Abstract 241: Up-regulation of PD-L1 in melanoma determines resistance to BRAF and MEK inhibitors, induces a more aggressive phenotype and is partially mediated through post-transcriptional mechanisms involving miR-17-5p
Authors: Brusa Davide , Stingi Aureliano , Audrito Valentina , Orso Francesca , Serra Sara , Buonincontri Roberta , Neri Francesco , Baroni Gianna , Merelli Barbara , Nassini Romina , Massi Daniela , Oliviero Salvatore , Taverna Daniela , Mandalà Mario , Deaglio Silvia 
Published: July 2016

Abstract
Introduction: The therapy of metastatic melanoma (MM) was radically changed by the introduction of BRAF inhibitors (BRAFi). Even if highly effective in the short term, patients invariably develop resistance in the long term. For this reason other inhibitors as well as alternative or complementary therapeutic strategies are being tested in these patients. Among the immune checkpoint targets of clinical importance is PD-1, which is expressed by T cells and which binds to the PD-L1 ligand, which may be expressed by melanoma cells. We and others showed that PD-L1 is an independent negative prognostic marker for patients with MM.
Methods: BRAFi-/MEKi-resistant melanoma cell lines were generated by treating cells with increasing concentrations of BRAFi or MEKi. Resistance, viability and aggressiveness were analyzed by MTT, migration and wound healing assays. Results were confirmed using xenograft models. Resistant cell lines were compared using RNAseq to identify enriched genetic pathways involved in the resistance. Luciferase reporter assay analysis was used to study the direct interactions between the PD-L1 and miR-17-5p.
Results: By comparing responses to BRAFi in PD-L1+ and PD-L1- variants of the A375 cell line, we found that PD-L1 expression conferred resistance to BRAFi or MEKi. Conversely, silencing of the molecule restored sensitivity to these drugs. Resistant melanoma cell lines acquired PD-L1 expression and were characterized by a specific genetic profile, with the modulation of genes controlling cell movement and immune responses. Consistently, these cells showed a more aggressive behavior both in vitro and in xenograft models. PD-L1 silencing in resistant cells decreased invasive properties and restored expression of HLA-II molecules.
PD-L1 up-regulation was only partly dependent on the paradoxical activation of the MAPK pathway, which characterized resistant cells. In addition, we found that resistance to BRAFi and MEKi down-modulated miR-17-5p, which showed an inverse correlation with PD-L1. Transfection of miR-17-5p into BRAFi-resistant cell lines induced the down-modulation of PD-L1, reduced the aggressive behavior of the cells and partially restored sensitivity to BRAFi. Finally, in the plasma of patients with MM, miR-17-5p was inversely correlated with expression of PD-L1 in the tumor tissue.
Conclusions: These data demonstrate a direct link between expression of PD-L1 and resistance to BRAFi, as well as to a more aggressive behavior of melanoma cells. Furthermore, we define a novel post-transcriptional circuit responsible for PD-L1 up-regulation, based on a direct interaction between miR-17-5p and PD-L1 mRNA. Lastly, miR-17-5p plasmatic levels show an inverse correlation with PD-L1 expression by tumor cells, suggesting that they may be useful in monitoring disease outcome and drug sensitivity.
Citation Format: Davide Brusa, Aureliano Stingi, Valentina Audrito, Francesca Orso, Sara Serra, Roberta Buonincontri, Francesco Neri, Gianna Baroni, Barbara Merelli, Romina Nassini, Daniela Massi, Salvatore Oliviero, Daniela Taverna, Mario Mandalà, Silvia Deaglio. Up-regulation of PD-L1 in melanoma determines resistance to BRAF and MEK inhibitors, induces a more aggressive phenotype and is partially mediated through post-transcriptional mechanisms involving miR-17-5p. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 241.