Abstract B06: Loss of SPRY1 expression in BRAF-mutant cutaneous melanoma inhibits cell proliferation and improves response to targeted therapy

Abstract

Abstract Background: Targeted therapy has proven efficacy in BRAF-mutant metastatic cutaneous melanoma (CM), but patients almost invariantly develop an early resistance. Although the underlying mechanisms are various and heterogeneous, resistance usually associates with an altered signaling. Sprouty 1 (SPRY1) has been described as an upstream mediator of MAPK signaling pathway, and although it has been found to be implicated in different biologic processes, its specific role in BRAF-mutant CM has been only partially addressed. Material and Methods: SPRY1 expression was assessed by quantitative real-time RT-PCR (qRT-PCR) and Western blotting (WB) analyses in BRAF-mutant CM cell lines and tissues. SPRY1 gene was knocked out using CRISPR-strategy. WB analyses evaluated the modulation of MAPK signaling pathways in SPRY1 knockout (SPRY1KO) clones. RNA-seq identified mRNAs that were differentially expressed in SPRY1KO clones respect to parental cells, and their expression was then validated by qRT-PCR. To further compare SPRY1KO clones to parental cells, cell proliferation was measured using xCELLigence instrument, whereas cell cycle progression, ROS induction, and apoptosis were assessed by flow cytometry. In vivo effects of SPRY1 silencing were investigated in xenografted mice treated with or without BRAF inhibitor (BRAFi). Results: SPRY1 was found highly expressed in a panel of BRAF-mutant CM cell lines and tissues, both at mRNA and protein level. To investigate the biologic role of SPRY1 in BRAF-mutant CM, SPRY1KO clones were obtained from 3 CM cell lines. RNA-seq identified 38 mRNAs that were significantly commonly modulated in SPRY1KO clones respect to parental cells. Ingenuity Pathway Analysis indicated that the top of the neighbor coding gene function of differentially expressed mRNAs was involved in cell proliferation and cell death. In addition, WB assays showed SPRY1 silencing was associated with i) decreased activation of p38 MAPK and beta-catenin signaling, ii) reduced expression of metalloproteinase 2, and iii) increased levels of p53. Consistent with these data, SPRY1 silencing markedly induced cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and impaired tumor growth in vivo. In addition, SPRY1 knockdown associated with improved response to BRAFi both in vitro and in vivo, which could be partly contributed by the higher levels of ROS observed in SPRY1KO clones. Interestingly, BRAFi treatment reduced mRNA and protein SPRY1 levels in parental BRAF-mutant CM cell lines, indicating that SPRY1 expression is downstream of the MEK-ERK signaling. Of note, some of the effects induced by BRAFi in parental cells were similar to those observed in SPRY1KO clones, suggesting that BRAFi activity may at least in part rely on SPRY1 downregulation. Conclusions: All together, our findings reveal important insights into SPRY1 role in BRAF-mutant CM and suggest its potential involvement in targeted therapy response. Citation Format: Barbara Montico, Giorgio Giurato, Francesca Colizzi, Alessia Covre, Elisa Comaro, Eliana Pivetta, Dania Benedetti, Alessandro Weisz, Agostino Steffan, Michele Maio, Luca Sigalotti, Elisabetta Fratta. Loss of SPRY1 expression in BRAF-mutant cutaneous melanoma inhibits cell proliferation and improves response to targeted therapy [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B06.