22C). Interestingly, EGRl binding significantly increases RNA mobility (sub-shifted complex),, as opposed to more commonly observed RNA retardation (super-shifted complex), possibly a result of altered (likely relaxed) RNA secondary structure upon protein binding. Unlabeled SLNCRl is able to compete for EGRl binding, observed as a loss of increased mobility {i.e. upward shift), confirming a direct and specific EGRl -SLNCRl interaction. Collectively, these data further confirm that endogenous SLNCR and EGRl directly interact in vitro and at endogenous levels in melanoma cells.

Given that SLNCRl binds to EGRl, and the EGRl motif is significantly enriched in AR-bound, -SZNCR-regulated genes, it is hypothesized that AR colocalizes and binds to a subset of -SZNCR-regulated genes in cooperation with EGRl . Thus, EGRl ChlP-seq was first used to determine global EGRl binding sites in A375 cells, identifying a total of 16,789 active regions (Table 11). Interestingly, despite a relatively small overlap (18.5%) between EGRl and AR bound regions overall (FIG. 23B), the majority (4,091 out of 6,960, or 58.8%) of EGRl active genes (i.e. active regions that are located within 10,000 bp of an annotated gene, Table 12) overlap with AR active genes. A higher proportional overlap between AR and EGRl binding-sites within gene regions indicates a functional relationship between the two transcription factors. EGRl binds a total of 8,373 active regions (Table 11), corresponding to a total of 6,960 active genes (Table 12). Consistent with expected genomic occupancy, EGRl ChlP-seq analysis identified many known EGRl -regulated genes, including CCDC28B, ATAD2, and the promoter of EGRl itself (FIG. 22H) (Arora, S., et al., Genome Biol, 2008. 9: p. R166; Kubosaki, A., et al., Genome Biol, 2009. 10: p. R41; Subbaramaiah, K., et al., The Journal of Biological Chemistry, 2004. 279: p. 12647- 12658). Unlike AR, EGRl appears to bind its known DNA binding sequence in A375 cells, as a sequence resembling this motif is the most significantly enriched in EGRl ChlP- seq peaks (p < lxlO ^"5 , FIG. 22D).

Surprisingly, a significant overlap between AR and EGRl binding sites was observed. Additionally, AR and EGRl frequently co-bound at -SZNCR-regulated genes.

Although AR and EGRl bound only 25.3% (9, 139 of 36,074) and 19.3% (6,960 of 36,074) of all genes, respectively, AR bound to 58.8% of EGRl-bound genes (4,091 out of 6,960 total EGRl active genes, Binomial Test p<0.0001, FIG. 23C). It is important to note that co-bound genes were identified through a stringent analysis of overlapping ChlP-seq reads. This was accomplished by directly integrating AR- and EGRl - ChlP-seq reads (spanning an average of only 747 bps) rather than extrapolating binding events occurring within 10,000 bps of an annotated gene. EGRl bound to 31% of -SZNCR-regulated genes (68 out of 222 genes, Binomial Test, p=0.0003) and 46% of -SZNCR-regulated, AR-bound genes (44 out of 96 genes Tables 13 A and 13B, Fisher Exact pO.0001, FIG. 22E). Consistent with cooperative transcription factor binding, AR and EGRl ChlP-seq peak read intensities overlapped within many of the 44 ffiNCR-regulated, AR- and EGR-bound genes, including PSAT1, SHF, SLC36A11, and SSU72, and the divergently-transcribed, SZNCR-regulated gene pair NAA50 and ATP6V1 A (FIG. 22G). Collectively, these data reveal that AR and EGR binding sites overlap more frequently than would be expected by chance, and that these sites are enriched among _^ STJVCR-regulated genes. Because AR and EGRl binding occurs at known or predicted EGRl DNA binding motif, these data indicate that EGRl is required for regulation of at least a subset of AR- and -SZNCR-regulated genes. If a functional interaction exists between SLNCR, AR and EGRl that dictates binding of these transcription factors to chromatin, a higher frequency of EGRl at SLNCR- regulated AR-bound genes would be expected. Thus, the identification of SLNCR- regulated genes with the AR and EGRl ChlP-seq data were integrated. Interestingly, while EGRl binds to roughly a third of SLNCR-regulated genes (68 out of 222 total, ~31%),

EGRl is bound to a higher proportion of SLNCR-regulated, AR-bound genes (44 out of 96 AR-bound, SLNCR-regulated genes, -46%, Table 13 A and Table 13B). Briefly, Table 13 A contains expression data for all SLNCR-regulated genes, as identified via RNA-seq. The last 3 columns contain information on whether a ChlP-peak was identified for AR with endogenous SLNCR (pNL7_minus_lncRNA, column L), AR with ectopic SLNCR expression (pNL97 _plus_lncRNA, column M), or EGR (column N). A T denotes the presence of a peak, while '0' denotes absence of a peak. Moreover, in contrast to the -58.8% of all EGRl active genes that overlap with AR genes, the percentage of overlap increases to 64.7% (44 out of 68 genes) when limited to only SLNCR-regulated genes. Collectively, these data indicates that EGRl is involved in AR- and SLNCR-mediated gene regulation of at least a subset of SLNCR-regulated genes. Since SLNCR binds to both AR and EGRl, AR and EGRl co-bind EGRl motifs within _^ SZNCR-regulated genes, and EGRl occupancy is proportionally higher at SLNCR-regulated and AR-bound genes, SLNCR might recruit AR to EGRl -occupied genomic regions. If true, SLNCR- and AR-based regulation of target would require EGRl . Specifically, EGRl should regulate expression of these genes, and SLNCR- and AR-based regulation would require an intact EGRl DNA binding site. In support of EGRl -mediated regulation, EGRl expression is significantly correlated with expression of over half of -SZNCR-regulated genes (65.3%, 145/222), while significant correlation is maintained for 71 of these genes after correcting for multiple hypothesis testing (Bonferroni correction, p < 0.00023, Table 6D). To first test if EGRl regulates p21, p21 mRNA and protein expression was quantified following siRNA- mediated knock down of EGRl . EGRl positively regulates expression of p21 because EGRl knockdown decreased p21 mRNA and protein levels (FIG. 24B-24C). Contrary to results seen upon knockdown of AR or SLNCR, knockdown of EGRl significantly decreases p21 mRNA and protein levels (FIG. 14A and 14B). Moreover, EGRl regulates p21 independent of p53, because EGRl knockdown in the p53-mutant SK-MEL-28 cells decreased p21 levels (FIG. 24B-24C and 25 A). Decreased p21 levels occurs

independently of p53, as this decrease is seen in the p53 mutant SK-MEL-28 cells (FIGs. 14A, 14B and 15A). Thus, EGRl increases CDKNlA/p21 expression, acting in opposition to AR and SLNCR. In other words, in contrast to SLNCR and AR which repress p21, EGRl activates p21 expression in a p53-independent manner.

It is next tested if AR- and -SZNCR-mediated CDKNIA regulation requires EGRl binding to the CDKNIA loci. To test whether SLNCR- and AR-mediated p21 regulation requires an intact EGRl binding site, a firefly luciferase reporter construct containing 4663 nucleotides (nts) of the CDKNIA promoter, spanning from the first transcription start site to 2966 nts upstream of the translation start codon and containing the AR- and EGRl -bound consensus EGRl DNA binding site, was generated (FIG. 24). Surprisingly, and contrary to increased endogenous mRNA and protein levels, knockdown of SLNCR or AR significantly decreases luciferase activity and the expression of the ectopic CDKNIA luciferase reporter in A375. This discrepancy is likely due to inherent differences between genomic DNA and ectopically-expressed plasmid DNA and is discussed in more detail later. Consistent with ligand-independent AR activation, SLNCR and AR knockdown also decreased expression of the CDKNIA reporter even in the absence of exogenous hormones (FIG. 25B). Most importantly, however, mutation of the EGRl binding site negates the ability of AR or SLNCR to regulate expression from the CDKNA promoter. These data strongly indicate that AR and SLNCR associate with the CDKNIA promoter through the EGRl binding site and EGRl DNA binding site is required for SLNCR- and AR-based regulation of CDKNIA.

Example 13: P53-deficient female melanomas express higher levels of p21 than male melanomas

Given that SLNCR mediates ligand-independent AR function in melanoma, and the gender-specific expression of both AR and SLNCR, TCGA data for gender-specific expression of p21 was interrogated. Due to the likelihood that p53 -dependent mechanisms of p21 regulation are likely dominant pathways of regulation, the analysis was limited to p53-deficient melanomas (as determined via RNA-seq). Consistent with the model that SLNCR-mediates AR repression of CDKNIA, females express significantly higher levels of p21 than males (FIG. 25D).

Example 14: AR and Brn3a binding sites reside within an unstructured region of SLNCR1, in vivo The in vitro data presented herein indicate that AR preferentially binds to unstructured, single-strand RNA in a sequence-specific manner. However, REMSA combined with sequence and structural prediction analyses do not accurately predict how an RNA sequence folds in the context of an entire transcript within a cell. Indeed, proteins and other cellular macromolecules can lead to changes in RNA structure that cannot be accurately predicted by in silico-o Ay calculations of RNA in solution. Therefore, it was sought to experimentally determine if SLNCRFs AR-binding motifs are (1) accessible to protein interactions and (2) predicted to be bound by protein, in vivo. Specifically, the secondary structure of SLNCR1 was modeled using measurements of RNA flexibility within patient-derived melanoma cells.

To understand the local RNA structure and nucleotide accessibility of SLNCR1 AR- binding motifs, in vivo, SHAPE and mutational profiling (MaP) was performed. SHAPE- MaP provides experimental evidence for RNA secondary structures at nucleotide resolution, including in living cells (Siegfried, N. A., et al., Nature Methods, 2014. 11 : p. 959-965; Smola, M.J., et al., Nat Protoc, 2015. 10: p. 1643-1669). The SHAPE-MaP approach was applied using the melanoma short-term culture (MSTC) WM1976, whose invasion potential can be disrupted through siRNA-mediated depletion of SLNCR1

(Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). Importantly, MSTCs have undergone limited passages outside of the patient and therefore closely reflect the genetics and relative expression patterns of the tumor, in situ.

The structure of SLNCR1 was probed using the SHAPE reagent 5-nitroisatoic anhydride (5NIA) and the methylating agent dimethyl sulfate (DMS). 5NIA has an extended half-life in solution (100 seconds), enabling superior structure probing in certain cell lines. DMS methylates nucleotide bases at a higher rate than 5NIA acylates nucleotide sugars, but DMS reacts most strongly with only adenosine and cytosine. Using 5NIA and DMS, the nucleotide flexibilities of SLNCR1 was probed both within WM1976 cells (in- cell) and after gentle extraction of the RNA from WM1976 cells into a protein-free environment (cell-free). The SHAPE reactivities of cell-extracted RNA (cell-free) can be used to model RNA in its native cellular structure, and in-cell SHAPE reactivities both provide further evidence in support of structured motifs and also identify RNA sequences whose structure changes in the presence of proteins. To discern whether SLNCR1 modifications or protein-mediated folding events lead to different stable RNA

conformations in the nucleus, where the SLNCR-AK complex can regulate transcription, versus in the cytoplasm, extracted RNA (cell-free) was separated into nuclear and cytoplasmic fractions before treatment with 5NIA or DMS. Sites of SHAPE and DMS modification within in-cell and cell-free RNAs were encoded as non-template-encoded nucleotides or deletions in the cDNA generated during reverse transcription, which can then be detected by massively parallel sequencing (Homan, P.J., et al., Proc. Nat. Acad, of Sci. of the U. S. A., 2014. I l l : p. 13858-13863; Siegfried, N. A., et al., Nature Methods, 2014. 11 : p. 959-965; Smola, M.J., et al., Nat Protoc, 2015. 10: p. 1643-1669). Using a gene- specific reverse transcription primer and gene-specific PCR amplification primers, single- nucleotide resolution structural information on nts 403-780 of SLNCRl (SLNCRl ^{403'1 0} ) was obtained. This region encompasses most of SLNCRl ^coas (nts 372-672), including the Brn3a binding region (SLNCRl ^462'512 ) and both AR binding motifs, required for mediating melanoma invasion. The frequency of observed sequence changes in synthesized cDNAs were converted into SHAPE or DMS reactivity values for each nucleotide (Siegfried, N. A., et al., Nature Methods, 2014. 11 : p. 959-965; Smola, M.J., et al., Nat Protoc, 2015. 10: p. 1643-1669).

Generally high SHAPE reactivity was observed across SLNCRl ^{403'1 0} in cell-free RNA (FIG. 18A and 18C), indicating some level of flexibility within the region and a lack of a single, well-defined structure, even in the absence of cellular components. To calibrate the reactivity scale, SHAPE reactivities were also collected for the highly structured Ul small nuclear RNA (snRNA) (FIG. 17-18 and 22A). While a similar range of SHAPE reactivities was observed for both SLNCRl and Ul snRNA, reactive nucleotides were more widespread and diffuse in SLNCRl ^403'780 with fewer regions of dense lowly or highly reactive nucleotides that define the Ul snRNA (FIG. 17C, 18 A, 18C, 22A, and 22B). The average SHAPE reactivity in SLNCRl is highest across nts 495-570, which overlap the SLNCRl region that is necessary for interaction with Brn3a (SLNCRl ^462'512 ) (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). The SHAPE reactivity profiles of SLNCRl when extracted from the nucleus and the cytoplasm are nearly identical, indicating that events in either compartment do not induce stable changes to the structure of this region of SLNCRl (FIG. 18A and 18C). When RNAs were treated with 5NIA in-cell, the reactivity profile largely resembled the cell-free profiles, with regions of average to low SHAPE reactivity flanking a more highly-reactive conserved region (FIG. 18B). DMS modification patterns for both extracted and in-cell SLNCRl were broadly comparable to our 5NIA data despite the differences in modification mechanism (FIG. 19 and 20). Together, these data indicate that the SLNCR1 RNA lacks extensive stable secondary structure, especially within the Brn3a-binding region (SLNCR1 ^462'512 ). The in-cell data presented herein indicate that the cellular environment does not impose any wide-ranging structural shifts in SLNCR ^{403' 78C} and further confirm the presence of a large flexible region accessible to single-stranded RNA-binding proteins.

Example 15: Proteins bind multiple regions of SLNCR1 ^C0I1S , including AR motifs, in vivo

Comparison of cell-free and in-cell SHAPE and DMS profiles enables discovery of nucleotides that are protected in living cells, often indicative of protein binding to these regions of RNA (Smola, M.J., et al., Biochemistry, 2015. 54: p. 6867-6875; Smola, M.J., et al., Proc. Nat. Acad, of Sci. of the U. S. A., 2016. 113 : p. 10322-10327). For example, loops in the Ul snRNA engaged by the 70K, Ul A, and Sm proteins are highly reactive to SHAPE in the cell-free state but become lowly reactive in-cell (FIG. 17 and 18) (Kondo, Y., et al., Elife, 2015. 4; Pomeranz Krummel, D.A., et al., Nature, 2009. 458: p. 475-480). Many in-cell protections from SHAPE and DMS reactivity throughout SLNCRl ^{403'1 0} was observed, consistent with interactions with RNA-binding proteins (FIG. 18). Two of the three largest decreases in in-cell SHAPE reactivity were observed within the Brn3a- interacting region (SLNCR1 ^462'512 ). One statistically significant protection (Z-factor = 0.59, Z-score = 1.21) was observed at the 5' end of the first AR motif and another adjacent to the 3' end of the second motif (Z-factor = 0.60, Z-score = 1.20), indicating that these motifs are protein-bound in vivo. Agreement between the two probing reagents was observed: all DMS protection sites are within 5 nucleotides of an in-cell SHAPE protection (FIG. 18). Importantly, DMS probing corroborated the in-cell SHAPE protection of the first AR motif, further confirming that this motif is indeed protein-bound in cells. Moreover, stronger observed in-cell protection at motif 1 is consistent with the data presented herein showing that AR binds the SLNCR1 motif 1 with higher affinity than motif 2 (FIG. 12G).

Example 16: SHAPE-guided modeling of SLNCR ^C0I1S reveals a high-flexibility region flanked by limited RNA structure

It was next sought to better understand the structural context of the Brn3a-binding region and the AR motifs that are critical for AR-mediated function. To do this, the SLNCR1 5NIA data were further analyzed using the SuperFold SHAPE analysis pipeline, which enables modeling of RNA structures using measured SHAPE reactivities (Smola, M.J., et al., Nat Protoc, 2015. 10: p. 1643-1669). In SHAPE-driven models of RNA structure, regions of high SHAPE reactivity are modeled as mostly unpaired, regions of low SHAPE reactivity are modeled as paired, and the resulting base-pairing probabilities can be used to generate a minimum free energy model that represents the dominant RNA structure conformation. As a control, 5NIA SHAPE data and SuperFold were used to model the Ul snRNA extracted from WM1976 cells and recovered the correct four-stem structure (FIG. 22A and 18G). The reactivities for the Ul snRNA and SLNCR ^403'780 were placed on the same SHAPE reactivity scale (see Methods). Compared to Ul, the distribution of SHAPE reactivities in SLNCR ^403'780 is strongly shifted to the right, indicating that this region of SLNCR1 is much more reactive and thus less structured than the Ul snRNA (FIG. 22B). Shannon entropies were also computed for both RNAs, which is a measure of structural well-determinedness and RNA conformational diversity: high Shannon entropy corresponds to an absence of a well-defined structure and many potential RNA states. SLNCR1 ^403'780 global median entropy (0.19) is much greater than that of the highly structured Ul snRNA (0.01), indicative of a lack of well-defined structure within the IncRNA region (FIG. 22A and 22F). The Brn3a-binding region (SLNCR1 ⁴⁶² - ⁵¹² ) has both high SHAPE reactivity and high entropy, indicating a highly-flexible region that samples many conformations. The AR motifs themselves are moderately reactive when compared to the rest of SLNCR ^403'780 , but have differing levels of entropy between them. SLNCR1 AR motif 1 has moderately low SHAPE reactivity (0.32) but high entropy (0.44), indicating that the motif samples multiple base-pairing states but is unlikely to exist in a stable conformation. AR motif 2 is both lowly reactive (0.32) and has low entropy (0.07), indicating that this motif potentially persists in a partially base-paired state.

One strategy to detect well-defined structure motifs is to identify regions of both low SHAPE reactivity and low Shannon entropy (Siegfried, N.A., et al., Nature Methods, 2014. 11 : p. 959-965). Setting thresholds of median SHAPE < 0.4 (likely to be base- paired) and median entropy < 0.03 (base-pairing probability > 93%) identifies a low

SHAPE, low entropy region within Ul snRNA that, when expanded to include intersecting helices (See Methods), includes the entirety of Ul in a well-defined structure motif (FIG. 18B), as expected for this RNA with a well-defined functional structure. Applying the same thresholds to SLNCR ^403'780 reveals no low SHAPE, low entropy regions (FIG. 22F). Relaxing these thresholds or looking for regions with reactivities and entropies below the global medians enable modeling of potential secondary structures (FIG. 22 and 23), but the relative high reactivity and entropy of SLNCRl ^403'780 indicate that these structures do not persist stably in vivo. Together, the REMSA and SHAPE data presented herein confirm a model wherein SLNCRl ^cons is unstructured, enabling interaction with AR, in vivo.

Example 17: AR binding to its cognate motif is required for SLNCRl-mediated melanoma invasion

To assess the consequences of AR binding to it cognate elements in vivo, the two identified AR binding motifs were mutated and tested for ,SZNCR7-mediated melanoma invasion. Ectopic expression of SLNCRl or SLNCRl ^coas significantly increased the invasion of A375 melanoma cells, as quantified by matrigel invasion assays (FIG. 24). Importantly, expression of SLNCRl harboring site-directed mutations in the AR binding motifs 1 or 2 (SLNCRl^ or SLNCRl respectively) enabled SLNCRl-mediated melanoma invasion, though diminished when compared to wildtype. However, mutation of both motifs (SLNCRl ^{m MUT 1+2} ) abolished SLNCRl-mediated invasion (FIG. 24). The additive suppression of the invasion phenotype indicates that both motifs contribute to SLNCRl- and AR-mediated melanoma invasion, and is consistent with in vitro REMSA results indicating that AR binding requires the presence of at least one CYUYUCCWS motif (FIG. 14 A).

Because SLNCRl -mediated invasion occurs through transcriptional upregulation of

MMP9, SLNCRl and MMP9 levels were quantified upon expression of the three AR mutant SLNCRl constructs. MMP9 levels are significantly increased with expression of SLNCRl, but not changed upon expression of SLNCRl^^ ¹ or SLNCRl**™ ^{71 1 and 2} (~3-fold increase, FIG. 25). Interestingly, mutation of AR motif 2 attenuated (but did not abolish) SLNCRl-mediated invasion while MMP9 levels significantly increased upon expression of SLNCRl ^{m m]T 2} (~4-fold increase, FIG. 24B and 25B). The discrepancy of increased MMP9 levels but not increased melanoma invasion with expression of SLNCRl**^ ¹ - ² may be due to differences in the kinetics oiMMP9 upregulation in the presence or absence of SLNCRL s higher affinity AR binding motif 1.

The work presented herein identifies a novel sequence-specific IncRNA motif responsible for mediating protein binding and thereby IncRNA function. This work demonstrates that AR NTD binds with high affinity to unstructured, pyrimidine-rich RNA in a sequence-specific manner. This work further demonstrates that the AR-RNA interactions observed herein occur in an androgen-independent manner. Moreover, AR NTD, which lacks the ligand-binding domain, is sufficient for RNA binding. The observation that IncRNAs regulate AR activity independently of androgens has important implications for the study of AR in the context of human cancers. For example, multiple AR-driven cancers show only limited response to anti-androgen therapies, indicating androgen-independent mechanisms for multiple AR-driven cancers (Morvillo, V., et al., Pigment Cell Res, 1995. 8: p. 135-141; Morvillo, V., et al., Melanoma Research, 2002. 12: p. 529-538; Rose, C, et al., Eur J Cancer Clin Oncol, 1985. 21 : p. 1171-1174). Increasing our understanding of novel RNA-mediated AR function may help reconcile these inconsistencies and explain the failure of anti-androgens in the treatment of these tumors. AR is capable of binding SLNCR1 independently of canonical androgen-mediated AR activation and dimerization, which is normally necessary for AR target recognition

(Brinkmann, A.O., et al., J Steroid Biochem Mol Biol, 1999. 69: p. 307-313). It is intriguing that SLNCR1 contains two AR binding motifs in close proximity (spanning 24 nts). AR appears to interact independently and uniquely with each site, as evidenced by altered REMSA migration patterns and phenotypic differences upon mutation of either motif 1 or motif 2 (FIG. 3G and 9B). In-cell protections and phenotypic data indicate that motif 1 is predominantly involved in AR binding (FIG. 4C, 5B and 10D), possibly due to the higher affinity of AR to this sequence (FIG. 1 and 3F). The second, low affinity binding site may either (1) accommodate or anchor AR dimers following canonical activation, or (2) independently bind a second AR monomer, forcing proximal dimerization and possibly initiating cooperative activity. It is noted that high concentrations of full- length AR, but not AR NTD, resulted in a secondary sub-shift of the WT-41 probe, indicating a change in protein:RNA stoichiometry (FIG. IB and 1C). Because AR NTD is unable to form head-to-tail dimers, this indicates that, at high protein concentrations, dimerized AR may bind SLNCR1. Forced dimerization of AR is not required for transcriptional upregulation of MMP9 and increased invasion, as mutation of AR binding motif 2 does not abolish activity (FIG. 5 and 10). However, it is possible that binding of two AR monomers, particularly in cells expressing high AR, may elicit a unique transcriptional response.

The results presented herein indicate that AR binds RNA independently of its DNA- binding domain. AR may also bind actively transcribed IncRNAs, recruiting AR in cis to particular genomic regions independently of DNA encoded AR response elements (AREs). The proximal recruitment of AR to these regions may be sufficient to elicit AR-mediated transcriptional regulation of the IncRNA or other neighboring genes. In this way, AR may regulate expression of many non-ARE containing genes. Indeed, many IncRNAs are expressed divergently or antisense to protein-coding genes, and expression of many of these IncRNAs have been linked to transcriptional changes in expression of cognate mRNA. In addition to AR, many other transcription factors also bind RNA independently of canonical DNA interactions (Long, Y., et al., Sci Adv, 2017. 3 : p. eaao2110). Identification of RNA- binding transcription factors and elucidation of their RNA sequence- or structure- requirements is critical to fully appreciate the complexities of regulated gene expression.

Identification of protein-associated IncRNA motifs is critical to establish methods for predicting IncRNA function from sequence analysis alone, as indicated herein by identification of a novel, AR-binding sequence in the HOXA11 AS-203 IncRNA (FIG. 6). It is observed herein that AR NTD directly binds SLNCR1, but not PCGEM1 or HOTAIR, independently of additional protein cofactors. This selective binding is consistent with previous work revealing that the AR-PCGEM1 interaction requires binding of the IncRNA PRNCRl to AR's carboxyl -terminus, recruitment of DOT1L, and methylation at AR K349 (Yang, L., et al., Nature, 2013. 500(7464): p. 598-602). The results presented herein also indicate that AR NTD binds SRA1 (FIG. 7A). The AR-SRA1 interaction appears weaker and/or more transient compared to the AR-SLNCR1 interaction (FIG. 7A), possibly due to the presence of only one CYUYUCCWS motif found within a non-pyrimidine-rich sequence, compared to the two motifs found in SLNCRFs highly pyrimidine-rich sequence.

Many IncRNAs, including SLNCR1, are likely composed of varying combinations of discrete domains that impart functional specificity through assembly of associated proteins, RNA and DNA (Guttman, M., and J.L. Rinn, Nature, 2012. 482: p. 339-346). These IncRNA domains might impart function through a variety of means, for example: (1) by acting as allosteric regulators, assuming complex secondary and tertiary structures that present a unique surface for interaction and impart new or enhanced activities on associated macromolecules (Shamovsky, I, et al., Nature, 2006. 440: p. 556-560; Wang, X., et al., Nature, 2008. 454: p. 126-130); (2) by acting as sponges that compete for binding to factors and thus inhibit their effects (Lee, S., et al., Cell, 2016. 164: p. 69-80); or (3) by providing protein-specific landing platforms to scaffold formation of higher order complexes, thereby coordinating the activities of multiple associated proteins through induced proximity (Cerase, A., et al., Genome Biol, 2015. 16: p. 166). SLNCR1 ⁴⁰³ ™ behaves most like option 3, serving as a flexible scaffold for AR and Brn3 to coordinate their transcriptional activities. The high-affinity AR-binding motif 1, which exists at the junction of the highly flexible Brn3a-binding domain and a lower SHAPE and entropy 3' region, may be more accessible than AR-binding motif 2, which resides within the lower SHAPE and entropy region (FIG. 4H). Ultimately, binding of Brn3a to the flexible scaffold may enhance recognition of downstream AR-binding motifs, and recognition of AR motif 1 might increase the accessibility of motif 2. In other cancer cell lines, SLNCR1 interacts with epigenetic regulators EZH2, D MT1, LSD1, though it is unknown if these interactions occur in the melanomas used in this study (Ba, M.C., et al., Oncotarget, 2017. 8: p. 95542- 95553; Huang, M., et al., Mol Ther, 2017. 25: p. 1014-1026; Ma, C, et al., Oncotarget,

2017. 8: p. 32696-32705; Shi, X., et al., Oncotarget, 2016. 7: p. 25558-25575). Given that the most significant SHAPE protection in-cell within SLNCRl ^{403'1 0} lies adjacent to the Brn3a and AR interacting domains (nts 672-678), it is likely that additional yet unidentified factors associate with SLNCR1 and guide ribonucleoprotein complex assembly.

Proteins that are scaffolded by IncRNAs may bind RNA structural elements, as suggested for HOTAIR and SRA1, and sequence motifs, as shown here for SLNCR1 (Guo, X., et al., Briefings in Functional Genomics, 2016. 15: p. 38-46). Identification of short RNA sequences or groups of RNA sequences predicted to have similar interactors may reveal sequence and/or structural requirements predictive of IncRNA biology. This high resolution functional information is necessary to improve our ability to predict IncRNA function from sequence analysis and secondary structure predictions, ultimately leading to the determination of the mechanisms of uncharacterized IncRNAs. The work presented herein demonstrates that identification and characterization of protein-lncRNA interactions enables prediction of novel IncRNA interactions which could function similarly in orthologous pathways.

Given their critical role in the regulation of complex gene expression patterns and their implication in many human cancers, IncRNAs are rapidly emerging as an attractive class of novel pharmacological targets. Despite several hurdles early in the development of oligonucleotide therapeutics, particularly surrounding oligonucleotide delivery and stability, several oligonucleotide-based therapeutics have since been approved (Juliano, R.L., Nucleic Acids Research, 2016. 44: p. 6518-6548; Lundin, K.E., et al., Hum Gene Ther, 2015. 26: p. 475-485; Stein, C.A., and D. Castanotto, Mol Ther, 2017. 25: p. 1069- 1075). It is demonstrated for the first time herein that chemically stable oligonucleotides that block the AR-SLNCR1 interaction inhibit <SZNCR7-mediated melanoma invasion, indicating that disrupting this interaction has potential therapeutic relevance. Thus, characterizing IncRNA-protein interactions presents a novel avenue for the design and implementation of IncRNA-targeting therapies. In addition to oligonucleotide-based approaches, whose systemic delivery strategies are currently limited by accumulation in the liver, small molecules could also be used to selectively inhibit the AR-SLNCR1 interaction. Importantly, the SLNCRI structural information generated here is critical to enable identification of small molecules that bind to SLNCRI and selectively inhibit AR binding Connelly, CM., et al., Cell Chem Biol, 2016. 23 : p. 1077-1090).

Example 18: SLNCR isoforms exhibit both unique and overlapping functions

Melanomas express at least 3 isoforms of SLNCR, SLNCRl-3 (FIG. 121). SLNCRI is the shortest and most prevalent isoform, while SLNCR2 and SLNCR3 differ only in the inclusion of an additional exon of varying length. It was previously demonstrated that SLNCRI binds to AR and recruits it to the MMP9 promoter, and that SLNCRI and AR are required for transcriptionally upregulating A4MP9 expression and promoting melanoma invasion (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). Surprisingly, unlike SLNCRI, neither SLNCR2 nor SLNCR3 upregulateAiMPP nor increase melanoma invasion, indicating that SLNCR isoforms have at least partially unique functions (FIG. 12J and 12K). Isoform-specific functions cannot be explained by differences in AR binding, as all 3 isoforms contain the RNA region required for AR binding and consistently bind AR in vivo (FIG. 121 and 12L). Thus, all three SLNCR isoforms likely regulate AR activity.

To investigate the role for isoform-overlapping SLNCR function in melanoma development, siRNAs were designed to knockdown all SLNCR isoforms (hereafter

'SLNCR ' refers to 'SLNCRl-3 ') (FIG. 121). These siRNAs knocked down SLNCR by -60- 80% in two patient-derived melanoma short-term cultures (MSTCs), WM1976 and

WM858, and by 50-70% in the immortalized malignant melanoma cell line A375 (FIG. 13C). Importantly, MSTCs have undergone minimal passages outside of the patient and provide an accurate genetic model of melanoma (Lin, W.M., et al., Cancer Res, 2008.

68(3): p. 664-73). WM1976 and WM858 exhibit moderate to high levels of SLNCR expression, and are amenable to genetic studies requiring transfection of DNA or RNA (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). Using RNA-sequencing (RNA- seq), melanoma cells were transcriptionally profiled before and after siRNA-mediated knockdown of SLNCR in WM1976, the MSTC expressing highest levels of SLNCR.

Depletion of SLNCR significantly dysregulated 222 genes compared to a scramble siRNA control, upregulating 131 genes and downregulating 91 genes (p < 0.01, FIG. 12E, Tables 6A-6C).

Analysis of the full melanoma dataset from The Cancer Genome Atlas (TCGA) revealed that SLNCR expression is significantly correlated with expression of 120 candidate target genes (p < 0.05, Table 6D) (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). Moreover, expression of SLNCR and 62 of these target genes is significantly correlated even when correcting for multiple hypothesis testing (Bonferroni correction, p < 0.00023), strongly indicating that the RNA-seq analysis faithfully identified many ffiNCR-regulated genes.

Next, SLNCR differentially-expressed genes (DEGs) identified herein were compared to isoform-specific SLNCRl DEGs identified previously (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37). Of the 222 SLNCR DEGs, 41 genes (18.5%) were also dysregulated upon knockdown of SLNCRl (Tables 6A-6C, p < 0.01 in both RNA-seq experiments). Moreover, the majority (35 out of 41) of SLNCR and SLNCRl DEGs displayed concordant dysregulation (Pearson r = 0.27, p < 0.0001, FIG. 13E), reflecting that SLNCRl and SLNCR similarly regulate these genes.

Importantly, 6 DEGs are discordantly regulated upon SLNCR or SLNCRl knockdown, supporting the conclusion that SLNCR isoforms have unique functions. Two of these discordantly regulated genes, fibronection (FN1) and integrin subunit beta-1 (ITGB1), regulate cell-matrix adhesion (Gene Ontology [GO] Enrichment analysis, GO category 0007161, p = 1.02 x 10 ^"06 , false discovery rate = 1.58 x 10 ^"02 ), while another two of these genes, transmembrane protein 45 A (TMEM45A) and Ras-related protein RAB31, have been implicated in cancer invasion and cell adhesion (Grismayer, B., et al., Molecular Cancer, 2012. 11 : p. 62; Guo, J., et al., Oncology Reports, 2015. 33 : p. 3124-3130).

Combined with the findings that (i) SLNCRl, but not SLNCR2 or SLNCR3, increase melanoma invasion (FIG. 12), and (ii) knockdown of SLNCRl, but not SLNCR,

significantly decreases MMP9 (Tables 6A-6C), it is concluded herein that SLNCR isoforms uniquely regulate melanoma invasion. Because knockdown of SLNCR or SLNCRl only similarly dysregulates the majority of DEGs, and all three SLNCR isoforms bind AR, isoform-overlapping regulation of AR was focused in the Examples presented herein. Example 19: Implications of SZJVCR-mediated AR activity and the melanoma gender bias

The results presented herein indicate that ^NCR-mediated repression of p21 requires AR and EGRl . AR has previously been implicated in the melanoma gender bias, in which men suffer more frequent and severe melanomas than females (Joosse, A., et al., The Journal of Investigative Dermatology, 2011. 131 : p. 719-726; Micheli, A., et al., European Journal of Cancer, 2009. 45: p. 1017-1027; Siegel, R.L., Miller, K.D., and A. Jemal, CA: a cancer journal for clinicians, 2015. 65: p. 5-29; Spanogle, J.P., et al., Journal of the American Academy of Dermatology, 2010. 62: p. 757-767). More recent studies have confirmed oncogenic AR activity in melanoma (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37; Wang, Y., et al., Oncogene, 2017. 36: p. 1644-1654). Combined with the fact that primary male melanomas express higher AR protein than female melanomas (p = 0.046, FIG. 13B), it is believed that oncogenic AR activity contributes to the observed gender differences.

To explore potential contributions of ,SZNCR-mediated AR activity to these gender differences, TCGA was interrogated to determine if p21 is expressed in a gender-specific manner. To avoid confounding the analysis with p53-dependent regulation of p21, the analysis was limited to p53-deficient melanomas (Table 15). Consistent with the model that SLNCR and AR cooperatively repress CDKN1A expression, and the fact that males express higher levels of AR, male p53-deficient melanomas express significantly lower levels of p21 than females (p = 0.045, FIG. 25E). The gender-specific expression of a known melanoma tumor suppressor lends credence to the hypothesis that transcriptional regulation by SLNCR and AR contributes to the melanoma gender bias.

Despite the long-held belief that AR contributes to melanomagenesis, there has been little progress in determining the role of AR in melanoma etiology. Moreover, the interpretation that AR acts as a melanoma oncogene has been confounded by the fact that AR expression is not associated with worse overall melanoma survival. It is

comprehensively interrogated herein the role of AR in melanoma gene regulation, identifying many AR-regulated tumor suppressors and oncogenes. This work indicates that SLNCR imparts androgen -independent oncogenic activity to AR, including repression of p21. The work presented herein highlights the importance of SLNCR in mediating AR's oncogenic effects in melanoma, particularly in the context of the melanoma gender bias. Collectively, the data presented herein is consistent with a model in which SLNCR recruits AR to chromatin-bound EGRl to inhibit EGRl -mediated transcriptional activation of p21 (FIG. 26). Under normal physiological conditions, EGRl binds directly to an EGRl consensus motif located within the CDKN1A promoter, increasing p21 expression. During melanomagenesis, SLNCR recruits AR to chromatin-bound EGRl to inhibit EGRl transcriptional activation of p21.

To further explore the relationship between p21 and known regulatory proteins and RNAs (identified here and elsewhere), possible associations between SLNCR, AR, EGRl, p53 and gender with p21 expression within the TCGA melanoma dataset (using available protein expression for AR, p53 and p21, n = 354) were investigated. Using hierarchical multiple regression it was identified herein a model containing a significant three-way interaction between EGRl mRNA, AR and p53 expression associated with p21 expression (estimate [95% CI] = 0.12 [0.03, 0.21], p = 0.008, Table 16). This finding is in line with data indicating that EGRl and p53 upregulate p21 (FIG. 22 and 23), and that AR and p53 are transcriptionally and functionally linked (Table 8). The regression model also identified a borderline nonsignificant three-way interaction between EGRl, AR, and SLNCR (estimate [95% CI] = -0.12 [-0.25, 0.001], p = 0.052) and a significant two-way interaction between SLNCR and EGRl (estimate [95% CI] = -0.10 (-0.19, -0.01), p = 0.024), indicating an inverse association between SLNCR and p21 expression dependent on the levels of EGRl and AR. This is consistent with the mechanism demonstrated herein that EGRl is required for _^ SZNCR-mediated repression of p21. Considering the data presented herein indicating that (i) SLNCR binds EGRl and AR (FIG. 22) (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37), (ii) SLNCR or AR knockdown increased p21 expression (FIG. 18 and 20), (iii) AR and EGRl bind to an EGRl DNA binding site in the p21 promoter, and (iv) the EGRl binding site is required for SLNCR and AR-mediated p21 regulation (FIG. 24D), identification of this possible interaction further confirms the conclusion that SLNCR- mediated repression of p21 requires AR and EGRl .

SLNCR- and AR-regulated gene expression appears to be gene-specific, as SLNCR- mediated recruitment of AR may either increase or decrease gene expression (FIG. 17 A). Intriguingly, SLNCR and AR can have opposing effects on a single loci, as seen with CXCL2 (FIG. 17A).

The observations described herein provide the first global characterization of a role for AR in melanoma biology and confirm that AR binds to many melanoma-relevant genes. Remarkably, AR is associated with these regions even in the absence of canonical hormone-mediated activation, indicating that traditional anti-androgen therapies are unlikely to inhibit oncogenic activities of AR. Instead, SLNCR likely recruits AR to many loci. It is important to note that SLNCR, but not AR, expression is associated with shorter overall melanoma survival (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37) and may be required for mediating gender-specific differences in AR activity. Collectively, the data presented herein demonstrates ^NCR-mediated AR function as a novel, oncogenic pathway, resulting in gender-specific differences in target gene expression. Moreover, this work is further proof that non-coding RNAs are critical regulators of human gene expression. Detailed mechanistic studies of the fundamental actions of IncRNAs and identification of their associated protein partners is critical to the design and

implementation of new therapeutics.

Table 6A. Differential expression of transcripts upon siRNA knockdown of SLNCR in WM1976

Table 6A lists all transcripts that show differential expression upon siRNA knockdown of SLNCR in WM1976.

Table 6B. Differential expression of transcripts upon siRNA knockdown of SLNCR in WM1976

Table 6B lists the transcripts whose expression was significantly regulated by knockdown of SLNCR (p-value < 0.01). SLNCR (LINC00511 according to current annotation) is the last item listed.

Table 6C. Differential expression of transcripts upon siRNA knockdown of SLNCR in WM1976

Table 6C lists differentially regulated transcripts and includes Log2 (fold change) from previous RNA-seq experiments in which only SLNCRI was depleted. SLNCR is italicized. Genes in white boxes are also significantly dysregulated upon SLNCRI knockdown (p- value < 0.01), and genes in grey are not significantly dysregulated. Genes highlighted in bold font are significantly dysregulated upon knockdown of both SLNCR and SLNCRI, but in opposing directions. log2(fold_change) log2(fold_change) si- gene si-SLNCR p_value SLNCR1 p-value

LINC00511/SLNCR -2.17377 5.00E-05 -1.285961044 7.22542E-15

ANK D52 -0.695213 0.00545 -1.167074788 4.27813E-14

CDK19 -0.895808 0.00665 -1.50760447 1.53237E-11

DESI2 0.941009 5.00E-05 1.028630413 1.25232E-10

NOV 1.27015 5.00E-05 0.924282425 4.37855E-09

COL15A1 -1.26554 0.00025 -1.38020291 1.38048E-08

ACVR2B -1.0825 0.0022 -0.95786338 1.79291E-08

SM IM 15 1.72303 5.00E-05 0.872592076 3.69797E-07

D AM 1 0.809324 0.0079 0.967167703 3.75789E-07

J UN 0.897808 0.00095 0.812247613 1.38357E-06

TM EM 2 0.829844 0.0073 0.70062641 3.60295E-06

PIGA -0.941392 0.0043 -0.714024654 6.81504E-05

GADD45A 1.03814 0.00035 0.676346577 7.24128E-05

AKAP12 -0.925125 0.0002 -0.627994089 7.27494E-05

NAA50 0.958582 0.00705 0.610151367 8.15928E-05

RAB31 0.81993 0.00635 -0.748412548 8.4259E-05

ITGB1 0.853703 0.00085 -0.616082372 0.000150832

ANXA1 1.6086 5.00E-05 0.579131421 0.000531153

FERMT2 0.839753 0.0023 0.617463973 0.000607989

SESN1 1.08837 0.0001 0.662583976 0.000630562

CXCL1 -1.28746 5.00E-05 0.572907828 0.000864034

SSU72 0.772586 0.00235 0.527320347 0.000881997

FN1 -0.857003 0.0037 0.472680187 0.001137893

AMOTL2 0.969322 0.0002 0.515514275 0.001168983

PABPC4 0.748888 0.008 0.489621381 0.001378736

KCNAB2 0.995352 0.00585 0.492931013 0.002036119

FAM 129A 0.753693 0.00225 0.457282085 0.002285646

KI F6 -1.02453 0.00555 -0.613100138 0.00287593

I L6 -1.7286 0.00095 -1.592393299 0.003290389

0.0011

ITGA4 -0.921764 -0.470147297 0.003930652

Table 6D. Correlation of SLNCR, AR and EGRl expression with SLNCR target genes

Table 6D lists spearman correlation coefficient, confidence intervals, and p-values for SLNCR, AR or EGRl gene expression with SLNCR target genes (identified via RNA-seq). Within AR gene correlations, genes that are bound by AR (as determined by ChlP-seq) are highlighted in bold font.

Table 7. AR active regions as determined via AR ChlP-Seq

Table 7 lists the AR active regions as determined via AR ChlP-Seq. AR ChlP-seq was performed from hormone-deprived A375 cells transfected with either an empty or SLNCR1- expressing plasmid. Active regions for both the vector only control and cells ectopically- expressing SLNCR1.

l_A375_pNL7_minus_lncRNA_AR = vector control

2_A375_pNL97_plus_lncRNA_AR = SLNCR1 -expressing vector

Table 8. AR active regions as determined via AR ChlP-Seq

Table 9A. AR active genes as determined via AR ChlP-Seq

Table 9A lists the AR active genes as determined via AR ChlP-SEQ.

Annotations:

Table 9B. Genes differentially bound by AR

TAble 9B lists the genes that are differentially bound by AR in the presence or absence of AR as determines via MACS.

Annotations:

Table 9C. Genes differentially bound by AR

TAble 9C lists the genes differentially bound by AR (Table 9B) with corresponding fold change and significance of differential gene expression values after expression of SLNCRl (Schmidt, K., et al., Cell Rep, 2016. 15(9): p. 2025-37).

Table 9C

Table 10. Genes bound by AR and regulated by SLNCR

Table 10 lists the genes bound by AR and regulated by SLNCR. RNA-Seq differential expression was taken from Tables 6A-6C, and AR ChlP-Seq values were taken from Table 9A. The table lists only those genes that are significantly dysregulated by SLNCR knockdown and bound by AR.

Annotations:

[1] l_A375_pNL7_minus_lncRNA:: l Present

[2] 2_A375_pNL97_plus_lncRNA: : l Present

[3] l_A375_pNL7_minus_lncRNA: : l AvgPeak

[4] 2_A375_pNL97_plus_lncRNA: : 1 AvgPeak

Table 11. AR and EGRl active regions as determined via ChlP-Seq

Table 11 lists the AR and EGRl active regions as determinied via ChlP-Seq. EGRl ChlP- Seq was performed from A375 melanoma cells. The table also contains AR ChlP-Seq active regions (Table 7) to enable comparative analyses.

l_A375_pNL7_minus_lncRNA_AR = AR ChlP-seq from vector control

2_A375_pNL97_plus_lncRNA_AR = AR ChlP-seq from SLNCR1 -expressing vector 4 A375 EGR1 = EGRl ChlP-seq

Table 11

Table 12. AR and EGRl active genes as determined via ChlP-Seq

Table 12 lists the AR and EGRl active genes as determinied via ChlP-Seq. EGRl ChlP- Seq was performed from A375 melanoma cells. The table also contains AR ChlP-Seq active genes (Table 9A) to enable comparative analyses.

l_A375_pNL7_minus_lncRNA_AR = AR ChlP-seq from vector control

2_A375_pNL97_plus_lncRNA_AR = AR ChlP-seq from SLNCR1 -expressing vector 4 A375 EGR1 = EGRl ChlP-seq

Table 12

Table 13A. Genes bound by AR or EGRl and regulated by SLNCR

Table 13 A lists the genes bound by AR or EGRl and regulated by SLNCR. RNA-seq differential expression was taken from Tables 6A-6C, AR ChlP-seq values were taken from Table 9A, and EGRl ChlP-seq values from Table 12. The table lists all genes that are differentially expressed by SLNCR knockdown.

1 A375 j)NL7_minus_lncRNA_AR = AR ChlP-seq peaks from vector control

2_A375_pNL97_plus_lncRNA_AR = AR ChlP-seq peaks from SLNCR1 -expressing vector

4_A375_EGR1= EGRl ChlP-seq peaks

Table 13A

Table 13B. Genes bound by AR or EGRl and regulated by SLNCR

Table 13B lists the genes bound by AR or EGRl and regulated by SLNCR. RNA-seq differential expression was taken from Tables 6A-6C, AR ChlP-seq values were taken from Table 9A, and EGRl ChlP-seq values from Table 12. The table lists only those genes from Table 13 A that are bound by AR, EGRl or both.

Table 14. Summary statistics for AR ChlP-seq active regions

Table 15. AR and p21 protein expression of p53-deficient TCGA melanomas.

P53-deficient melanomas were defined as (i) primary melanomas or metastases of known melanoma origin, (ii) patients with no prior treatment, and (iii) harboring nonfunctional p53 mutations, as defined by the TP53 database (p53.fr) (Leroy, B., et al., Hum Mutat, 2014. 35: p. 756-765). Three additional patients containing R248W or Y220C gain of function p53 mutations were excluded based on reported regulation of p21 (Di Fiore, R., et al., Bone, 2014. 60: p. 198-212; Song, H., Hollstein, M, and Y. Xu, Nature Cell Biol, 2007. 9: p. 573-580; Xu, J., et al., Cell Death Dis, 2014. 5: p. el 108). Table 16. Final model determined from hierarchical multiple regression analysis of p21 protein expression in TCGA melanomas (n=354). Adjusted R-squared = 0.06,

Model (F-statistic) p = 0.0009

Variables assessed as part of the multiple regression analysis of p21 expression included patient gender, SLNCR and EGR1 mRNA log2 expression, and AR and p53 protein expression (from TCP A) and all possible interactions. All continuous variables were converted to z-scores in order to improve interpretability of the model output. In order to determine the set of parameters present in the final model, standard reduction techniques were used, including iterative removal of the least significant parameter along with evaluation of the Akaike's information criterion (AIC) and ANOVA comparisons of model fit. Non-significant parameters remain in the final model due to the presence of significant two- and three-way interactions. P-values have not been adjusted for multiple hypothesis testing. Table 17. siRNA targets and oligo sequences used in this study.

Bold italicized font denotes mutated nucleotides. Incorporation by Reference

The contents of all references, patent applications, patents, and published patent applications, as well as the Figures and the Sequence Listing, cited throughout this application are hereby incorporated by reference. Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the following claims.