MELANOLOCK

Immunotherapy with Immune Checkpoint Inhibitors (ICIs) has become the frontline therapy for patients with metastatic melanoma independent of the BRAF-mutational status. Despite the clinical efficacy, 40–60% of patients do not achieve significant therapeutic response and a substantial proportion of responders relapse within 2 years. Reprogramming towards an immunosuppressive phenotype of the tumour immune microenvironment (TIME) has emerged as one of the main determining factors of resistance to ICIs. Strategies that aim to reprogram various facets of TIME may help to tackle both innate and acquired immune-resistance mechanisms. Over the last few years, microRNAs (miRNAs) have emerged as orchestrators of therapy resistance in melanoma. In this project, we plan to apply a strategy based approach on Locked Nucleic Acids (LNA) that targets specific miRNAs involved in TIME shaping towards an immune evasive phenotype and to show their therapeutic efficacy in innovative pre-clinical models. Thanks to this approach, we aim to tackle resistance to immunotherapy with ICI in advanced melanoma. The miRNAs that will be targeted in this project proposal have been recently identified by miRNA-seq profiling, performed on serum samples from melanoma patients before starting ICI therapy. This allowed to identify a subset of 19 oncogenic miRNAs (oncomiRs) highly up-regulated in non-responder patients. Starting from these data, 4 oncomiRs were selected according to the i) higher level of deregulation and ii) association with melanoma patients’ prognosis based on TCGA data. Moreover, digital PCR (dPCR) performed on the RNAs extracted from extracellular vesicles (EVs) from melanoma cells have underscored the presence of these oncomiRs, thus suggesting their role in TIME shaping. From here, the leading hypothesis postulated is that these circulating miRNAs are released by melanoma cells and regulate a plethora of post-transcriptional mechanisms responsible for the shaping of an immune evasive TIME. According to this hypothesis, preliminary results revealed that these miRNAs govern molecular pathways like i) CXCR3-CXCL9/CXCL10 axis, which regulates the recruitment and positioning of effector T cells within the TIME and ii) GZMB and PRF1 genes expression, which are crucial for the cytotoxic activities of CD8 T lymphocytes (CTL). More recently, we have demonstrated that the inhibition of one of these four miRNAs by LNAs is able to increase the release of CXCL9 cytokine from human macrophages. In turn, the culture media (CM), isolated from macrophages treated with these LNAs, are able to better attract T lymphocytes as compared to CM from macrophages treated with control LNAs. Given these preliminary results, the aim of this project is to target the above mentioned oncomiRs using LNAs, to potentiate immunotherapy in advanced melanomas exploiting different immunocompetent melanoma preclinical models namely 3D organotypic models and humanized PDX models.