831 Generation of thioredoxin reductase 1 (TR1) knockdowns in human melanoma cell lines using CRISPR Cas9 C Kline 1 , M Honeggar 1 , M Laws 2 , S Leachman 3 and P Cassidy 1 1 Oregon Health & Science University, Portland, OR, 2 Oregon Health and Science University, Portland, OR and 3 OHSU, Portland, OR UV-induced antioxidant responses are involved in various cell signaling and redox sensitive pathways which play an important role in maintaining the health of a melanocyte. Detoxi- fication of ROS produced by exposure of the skin to the sun’s UV radiation is controlled in part by the antioxidant networks glutathione (GSH) and thioredoxin reductase 1 (TR1). Recently our lab found that TR1 expression levels varied in human melanoma tissues, with increased expression being associated with advanced stages of melanoma and increased metastasis. Based on these observations we believe that a melanocyte’s ability to respond to UV-induced oxidative stress might be the basis of increased risk for melanoma, while hyper activation of the TR1 antioxidant system facilitates progression of disease in melanomas. In order to better understand how antioxidants can be protective, and yet accelerate melanomas we have started to investigate the importance of the antioxidant network(s) in the human melanoma cell line M-14, which has one of the highest level of TR1 in all of the melanoma lines in the NCI 60 panel. In order to directly evaluate the role of TR1 in M-14 melanoma cells we are using the genome editing tool CRISPR Cas9 to generate TR1 knockouts and assess the effects on the antioxidant response to simulated solar radiation. In this study, we hope to elucidate the mechanisms by which TR1 levels effect response of the complex antioxidant networks of the melanocytes to UV-induced oxidative stress and oncogenic transformation. This information is vital for developing effective chemoprevention agents in addition to un- derstanding the mechanism of melanoma progression. 832 MAPK inhibition modulates the induction of MHC molecules by IFN-a2b and TNF-a in human melanoma cells M Sasaki 1 , B Sapkota 2 and B Pollack 3 1 Atlanta VAMC, Atlanta, GA, 2 Emory University, Atlanta, GA and 3 Emory University / Atlanta VAMC, Atlanta, GA Aberrant signaling via the mitogen-activated protein kinase (MAPK) pathway plays a central role in melanoma development. Inhibitors of enzymes such as BRAF V600E and MEK are currently used in the treatment of melanoma patients. While these inhibitors were initially developed to block tumor cell proliferation and survival, there is an increasing recognition that these inhibitors also influence processes relevant to anti-tumor immunity. To this end, we examined the impact of vemurafenib and trametinib on the induction of MHC molecules by interferons and TNF-a. We found that both vemurafenib and trametinib enhanced the in- duction of cell surface MHC class I molecules by IFN-a2b in BRAF-mutant melanoma cells. In addition, trametinib also enhanced MHC class I induction in BRAF wildtype melanoma cells which are unresponsive to vemurafenib. These changes were associated with increases in the expression of the MHC class I transcriptional co-activator NLRC5. In addition, we found that trametinib blocked the repression of MHC class II by TNF-a in A375 melanoma cells. Using chromatin immunoprecipitation (ChIP), we found that vemurafenib enhanced levels of histone H3 lysine 4 trimethylation within promoter III of CIITA. These studies suggest that MAPK inhibitors may modulate anti-tumor immune responses in melanoma by altering the cellular response to cytokines and the expression of genes regulating antigen presentation. 833 Ablation of b-catenin signaling in stromal fibroblasts inhibits dynamic tumor- stromal interactions K Yang 1 , L Zhou 1 , A Kadekaro 1 and Y Zhang 2 1 University of Cincinnati, Cincinnati, OH and 2 University of Cincinnati, Cinicnnati, OH Cancer-associated fibroblasts (CAFs) are one of the most abundant non-cancer cells in tumor microenvironment that support melanoma to grow, migrate and develop drug resistance. b- catenin signaling is important for fibroblast to maintain biological functions. To determine whether b-catenin signaling is required for tumor-stromal interactions, we knocked down b- catenin expression in primary human fibroblasts using inducible lentiviral shRNAs (bcat-GFP/ Fbs). WM115 or SK-MEL-24 melanoma cells were cocultured with either bcat-GFP/Fbs or fibroblasts transduced with scramble shRNA (GFP/Fbs) and then stained for CAF marker expression. Interestingly, the expression of a-SMA, S100A4 and YAP nuclear translocation were all lost in bcat-GFP/Fbs. In addition, our qPCR data showed that the expression of extracellular matrix proteins, cytokines and growth factors, such as MMP8, fibronectin, TGF-b and VEGF, were higher in GFP/Fbs than in bcat-GFP/Fbs when cultured with melanoma- conditioned media. The data suggested that stromal fibroblasts require b-catenin signaling to respond to direct and indirect stimulation of human melanoma cells. To determine whether b- catenin signaling is required by stromal fibroblasts to support the growth of melanoma cells, bcat-GFP/Fbs and GFP/Fbs were cocultured with melanoma cells in a 3D spheroids tumor model. The size of tumor-GFP/Fbs spheroids was consistently bigger than that of tumor-bcat- GFP/Fbs spheroids during culture. Flow analysis showed increased cell cycle arrest and cell death in melanoma cells in spheroids mixed with bcat-GFP/Fbs, suggesting that stromal fi- broblasts lost the ability to support melanoma cell growth after b-catenin ablation. Overall, our data revealed an important role of b-catenin signaling in CAFs to maintain reciprocal tumor-stroma interactions. Furthermore, targeting b-catenin in CAFs has the potential to be an effective approach to improve melanoma treatment. 834 Exploiting oxidative stress to target melanoma A Kadekaro 1 , K Ernest 2 , F Thowfeik 3 , A Vadukoot 3 , L Zhou 4 , K Yang 5 , Y Zhang 4 and E Merino 2 1 University of Cincinnati, Cincinnati, OH, 2 Univ. Cincinnati, Dept. Chemistry, Cincinnati, OH, 3 Dept. Chemistry, Cincinnati, OH, 4 Univ. Cincinnati, Div. Pharmaceutical Sciences, Cincinnati, OH and 5 Div. Pharmaceutical Sciences, Cincinnati, OH Unlike normal cells, melanoma cells are distinguished by their high production of reactive oxygen species (ROS), a consequence of changes in metabolism associated with their ma- lignant transformation. The difference in redox state between normal and malignant cells opens an important therapeutic window that can be exploited for melanoma treatment. We have designed a cell-permeable pro-drug named RAC-1 that, upon intracellular activation by ROS induces DNA damage. We hypothesized that RAC-1 differentially target melanoma cells while sparring normal tissue. Primary melanoma tumors, derived from the Pathology lab arquives, were examined for expression of 8-OxodG, the most recognized form of oxidative DNA damage. Histological analysis of the sections revealed that all tumors examined showed extensive positive staining for 8-OxodG. In contrast, in the adjacent tumor-free area only a few cells showed positivity, suggesting reduced oxidative stress compared to melanoma cells. Next, a panel of human melanoma cell lines and normal melanocytes (NMCs) was used to determine their sensitivity to RAC-1. Out of 7 melanomas 5 displayed higher sensitivity to RAC1 (IC50 11.5+ 3; 19+ 3; 12+ 3;18+ 4; 5+ 1) compared to NMCs (50+ 7). The sensitivity to RAC1 correlated with the determined ROS levels, about ten times higher than NMCs and DNA damage determined by single cell gel electrophoresis (tail moment 22.1+ 0.7 Vs.11.5+2.3). Western blot analysis demonstrated accumulation of p53 and reduction of Bcl2. In agreement with the in vitro findings, C57BL/6J mice induced with B16F10 melanoma developed smaller tumors when treated with RAC1. These results demonstrate that targeting melanoma cells with RAC1 may be an effective alternative to selectively eliminate malignant cells. 835 A role for the unfolded protein response in the pathogenesis of vitiligo P Manga, OA Arowojolu and SJ Orlow The Ronald O. Perelman Dept. of Dermatology, New York University School of Medicine, New York, NY Vitiligo is a common skin condition characterized by progressive depigmented lesions following autoimmune-mediated destruction of melanocytes. While recent studies greatly improved our understanding of the autoimmune and genetic components, the mechanisms that initiate vitiligo are poorly understood. We hypothesized that melanocytes from vitiligo patients (VMs) are more susceptible to vitiligo-inducing triggers, such as the phenol mono- benzone (MB), than melanocytes from normally pigmented individuals (NHMs) because key cytoprotective responses are dysfunctional. We therefore performed a microarray analysis experiment to identify pathways activated by NHMs dosed with MB, then determined if they were disrupted in VMs. NHMs exposed to MB activate NRF2-regulated antioxidant responses and the unfolded protein stress response (UPR). PERK, a UPR initiator, phosphorylates NRF2 as well as the translation initiation factor eIF2a. NRF2 knockdown sensitized NHMs to MB (p < 0.0001). Conversely, NRF2 activation by knockdown of its repressor KEAP significantly increased resistance (p < 0.0001). PERK down-regulation significantly reduced melanocyte viability (p < 0.0001), though some melanocytes adapted and were maintained in culture (shPERK LT ). Survival correlated with a paradoxical increase in phospho-eIF2a and reduced sensitivity to MB (PARP cleavage/cPARP at 500mM MB in shPERK LT /400mM in controls). Furthermore, the PERK kinase inhibitor, GSK2606414, sensitized melanocytes to MB (cPARP at 250mM MB with GSK2606414/400mM without). VMs were more sensitive to MB than NHMs (p < 0.0001). Furthermore, the MB-induced increase in expression of HO1 (an NRF2- regulated antioxidant) was higher in NHMs compared to VMs. Congruently, MB-induced phospho-eIF2a was evident in NHMs, but not VMs. The PERK-NRF2 and -eIF2a axes thus determine melanocyte sensitivity to MB. The UPR, already implicated in several autoimmune disorders, may link exposure to vitiligo-inducing triggers with disease onset. Pigmentation and Melanoma | ABSTRACTS www.jidonline.org S143