RMEL3 long noncoding RNA as a modulator of the immune response in melanoma

Abstract

In recent years, the treatment of cutaneous melanoma has experienced a revolution with theintroduction of targeted therapy (BRAF V600E and MEK inhibitors) and immunotherapy (anti-CTLA4, anti-PD1, and anti-LAG3). Even though most patients still show poor response ortumor progression due to acquired resistance, a small set of patients show extraordinarybenefit with the immunotherapy. These scenarios indicate the need to find efficient markers topredict the responsive patients and understand the mechanisms of resistance so that optimalbenefit could be extended to all patients. The highly tissue- and condition-specific expressionof long noncoding RNAs (lncRNAs) make them potential therapeutic targets and diagnosticmarkers. Several lncRNAs play roles in cancer and one of them, RMEL3, identified in ourlaboratory, shows enriched expression in melanoma, particularly associated with BRAF V600Emutation and the invasive phenotype. Interaction studies in our laboratory showed associationof RMEL3 with molecules of the MAPK (ARAF and a CRAF regulatory protein, FAM83D)and the HIPPO kinase (MST2) pathways, and in silico analysis allowed us to map theconserved interaction domains on the RMEL3 sequence. These domains are predicted to formstem loops that are appropriate structures for RNA-protein interaction. In a recent RNAseqstudy performed in A375 melanoma cell line, we linked RMEL3 with the positive regulationof many genes involved in immunogenicity and immunosuppression, including all MHC classII and many MHC class I genes of the HLA gene cluster located in chromosome 6p. Theseresults, together with functional studies, allowed us to hypothesize that RMEL3 plays anoncogenic role and modulates the melanoma tumor microenvironment towards immuneevasion and immunosuppression, making it a potential therapeutic target for melanomatreatment. In this BEPE project, we propose to design isoform and domain-specific antisenseoligonucleotides (ASOs) targeting RMEL3 lncRNA to be tested for their in vitro and in vivoanti-tumoral activity with strategies to determine whether the anti-tumoral activity is due tochanges in the melanoma cell susceptibility to CD8+ T cell activity and cytotoxicity, in a ASOdose-dependent manner. The activity of CD8+ T cells will be measured by quantification ofsecreted cytokines and killing assays against ASO or vehicle-treated co-cultured melanomacells. For in vivo assays, we will test the anti-tumoral effect of ASOs in immunocompetent andimmunodeficient mice. Thus, we expect that this project will contribute toward defining thetherapeutic usage of RMEL3 lncRNA inhibitors.