Abstract 2687: Receptor tyrosine kinases can mediate compensatory signaling and phenotype-switching associated with resistance to BRAF inhibitors

BRAF/ERK is the most frequently activated oncogenic signaling pathway in metastatic melanoma. Although clinical studies have demonstrated initial efficacy of BRAF inhibitors (BRAFi), the therapeutic responses are short-lived in many patients due to the acquisition of resistance. Inhibition of mutant BRAF in melanoma has been reported to induce epithelial-mesenchymal transition (EMT)-like phenotype switching. One mechanism of phenotype-switching associated with resistance to BRAFi is down-regulation of MITF and increased levels of WNT5A. Across a panel of 22 BRAFV600E melanoma lines, those intrinsically resistant to BRAFi were found to express significantly higher levels of mesenchymal markers whereas the sensitive lines exhibited more epithelial-like characteristics. The intrinsically resistant lines featured high expression of multiple receptor tyrosine kinases (RTKs) including EGFR, but low levels of other RTKs such as IGFR. RTKs are known able to mediate melanoma phenotype-switching and can confer BRAFi resistance via reactivation of ERK signaling. Hence, we further assessed the link between specific RTKs and drug resistance, ERK signaling and phenotype-switching. In A375 and other drug sensitive cell lines, specific RTKs constitutively expressed and activated with ligands, differed in their ability to confer a proliferative advantage during drug treatment, which was associated with variation in signaling. Activated EGFR was able to drive compensatory ERK signaling whereas activated IGFR strongly activated AKT signaling. In the absence of RTK expression, BRAFi-treated A375 cells displayed an EMT-like phenotype switch. Drug adaptation induced resistance to BRAFi, a mesenchymal-like phenotype and increased migration in A375 cells, which were reversible by removal of the drug. During this period, we found reversible hyper-activation of ERK signaling due to relief of feedback regulation by BRAFi, and an altered composition of the AP-1 complex with down-regulated expression of FRA-1. Interestingly this was associated with increased MITF and SLUG in A375 cells. Expression and activation of EGFR in A375 cells treated with BRAFi maintained ERK signaling, expression of feedback regulator Sprouty2 (SPRY2) and FRA-1. Enforced expression of SPRY2 was able to reverse BRAFi resistance induced by EGFR signaling. EGFR activation also suppressed BRAFi induction of MITF, expression of EMT-inducers, including SLUG and ZEB2, with cells retaining the morphology similar to t