Abstract 1794: Loss of Spry1 reduces growth of BRAFV600-mutant cutaneous melanoma and improves response to targeted therapy
Authors: Montico Barbara , Colizzi Francesca , Giurato Giorgio , Rizzo Aurora , Salvati Annamaria , Baboci Lorena , Benedetti Dania , Pivetta Eliana , Covre Alessia , Bo Michele Dal , Weisz Alessandro , Steffan Agostino , Maio Michele , Sigalotti Luca , Fratta Elisabetta 
Published: September 2020

Abstract
Introduction: Targeted therapies have recently revolutionized the treatment of BRAFV600-mutant metastatic cutaneous melanoma (CM). However, most patients do not achieve tumor regression at all due to the occurrence of resistance mechanisms. Mitogen-activated protein kinase (MAPK) pathway activation is a central step in BRAFV600-mutant CM pathogenesis and a better understanding of molecular mechanisms involved MAPK modulation is necessary to develop more effective therapeutic strategies. In the last years, Spry1 has been frequently described as an upstream regulator of MAPK signaling pathway, but its specific role in BRAFV600-mutant CM is still poorly defined.
Materials and methods: Autologous BRAFV600-mutant CM cell cultures were generated from CM patients resected in our institution. The expression of Spry1 was evaluated by quantitative real-time PCR (qRT-PCR) and Western blot (WB) analyses. Spry1 gene was knock out using CRISPR. RNA-seq identified mRNAs commonly differentially expressed in Spry1 knock-out (Spry1KO) clones, and their expression was further validated by qRT-PCR. WB analyses evaluated the modulation of MAPK signaling pathways in Spry1KO clones. 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 xenograft mice treated with or without BRAF inhibitor (BRAFi).
Results: The expression of Spry1 in human CM was explored using publicly available cancer gene expression profiling and transcriptome sequencing data. mRNA expression of Spry1 was significantly elevated in metastatic CM respect to primary tumors and mRNA levels of Spry1 were significantly up-regulated in metastatic CM compared with primary lesions. The function of Spry1 was evaluated by comparing three BRAFV600-mutant CM cell lines and the respective Spry1KO cells. We found that Spry1KO induced cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and impaired tumor growth in vivo. Moreover Spry1KO was found to impair the expression of several markers of epithelial-mesenchimal transition. In addition, ERK1/2 was found hyperactivated together with p38 leading to an increase in basal ROS levels in Spry1KO clones. This suggests that BRAFV600-mutant CM may restrain the activity of Spry1 to avoid oncogenic stress and to enable tumor growth. Indeed, treatment with the BRAFi vemurafenib down-regulated Spry1 levels in parental CM cell lines, indicating that Spry1 expression is sustained by MAPK/ERK signaling pathway in a positive feedback loop that safeguards cells from the potentially toxic effects of ERK1/2 hyperactivation. Disruption of this feedback loop rendered Spry1KO cells more susceptible to apoptosis and markedly improved response to BRAFi both in vitro and in vivo, as a consequence of the detrimental effect of ERK1/2 hyperactivation observed upon Spry1 abrogation.
Conclusions: All together, our data strongly suggest that targeting Spry1 might offer a treatment strategy for BRAFV600-mutant CM by inducing the toxic effects of ERK-mediated signaling.
Citation Format: Barbara Montico, Francesca Colizzi, Giorgio Giurato, Aurora Rizzo, Annamaria Salvati, Lorena Baboci, Dania Benedetti, Eliana Pivetta, Alessia Covre, Michele Dal Bo, Alessandro Weisz, Agostino Steffan, Michele Maio, Luca Sigalotti, Elisabetta Fratta. Loss of Spry1 reduces growth of BRAFV600-mutant cutaneous melanoma and improves response to targeted therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1794.