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Purpose: To identify a melanoma microRNA (miRNA) expression signature that is predictive of outcome and then evaluate its potential to improve risk stratification when added to the standard-of-care staging criteria.Experimental Design: Total RNA was extracted from 59 formalin-fixed paraffin-embedded melanoma metastases and hybridized to miRNA arrays containing 911 probes. We then correlated miRNA expression with post-recurrence survival and other clinicopathologic criteria.Results: We identified a signature of 18 miRNAs whose overexpression was significantly correlated with longer survival, defined as more than 18 months post-recurrence survival. Subsequent cross-validation showed that a small subset of these miRNAs can predict post-recurrence survival in metastatic melanoma with an estimated accuracy of 80.2% (95% confidence interval, 79.8−80.6%). In contrast to standard-of-care staging criteria, a six-miRNA signature significantly stratified stage III patients into “better” and “worse” prognostic categories, and a multivariate Cox regression analysis revealed the signature to be an independent predictor of survival. Furthermore, we showed that most miRNAs from the signature also showed differential expression between patients with better and worse prognoses in the corresponding paired primary melanoma.Conclusions: MiRNA signatures have potential as clinically relevant biomarkers of prognosis in metastatic melanoma. Our data suggest that molecularly based models of risk assessment can improve the standard staging criteria and support the incorporation of miRNAs into such models. Clin Cancer Res; 16(5); 1577–86

Supplementary Data from Melanoma MicroRNA Signature Predicts Post-Recurrence Survival

IntroductionWe are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. We also evaluated tissue-derived predictors of extracted nucleic acids’ quality and success in downstream testing.MethodsFollowing a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACT™ assay, methylation-profiling (array), and miRNA expression (Nanostring nCounter).ResultsSufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (pConclusionOur experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma.

Five years ago, the Melanoma Research Foundation (MRF) conducted an assessment of the challenges and opportunities facing the melanoma research community and patients with melanoma. Since then, remarkable progress has been made on both the basic and clinical research fronts. However, the incidence, recurrence, and death rates for melanoma remain unacceptably high and significant challenges remain. Hence, the MRF Scientific Advisory Council and Breakthrough Consortium, a group that includes clinicians and scientists, reconvened to facilitate intensive discussions on thematic areas essential to melanoma researchers and patients alike, prevention, detection, diagnosis, metastatic dormancy and progression, response and resistance to targeted and immune-based therapy, and the clinical consequences of COVID-19 for patients with melanoma and providers. These extensive discussions helped to crystalize our understanding of the challenges and opportunities facing the broader melanoma community today. In this report, we discuss the progress made since the last MRF assessment, comment on what remains to be overcome, and offer recommendations for the best path forward.

By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 enhances myogenesis by contributing to the derepression of MHC transcription.

Melanoma patients with BRAFV600E -mutant tumors display striking responses to BRAF inhibitors (BRAFi); however, almost all invariably relapse with drug-resistant disease. Here, we report that microRNA-125a (miR-125a) expression is upregulated in human melanoma cells and patient tissues upon acquisition of BRAFi resistance. We show that miR-125a induction confers resistance to BRAFV600E melanoma cells to BRAFi by directly suppressing pro-apoptotic components of the intrinsic apoptosis pathway, including BAK1 and MLK3. Apoptotic suppression and prolonged survival favor reactivation of the MAPK and AKT pathways by drug-resistant melanoma cells. We demonstrate that miR-125a inhibition suppresses the emergence of resistance to BRAFi and, in a subset of resistant melanoma cell lines, leads to partial drug resensitization. Finally, we show that miR-125a upregulation is mediated by TGFβ signaling. In conclusion, the identification of this novel role for miR-125a in BRAFi resistance exposes clinically relevant mechanisms of melanoma cell survival that can be exploited therapeutically.

Next-generation sequencing has enabled genetic and genomic characterization of melanoma to an unprecedent depth. However, the high mutational background plus the limited depth of coverage of whole-genome sequencing performed on cutaneous melanoma samples make the identification of novel driver mutations difficult. We sought to explore the somatic mutation portfolio in exonic and gene regulatory regions in human melanoma samples, for which we performed targeted sequencing of tumors and matched germline DNA samples from 89 melanoma patients, identifying known and novel recurrent mutations. Two recurrent mutations found in the RPS27 promoter associated with decreased RPS27 mRNA levels in vitro. Data mining and IHC analyses revealed a bimodal pattern of RPS27 expression in melanoma, with RPS27-low patients displaying worse prognosis. In vitro characterization of RPS27-high and RPS27-low melanoma cell lines, as well as loss-of-function experiments, demonstrated that high RPS27 status provides increased proliferative and invasive capacities, while low RPS27 confers survival advantage in low attachment and resistance to therapy. Additionally, we demonstrate that 10 other cancer types harbor bimodal RPS27 expression, and in those, similarly to melanoma, RPS27-low expression associates with worse clinical outcomes. RPS27 promoter mutation could thus represent a mechanism of gene expression modulation in melanoma patients, which may have prognostic and predictive implications.

Despite major advances in the treatment of metastatic melanoma, treatment failure is still inevitable in most cases. Manipulation of key epigenetic regulators, including inhibition of Bromodomain and extra‐terminal domain (BET) family members impairs cell proliferation in vitro and tumor growth in vivo in different cancers, including melanoma. Here, we investigated the effect of combining the BET inhibitor JQ1 with the BRAF inhibitor Vemurafenib in in vitro and in vivo models of BRAF‐mutant melanoma. We performed cytotoxicity and apoptosis assays, and a xenograft mouse model to determine the in vitro and in vivo efficacy of JQ1 in combination with Vemurafenib against BRAF‐mutant melanoma cell lines. Further, to investigate the molecular mechanisms underlying the effects of combined treatment, we conducted antibody arrays of in vitro drug‐treated cell lines and RNA sequencing of drug‐treated xenograft tumors. The combination of JQ1 and Vemurafenib acted synergistically in BRAF‐mutant cell lines, resulting in marked apoptosis in vitro, with upregulation of proapoptotic proteins. In vivo, combination treatment suppressed tumor growth and significantly improved survival compared to either drug alone. RNA sequencing of tumor tissues revealed almost four thousand genes that were uniquely modulated by the combination, with several anti‐apoptotic genes significantly down‐regulated. Collectively, our data provide a rationale for combined BET and BRAF inhibition as a novel strategy for the treatment of melanoma.

Metastasis is the primary cause of death of cancer patients. Dissecting mechanisms governing metastatic spread may uncover new biology and/or yield promising therapeutic insights. Here we investigated the role of circular RNAs (circRNAs), a class of noncoding RNAs lacking characterized functions, in metastasis, using melanoma as a model aggressive tumor. We analyzed RNA-seq of melanocytes and melanoma short-term cultures to characterize the landscape of circRNA in melanocytic cells. We observed silencing of Cerebellar Degeneration Related 1 (CDR1as), a neuronal-enriched circRNA and known regulator of the microRNA miR-7, in melanoma cell lines and short-term cultures compared to cultured melanocytes, and progressive loss from primary to metastatic disease in melanoma patient samples. Moreover, low CDR1as expression in primary tumors associates with poor prognostic histopathologic indicators and patient outcomes. We find that CDR1as loss results from H3K27me3-mediated silencing of its originating lincRNA LINC00632. We observed abundant H3K27me3, the repressive chromatin mark deposited by the PRC2 complex, in regulatory regions of LINC00632 in cell lines lacking CDR1as expression, and pharmacologic inhibition of EZH2 restored expression of both transcripts. Phenotypically, CDR1as depletion in vitro and in vivo enhances melanoma invasion and metastasis, with limited effect on cell proliferation or tumor growth. Surprisingly, we did not observe miR-7 regulation to be a catalyst of the proinvasive effects of CDR1as depletion in our models. Instead, we identified IGF2BP3 as a novel interactor of CDR1as and critical mediator downstream of CDR1as silencing. Finally, we find that CDR1as abundance associates with previously defined cellular states that engender distinct therapeutic responses to MAPK and GPX4 inhibitors. Collectively, our study reveals CDR1as silencing as a hallmark of melanoma progression; documents novel functional, prognostic and predictive roles for CDR1as; and exposes circRNAs as key players in metastasis. This abstract is also being presented as Poster A29. Citation Format: Douglas Hanniford, Alejandro Ulloa-Morales, Beatriz Sánchez-Sendra, Alcida Karz, Rana Moubarak, Tommaso Tabaglio, Maria Gabriela Berzoti-Coelho, Veronica Davalos, Pamela Wu, Varshini Vasudevaraja, Andreas Kloetgen, Karin Lilja, Jochen Imig, Carlos Monteagudo, Ernesto Guccione, Aristotelis Tsirigos, Iman Osman, Iannis Aifantis, Eva Hernando. Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR04.

Summary Metastasis is the primary cause of death of cancer patients. Dissecting mechanisms governing metastatic spread may uncover important tumor biology and/or yield promising therapeutic insights. Here, we investigated the role of circular RNAs (circRNA) in metastasis, using melanoma as a model aggressive tumor. We identified silencing of cerebellar degeneration-related 1 antisense (CDR1as), a regulator of miR-7, as a hallmark of melanoma progression. CDR1as depletion results from epigenetic silencing of LINC00632, its originating long non-coding RNA (lncRNA) and promotes invasion in vitro and metastasis in vivo through a miR-7-independent, IGF2BP3-mediated mechanism. Moreover, CDR1as levels reflect cellular states associated with distinct therapeutic responses. Our study reveals functional, prognostic, and predictive roles for CDR1as and expose circRNAs as key players in metastasis.

The American Joint Committee on Cancer staging system for cutaneous melanoma is based on primary tumor thickness and the presence of ulceration, mitoses, lymph node spread, and distant metastases as determinants of prognosis. Although this cutaneous melanoma staging system has evolved over time to more accurately reflect patient prognosis, improvements are still needed, because current understanding of the particular factors (genetic mutation, expression alteration, host response, etc) that are critical for predicting patient outcomes is incomplete. Given the clinical and biologic heterogeneity of primary melanomas, new prognostic tools are needed to more precisely identify patients who are most likely to develop advanced disease. Such tools would affect clinical surveillance strategies and aid in patient selection for adjuvant therapy. The authors reviewed the literature on prognostic molecular and immunologic markers in primary cutaneous melanoma, their associations with clinicopathologic and survival outcomes, and their potential for incorporation into current staging models. Overall, the studies considered in this review did not define prognostic markers that could be readily incorporated into the current staging system. Therefore, efforts should be continued in these and other directions to maximize the likelihood of identifying clinically useful prognostic biomarkers for cutaneous melanoma.

et al., SPROUTY-2 (SPRY2) is a modulator of tyrosine kinase receptor signaling with receptor- and cell type-dependent inhibitory or enhancing effects. Studies on the action of SPRY2 in major cancers are conflicting and its role remains unclear. Here we have dissected SPRY2 action in human colon cancer. Global transcriptomic analyses show that SPRY2 downregulates genes encoding tight junction proteins such as claudin-7 and occludin and other cell-to-cell and cell-to-matrix adhesion molecules in human SW480-ADH colon carcinoma cells. Moreover, SPRY2 represses LLGL2/HUGL2, PATJ1/INADL and ST14, main regulators of the polarized epithelial phenotype, and ESRP1, an epithelial-to-mesenchymal transition (EMT) inhibitor. A key action of SPRY2 is the upregulation of the major EMT inducer ZEB1, as these effects are reversed by ZEB1 knock-down by means of RNA interference. Consistently, we found an inverse correlation between the expression level of claudin-7 and those of SPRY2 and ZEB1 in human colon tumors. Mechanistically, ZEB1 upregulation by SPRY2 results from the combined induction of ETS1 transcription factor and the repression of microRNAs (miR-200 family, miR-150) that target ZEB1 RNA. Moreover, SPRY2 increased AKT activation by epidermal growth factor, whereas AKT and also Src inhibition reduced the induction of ZEB1. Altogether, these data suggest that AKT and Src are implicated in SPRY2 action. Collectively, these results show a tumorigenic role of SPRY2 in colon cancer that is based on the dysregulation of tight junction and epithelial polarity master genes via upregulation of ZEB1. The dissection of the mechanism of action of SPRY2 in colon cancer cells is important to understand the upregulation of this gene in a subset of patients with this neoplasia that have poor prognosis., This study was supported by the Ministerio de Economía y Competitividad of Spain and Fondo Europeo de Desarrollo Regional (FEDER) (grant SAF2013-43468-R to AM, SAF2011-29530 to FXR); FEDER-Instituto de Salud Carlos III (RD12/0036/0021 to AM and JMR, RD12/0036/0034 to FXR, RD12/0036/0016 to MS, RD12/0036/0012 to HGP, RD06/0020/0003, PS09/00562 and PI13/00703 to JMR); Comunidad de Madrid (S2010/BMD-2344 Colomics2 to AM); Fundación Científica de la Asociación Española contra el Cáncer (to JMR); US Department of Defense (CA093471 and CA110602 to EH); National Institutes of Health/National Cancer Institute (1R01CA155234-01 to EH); National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (1R21AR062239-01 to EH); and the Melanoma Research Alliance (to EH).

In this study, we investigated the mechanism(s) of altered expression of protooncogene SKP2 in metastatic melanoma and its clinical relevance in patients with metastatic melanoma. The genomic status of SKP2 was assessed in cell lines by sequencing, single nucleotide polymorphism array, and genomic PCR. Copy number status was then evaluated for concordance with SKP2 mRNA and protein expression. SKP2 protein was further evaluated by immunohistochemistry in 93 human metastatic tissues. No mutations were identified in SKP2. Increased copy number at the SKP2 locus was observed in 6/14 (43%) metastatic cell lines and in 9/22 (41%) human metastatic tissues which was associated with overexpression of SKP2 protein. Overexpression of SKP2 protein in human tissues was associated with worse survival in a multivariate model controlling for the site of metastasis. Copy number gain is a major contributing mechanism of SKP2 overexpression in metastatic melanoma. Results may have implications for the development of therapeutics that target SKP2.

Purpose: Brain metastasis is the major cause of mortality among patients with melanoma. A molecular prognostic test that can reliably stratify patients at initial melanoma diagnosis by risk of developing brain metastasis may inform the clinical management of these patients. Experimental Design: We performed a retrospective, cohort-based study analyzing genome-wide and targeted microRNA expression profiling of primary melanoma tumors of three patient cohorts (n = 92, 119, and 45) with extensive clinical follow-up. We used Cox regression analysis to establish a microRNA-based signature that improves the ability of the current clinicopathologic staging system to predict the development of brain metastasis. Results: Our analyses identified a 4-microRNA (miR-150-5p, miR-15b-5p, miR-16-5p, and miR-374b-3p) prognostic signature that, in combination with stage, distinguished primary melanomas that metastasized to the brain from nonrecurrent and non–brain metastatic primary tumors (training cohort: C-index = 81.4%, validation cohort: C-index = 67.4%, independent cohort: C-index = 76.9%). Corresponding Kaplan–Meier curves of high- versus low-risk patients displayed a clear separation in brain metastasis-free and overall survival (training: P < 0.001; P < 0.001, validation: P = 0.033; P = 0.007, independent: P = 0.021; P = 0.022, respectively). Finally, of the microRNA in the prognostic model, we found that the expression of a key lymphocyte miRNA, miR-150-5p, which is less abundant in primary melanomas metastatic to brain, correlated with presence of CD45+ tumor-infiltrating lymphocytes. Conclusions: A prognostic assay based on the described miRNA expression signature combined with the currently used staging criteria may improve accuracy of primary melanoma patient prognoses and aid clinical management of patients, including selection for adjuvant treatment or clinical trials of adjuvant therapies. Clin Cancer Res; 21(21); 4903–12. ©2015 AACR.

Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistaminehistamine and that the rank order of potency for H3 antagonists was clobenpropitiodophenpropitthioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.

9567 Background: Recently, tumor mutation burden (TMB) has been shown to increase the presentation of neoantigens that stimulate immune tumor recognition, resulting in improved immunotherapy (IT) outcomes in melanoma and other cancers. As melanoma is highly immunogenic, here we tested whether TMB associates with immune recognition during tumor progression, hence impacting melanoma overall survival (OS), independently of IT treatment. Methods: We have generated somatic mutation data from 314 IT-naive metastatic melanomas from The Cancer Genome Atlas (TCGA). In the TCGA cohort, TMB has been calculated for 210 genes (200GS) previously established from TMB studies of anti-CTLA4 and anti-PD1/PD-L1 IT. For validation, we have sequenced exonic regions of 20 genes (20GS) with the highest TMB among 200GS in 89 IT-naive metastatic melanomas ascertained at New York University Langone Medical Center. The TMB was defined using total number of somatic, non-synonymous mutations in either 200GS (TCGA discovery) or 20GS (validation), respectively. For discovery and validation cohorts, OS from primary diagnosis of samples with high TMB was compared against low TMB, using thresholds established in previous studies. Results: We found that total TMB predicts better OS (p = 0.03, HR = 2.64) in TCGA melanomas. Restricting the analysis only to the established 200GS, this association became more significant in all patients (p = 0.01, HR = 2.67) as well as in patients without IT (p = 0.01, HR = 2.67). In the validation stage of 89 melanomas without prior IT treatment, a high TMB in a subset of 20GS accurately determined favorable OS (p = 0.02, HR = 2.69) and confirmed TCGA observations from the 200GS. Conclusions: Here we show, for the first time, that in addition to IT, high TMB predicts more favorable OS in patients that never received IT, potentially serving as a novel marker of prognosis of melanoma and likely other immunogenic tumors at early stages. In addition, our study suggests that TMB test can be robust when applied to only a small subset of genes that trigger significantly higher immunogenicity. This may also eventually assist with accurate sub-selection of early stage patients likely to respond to IT regimens.

In this study, we investigated the mechanism(s) of altered expression of protooncogene SKP2 in metastatic melanoma and its clinical relevance in patients with metastatic melanoma. The genomic status of SKP2 was assessed in cell lines by sequencing, single nucleotide polymorphism array, and genomic PCR. Copy number status was then evaluated for concordance with SKP2 mRNA and protein expression. SKP2 protein was further evaluated by immunohistochemistry in 93 human metastatic tissues. No mutations were identified in SKP2. Increased copy number at the SKP2 locus was observed in 6/14 (43%) metastatic cell lines and in 9/22 (41%) human metastatic tissues which was associated with overexpression of SKP2 protein. Overexpression of SKP2 protein in human tissues was associated with worse survival in a multivariate model controlling for the site of metastasis. Copy number gain is a major contributing mechanism of SKP2 overexpression in metastatic melanoma. Results may have implications for the development of therapeutics that target SKP2.

The highly aggressive character of melanoma makes it an excellent model for probing the mechanisms underlying metastasis, which remains one of the most difficult challenges in treating cancer. We find that miR-182, member of a miRNA cluster in a chromosomal locus (7q31-34) frequently amplified in melanoma, is commonly upregulated in human melanoma cell lines and tissue samples; this up-regulation correlates with gene copy number in a subset of melanoma cell lines. Moreover, miR-182 ectopic expression stimulates migration of melanoma cells in vitro and their metastatic potential in vivo, whereas miR-182 down-regulation impedes invasion and triggers apoptosis. We further show that miR-182 over-expression promotes migration and survival by directly repressing microphthalmia-associated transcription factor-M and FOXO3, whereas enhanced expression of either microphthalmia-associated transcription factor-M or FOXO3 blocks miR-182's proinvasive effects. In human tissues, expression of miR-182 increases with progression from primary to metastatic melanoma and inversely correlates with FOXO3 and microphthalmia-associated transcription factor levels. Our data provide a mechanism for invasion and survival in melanoma that could prove applicable to metastasis of other cancers and suggest that miRNA silencing may be a worthwhile therapeutic strategy. © 2009 by The National Academy of Sciences of the USA., This work was funded by grants of the NYU Cancer Center (National Institutes of Health–National Cancer Institute Cancer Center Support Grant P30CA016087) and the Elsa U. Pardee Foundation. M.F.S. is supported by an Alfonso Martin-Escudero fellowship.

Purpose: Despite major advances in the treatment of metastatic melanoma, treatment failure is still inevitable in most cases. Manipulation of key epigenetic regulators, including inhibition of Bromodomain and extra-terminal domain (BET) family members impairs cell proliferation in vitro and tumor growth in vivo in different cancers, including melanoma. BET inhibition is thought to impair downstream transcriptional activity of the MAPK (MAPK) pathway and other key oncogenic signaling pathways, suggesting complementarity with MAPK inhibition. Here, we investigated the effect of combining the BET inhibitor JQ1 with the BRAF inhibitor Vemurafenib in in vitro and in vivo models of BRAF-mutant melanoma. We additionally screened potential synergism of JQ-1 with 27 additional compounds including inhibitors of the proteasome, histone deacetylases (HDACi) and MEK. Methods: We performed cytotoxicity and apoptosis assays, and a xenograft mouse model to determine the in vitro and in vivo efficacy of JQ1 in combination with Vemurafenib against BRAF-mutant melanoma cell lines. Further, to investigate the molecular mechanisms underlying the effects of this combined treatment, we conducted antibody arrays of in vitro drug-treated cell lines and RNA sequencing of drug-treated xenograft tumors. Synergism between JQ1 and other targeted compounds was examined by in vitro cytotoxicity assays. Results: The combination of JQ1 and Vemurafenib acted synergistically in BRAF-mutant cell lines. Additional combinations that were found synergistic included JQ-1 plus proteasome inhibitors (carfilzomib and marizomib, HDAC inhibitors (romidepsin and panobinostat), or anti-Bcl2 small molecules (Obatoclax and TW-37). The combination of JQ-1 and Vemurafenib resulted in marked apoptosis in vitro, with up-regulation of several pro-apoptotic proteins involved in both the intrinsic and extrinsic pathways of apoptosis. In vivo, this combination treatment suppressed tumor growth and significantly improved survival compared to either drug alone. RNA sequencing of tumor tissues revealed almost four thousand genes that were uniquely modulated by the combination. Furthermore, several anti-apoptotic genes such as MCL-1, BCL-XL and BCL-2 were significantly down-regulated. Conclusions: Collectively, our data provide a rationale for combinations of BET inhibitors with additional compounds including BRAF inhibitors, as a novel strategy for the treatment of melanoma. Citation Format: Luca Paoluzzi, Douglas Hanniford, Elena Sokolova, Iman Osman, Farbod Darvishian, Jinhua Wang, James E. Bradner, Eva Hernando. BET and BRAF inhibitors act synergistically against BRAF mutant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3522. doi:10.1158/1538-7445.AM2015-3522

The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z' values of the agonist binding assays in SPA format (Z'0.70). The Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.

9046 Background: The improvement of recurrence risk assessment at the time of primary melanoma diagnosis and the development of better surveillance strategies to monitor recurrence remain critical challenges in melanoma patient care. In the current study, we examine the ability of serum-based miRNAs to 1) improve patient risk stratification and 2) improve early detection of melanoma recurrence. Methods: Sera levels of 12 miRNAs were measured using qRT-PCR (Exiqon) in serum prospectively collected at diagnosis from 201 melanoma patients (median FU of survivors 91 months). A multivariate logistic regression model that includes miRNAs in combination with stage was built using the training cohort and evaluated in an independent validation cohort of 82 patients (median FU of survivors 39 months). Sera levels of the most informative miRNA signature were assessed longitudinally in a subset of patients (n=82) with samples drawn pre- and post-recurrence when applicable (n=225 samples). An L1 penalized regression a...

9089 Background: Estimating the risk of recurrence in patients with melanoma is extremely challenging. Standard of care is AJCC staging system, but the accuracy and robustness of this method is still under development. We conducted a proof of concept study exploring the use of machine-learned Bayesian Belief Networks (ml-BBNs) using a miRNA profiled cohort of melanoma patients with extended follow up to create Bayesian Biological Systems Models (BBSMs). We sought to determine if ml-BBNs could describe the biological system and if we could use the model to identify new cases with higher risk of recurrence. Methods: Our study cohort consisted of 89 patients (42 of which recurred) with a median follow up time of 118 months, that were examined for 869 miRNAs. Prior to modeling we segmented the data into training data (72 cases/80%) and testing data (17 cases/20%) at random. We recursively trained ml-BBNs on the training set, using all miRNAs. We used the directed graph structure of the ml-BBNs to identify miRNAs that consistently had more connectivity and goodness of fit as determined by Bayesian Information Criteria (BIC) scoring. MiRNAs that were in the top 50 BIC-scoring nodes across all models were selected for use to train the recurrence BBSMs. To compensate for a small number, bootstrapping was used to increase the sample to 100 records. We then compared our test set cases to our recurrence model, and used a similarity scoring algorithm to evaluate the similarity of values in each test instance to our biological models. For comparison we also trained an ml-BBN using clinical data from the same cohort. We then evaluated the scores against known recurrence outcome using Receiver Operating Characteristic (ROC) curve analysis. Results: BIC-scoring analysis selected 35 miRNAs for use in BBSM modeling. Area Under the Curve (AUC) for detection of recurrence is 0.76 in the training set and 0.62 in the testing set, while the clinical data yielded an AUC of 0.5 in this cohort. Conclusions: Our data suggest that ml-BBNs can be used to describe a biological model of melanoma recurrence. Additional data and refinement need to be made using independent datasets to be of a level that is clinically useful.

9091 Background: Manipulation of key epigenetic regulators in melanoma proliferation is emerging as a new therapeutic strategy. Bromodomain-containing proteins such as the extraterminal domain (BET) family are components of transcription factor complexes and determinants of epigenetic memory. We investigated the expression of BRD4, a BET family member in melanoma cell lines and tissues, and the effects of its inhibition with the small molecule compounds MS436 and MS417 in in vitro and in vivo models of melanoma. Methods: BRD2 and BRD4 expression were analyzed by immunohistochemistry. We tested the effects of pharmacological or RNAi-mediated inhibition of BRD4 in melanoma cells using crystal violet-based assays for proliferation/colony formation and flow-cytometry for cell cycle analysis. The molecular effects of BRD4 suppression were examined using RNA sequencing, Real-Time quantitative PCR and western blots for p27, p21, MYC, ERK1 and SKP2. In the in vivo xenograft experiments NOD/SCID/IL2γR-/-mice were injected with melanoma cells and treated with MS417. Statistical significance was determined by unpaired t-test (GraphPad). Results: BRD4 was found significantly upregulated in primary and metastatic melanoma tissues compared to melanocytes and nevi (p

8550 Background: Melanoma is curable for most patients whose tumors are surgically removed early in disease progression; however, many primary melanomas recur and progress to metastasis. Clinical staging is insufficient to robustly classify patients at highest risk of recurrence, and prognostic molecular biomarkers have not yet been identified. Methods: We performed miRNA profiling of 92 FFPE primary melanomas to discover metastasis relevant miRNAs and develop predictive models of recurrence, which were then validated in an independent cohort. We identified miRNAs differentially expressed between primary tumors that did and did not recur (3-year minimum follow-up) and between thick and thin lesions. We selected candidate miRs for screening in a fluorescence-based in vitro invasion assay, and prioritized a subset for in vivo testing. Potential downstream mediators of these miRNAs were selected by mRNA array analysis and tested in a secondary invasion screen. mRNA candidates that mimicked the miR’s invasion-suppressive effect were tested in 3’UTR reporter assays to confirm them as direct targets. Results: Using the discovery cohort we identified a 20 miRNA signature that can distinguish Stage I/II primary tumors (n=70) that did from those did not recur with 3-year minimum follow-up with an AUC=94%, 95% CI: (0.88, 0.99). Applying this model to predict risk for recurrence in the independent validation cohort yielded an AUC = 96%, 95% CI: (0.90, 1) in discriminating recurrent versus non-recurrent stages I/ II patients (n=45). From the discovery cohort, 40 candidates were selected for invasion assay screening, of which 5 miRNAs robustly inhibit in vitro invasion in 5 melanoma cell lines. Three miRs (miR-382, miR-516b, and miR-7) strongly suppressed metastasis in a mouse model. Moreover, multiple mRNAs tested as potential mediators mimicked the invasion-suppressive effects of candidate miRs. Of those, NCAPG2 and CDC42 were identified as miR-516b targets, CTTN as a miR-382 target, and PIK3CD as a miR-7 target. These genes have been linked to metastasis in melanoma or other tumors. Conclusions: Our data demonstrate that aberrant expression of specific miRNAs at diagnosis is predictive of and functionally impacts melanoma progression.

8565 Background: We hypothesize that BRAF mutations result in microRNA (miRNA) alterations which contribute to orchestrating the mutant BRAF’s oncogenic effects in melanoma. Our study is the first to examine the association between the BRAF mutation status in primary melanomas and the expression of miRNAs that target known tumor suppressors. Methods: 84 prospectively accrued melanoma patients at New York University Langone Medical Center were studied. DNA and total RNA were extracted from consecutive sections of formalin-fixed paraffin-embedded primary tissues. BRAF mutation status was determined by DNA sequencing. RNA was hybridized to miRCURY miRNA microarrays containing 1314 probes. Normalized miRNA data were analyzed using the t-test (p 2 were selected for predicted (TargetScan, PicTar) and validated (miRWalk) gene target analysis, and overlapping genes targeted by ≥ 2 miRNAs were analyzed using pathway-mapping algorithms (KEGG, BioCarta, PANTHER). Results: 48 (57%) primaries were BRAFwt and 36 (43%) were BRAFmut (26 V600E, 4 V600K, 1 V600R, 1 V600D, 4 other). 30 miRNAs met the criteria for statistically significant differential expression and FC thresholding: let-7i, miR-23c, -26a/b, -27b, -34a, -98, -126*, -141, -148a, -181b, -195, -199a-3p, -199a/b-5p, -200a/b/c, -203, -205, -455-3p, -491-3p, -606, -641, -646, -1297, -4301; miRPlus-C1070, -C1110, -G1246-3p (average FC: 2.3-3.5, all increased in BRAFmut vs. BRAFwt). Predicted and validated target gene analysis revealed 317 genes, of which 110 (35%) were convergent targets of ≥ 2 miRNAs. Pathway analyses of the predicted, validated, and convergent target genes pointed to the potential impact of BRAFmut-associated miRNAs on known tumor suppressors FAS, PTEN, and TNF and the p53 pathway. Conclusions: Differentially expressed miRNAs in BRAFmut vs. BRAFwt primaries target genes with known roles in melanoma biology and/or treatment response. These miRNAs warrant further study as potential effectors of the BRAFmut oncogenic program.

8540 Background: Distinct molecular alterations in acral, mucosal and uveal melanoma subtypes have been recently reported and have prompted the development of tailored treatment approaches. Superficial spreading melanoma (SSM) and nodular melanoma (NM), the 2 most common types of cutaneous melanoma (70% and 20% respectively), are currently perceived as sequential phases of linear progression from radial to vertical growth. However, clinicopathological data from our group and others suggest that SSM and NM might be two biologically different entities. In this study, we used microRNA profiling to identify the signature that distinguishes SSM from NM. We further determined the association between decreased expression of SSM-specific microRNAs and loss of the corresponding genomic loci. Methods: miRCURY LNA microRNA Arrays were used to profile RNA isolated from 82 micro and macrodissected primary melanomas (26 SSM and 56 NM) and 9 congenital nevi. Differentially expressed microRNAs were identified using T tes...

11034 Background: Skp2, a known oncogene, is overexpressed in several types of tumors and is associated with worse recurrence rate and overall survival in primary melanoma patients. Moreover, the anti-proliferative effects of Skp2 siRNA on various tumor cell lines have prompted the preclinical testing of Skp2 small molecule inhibitors. In this study, we assessed the clinical relevance and molecular mechanism(s) underlying Skp2 overexpression in metastatic melanoma patients. Methods: Skp2 protein levels were measured in 122 metastatic melanoma specimens using immunohistochemistry (IHC), and the association between Skp2 overexpression and post-recurrence survival was examined. Moreover, 22 cell lines (2 normal primary melanocytes, 2 primary immortal melanocytes, 4 primary melanoma cell lines, and 18 metastatic melanoma cell lines) were evaluated for Skp2 genomic amplification using Single Nucleotide Polymorphism (SNP) arrays (Affymetrix 6.0) and Skp2 gene expression using mRNA arrays (Affymetrix U133A 2.0) and quantitative RT-PCR. We also screened 18 cell lines for Skp2 mutation by sequencing. Results: Skp2 overexpression, defined as >25% tumor cells, was associated with shorter 3-yr post-recurrence survival (37%) compared to Skp2 expression ≤25% (55%) (HR=1.89, 95%, CI= 1.04, 3.42, p=0.04). Skp2 overexpression was significantly associated with the site of melanoma metastasis: visceral (n= 12; 89%), lymph node (n=49; 36%), brain (n=15; 14%), and soft-tissue (n=36; 6%) (p No significant financial relationships to disclose.

9006 Background: We recently reported the identification of a specific miRNA cluster frequently overexpressed in melanoma which enhances the metastatic behavior (Segura et al., 2009. PNAS. In press). In this study, we aim to define the clinical relevance of miRNA expression, in particular whether miRNAs can serve as prognostic biomarkers for melanoma patients. Methods: Total RNA was extracted from formalin-fixed paraffin embedded (FFPE) tissue samples from 61 metastatic melanoma specimens (25 lymph node, 16 soft tissue/skin, 13 brain and 7 visceral) accrued by the NYU Interdisciplinary Melanoma Cooperative Group (IMCG). RNA was hybridized into miRNA arrays (miRdicatorTM, Rosetta Genomics, Inc) containing probes for more than 600 miRNA sequences, including all the human miRNAs in the 9.0 version of miRBase. The Significance Analysis of Microarrays (SAM) was used to identify miRNAs significantly associated with survival, and the False Discovery Rate (FDR) approach was used to adjust for multiple comparisons. Results: We identified a signature of 18 miRNAs, whose up-regulation significantly associates with better prognosis (increased overall survival and post-recurrence survival) using the FDR of 0%. Quantitative RT-PCR on the same tissues has verified the array results in 15 out of 15 miRNAs analyzed. Some of these miRNAs were significantly associated with stage at recurrence, while others significantly correlated with the site of metastasis (i.e. visceral, brain, soft-tissue). Using cross-validation, we selected a miRNA signature consisting of 10 of these significant miRNAs with lowest misclassification error in predicting 1.5-year post-recurrence survival. We are currently validating these findings in a separate cohort of metastatic patients. Furthermore, we are investigating whether these ‘protective' miRNAs can be detected in the corresponding primary lesions and thus, whether they already hold prognostic value at early stages of the disease. Conclusions: Our data suggest a role for miRNAs as melanoma biomarkers, and support the development of miRNA- based monitoring assays. These analyses are also expanding our understanding of the molecular alterations underlying melanoma progression, by revealing miRNAs whose down-regulation associates with a more aggressive biological behavior. [Table: see text]

SummaryTo metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression.

We examined the microRNA signature that distinguishes the most common melanoma histological subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM). We also investigated the mechanisms underlying the differential expression of histology-specific microRNAs. MicroRNA array performed on a training cohort of 82 primary melanoma tumors (26 SSM, 56 NM), and nine congenital nevi (CN) revealed 134 microRNAs differentially expressed between SSM and NM (P