Co-expression of stimulators and inhibitors of T-cell activation in melanoma

Melanoma, one of the most aggressive skin cancers, has steadily been on the rise over the last three decades. With limited treatment options, the recent impetus in immuno-oncology drugs, especially checkpoint inhibitors, has changed the treatment landscape. While anti-CTLA4 and anti-PD1 demonstrated success in clinic, the search for additional targets is needed to enable potent comprehensive curative combination therapies. Our laboratory has characterized and screened five primary patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2. While immunomodulatory molecules are canonically present on antigen presenting cells and T-cells, increasing evidence suggests that they are not expressed in isolation. In an effort to identify immunomodulatory molecules expressed on our primary melanoma cells, a comprehensive RT-PCR screen of 29 co-inhibitory and co-stimulatory molecules was carried out. Several molecules including CD160, CD226, TIM1, HVEM, and BTLA were seen to be differentially expressed in melanoma cells compared to normal melanocytes at the mRNA level. Western blots and immunocytochemistry validated the differential expression of these molecules at the protein level. 50-80% of melanoma cases are positive for the BRAFV600E mutation, and are treated with a small molecule inhibitor of the mutated BRAF, vemurafenib (PLX4032). Treatment of these cells with PLX4032 led to an upregulation of transcription factors MITF and AP-1, as well as immunomodulatory molecules, CD160, CD226, TIM1, HVEM, and BTLA, a phenomenon seen only in cells positive for the BRAFV600E lesion. MITF and AP-1, owing to the binding sites present in the promoter regions of these molecules, can drive their expression upon treatment with PLX4032. These additional immune-regulatory molecules of T-cell activation and/or immune tolerance mechanisms are potential targets for a combination therapy with PLX4032 in melanoma patients positive for the BRAFV600E genetic lesion. Our future directions aim to elucidate the role of these molecules on the tumor cells and devise an effective combination with small-molecule inhibitors and immunotherapies. Citation Format: Rachana Maniyar, Robert Freund, Aryan Malhotra, Sanjukta Chakraborty, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Co-expression of stimulators and inhibitors of T-cell activation in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B174.