Background: About 50% of cutaneous melanoma (CM) harbor the activating BRAFV600E mutation, which exerts most of the oncogenic effects through the activation of the MAPK signaling pathways. BRAF inhibitors (BRAFi) have showed important clinical activities in CM, but their effectiveness is impaired by the emergence of an early drug resistance. Accordingly, a better understanding of the molecular basis of BRAFi resistance may provide insights useful to develop new therapeutic strategies in CM. Spry gene family includes four members (Spry1-4), which differ in their tissue distribution, activity and interaction partners, thus suggesting nonredundant functions. Although interactions between Spry proteins and several MAPK pathway components have been reported, the specific role of Spry1 in CM has yet to be defined.

Methods: Bioinformatic analysis on data available on TCGA was performed using the pipeline suggested on the TCGA portal. Spry1 expression levels were measured in BRAFV600 mutant CM cell lines using quantitative real-time PCR (qRT-PCR) and Western blotting. CRISPR based strategy was used to knock out Spry1 gene in BRAFV600 mutant CM cell lines established in our Institution. Transfected cells were sorted into single cells and subsequently expanded. Spry1 knockout (Spry1KO) clones were validated by Sanger sequencing and Western blotting, respectively. Cell viabilities before and after BRAFi treatment were evaluated by MTT and clonogenic assays. Apoptosis was assessed by Annexin V/PI staining. Modulation of MAPK signaling pathways and apoptosis were examined by qRT-PCR and Western blotting. P53 nuclear translocation was evaluated through multispectral imaging flow cytometry analysis.

Results: Specific bioinformatic analysis, using TCGA, was performed to provide more information regarding pathways associated with MAPK signaling. Our analysis revealed that Spry proteins are differentially expressed in CM. In line with these findings, Spry1 was found high expressed in a panel of CM cell lines with BRAFV600 mutation both at mRNA and protein level. Preliminary data demonstrated that the expression and/or phosphorylation of key proteins involved in MAPK signaling were modulated in Spry1KO clones with respect to the parental cell line. Furthermore, Spry1 inactivation associated with: i) enhanced activation and nuclear translocation of the tumor suppressor p53, and ii) decreased mRNA and protein levels of several antiapoptotic proteins. Of note, the treatment of Spry1KO clones with BRAFi reduced CM cell survival in clonogenic assays, and induced apoptosis in a dose- and time-dependent manner. Further studies are ongoing in order to determine the molecular changes involved in Spry1KO clones in response to BRAFi treatment.

Conclusions: Our results suggest that Spry1 gene may exert oncogenic functions in CM and its upregulation can potentially contribute to BRAFi resistance.

Citation Format: Giorgio Giurato, Francesca Colizzi, Aurora Rizzo, Debora Martorelli, Barbara Montico, Katy Mastorci, Dania Benedetti, Alessandro Weisz, Riccardo Dolcetti, Sigalotti Luca, Elisabetta Fratta. Suppression of Spry1 sensitizes cutaneous melanoma to BRAF-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1839.