Most patients with BRAF-mutant metastatic melanoma display remarkable but incomplete and short-lived responses to inhibitors of the BRAF kinase or the mitogen-activated protein kinase kinase (MEK), collectively BRAF/MEK inhibitors. We found that inherent resistance to these agents in BRAF(V600)-mutant melanoma cell lines was associated with high abundance of c-JUN and characteristics of a mesenchymal-like phenotype. Early drug adaptation in drug-sensitive cell lines grown in culture or as xenografts, and in patient samples during therapy, was consistently characterized by down-regulation of SPROUTY4 (a negative feedback regulator of receptor tyrosine kinases and the BRAF-MEK signaling pathway), increased expression of JUN and reduced expression of LEF1. This coincided with a switch in phenotype that resembled an epithelial-mesenchymal transition (EMT). In cultured cells, these BRAF inhibitor-induced changes were reversed upon removal of the drug. Knockdown of SPROUTY4 was sufficient to increase the abundance of c-JUN in the absence of drug treatment. Overexpressing c-JUN in drug-naïve melanoma cells induced similar EMT-like phenotypic changes to BRAF inhibitor treatment, whereas knocking down JUN abrogated the BRAF inhibitor-induced early adaptive changes associated with resistance and enhanced cell death. Combining the BRAF inhibitor with an inhibitor of c-JUN amino-terminal kinase (JNK) reduced c-JUN phosphorylation, decreased cell migration, and increased cell death in melanoma cells. Gene expression data from a panel of melanoma cell lines and a patient cohort showed that JUN expression correlated with a mesenchymal gene signature, implicating c-JUN as a key mediator of the mesenchymal-like phenotype associated with drug resistance.

The majority of melanoma patients treated with a BRAF inhibitor (BRAFi) eventually acquire drug resistance. A major mechanism for acquired resistance is the activation of specific receptor tyrosine kinases (RTKs). We have recently shown that inherent and early adaptive resistance is associated with activation of the JNK-JUN pathway, decreased levels of the SPROUTY negative regulators and a mesenchymal-like phenotype. RTKs have been reported to be regulated by SPROUTY proteins and are able to mediate cell migration and invasion. In this study, we compared the expression and activation of specific RTKs previously linked to BRAFi resistance, within the same cellular background. EGFR, FGFR1, MET and IGF1R were constitutively expressed in the BRAFi-sensitive melanoma cell line A375. The impact of ligand activation of each of these receptors in the presence of BRAFi on proliferation, survival, signalling, morphology, expression of phenotype markers and cell migration was assessed. We also over-expressed SPROUTY2 using an inducible system, to study its role in regulating RTK-mediated BRAFi resistance. In A375 cells, overexpression and activation of either EGFR, FGFR1 or MET were able to reverse inhibition of cell proliferation induced by BRAFi, whereas IGF1R expression and activation was able to reduce cell death induced by high doses of BRAFi, but was unable to reverse inhibition of cell proliferation. The RTKs differed in their ability to activate signalling following treatment with BRAFi. In A375 cells, EGFR, FGFR1 and MET activated by their respective ligands, predominantly induced ERK signalling, whereas ligand activated IGF1R predominantly induced AKT signalling. EGFR activation conferred resistance to BRAFi by overcoming RAF-MEK-ERK pathway inhibition. Activation of EGFR was also able to prevent the early drug adaptive phenotype and reverse the BRAFi-induced changes in the expression of c-JUN, SNAI2, MITF and ZEB2, In contrast activation of IGF1R did not reverse the early drug adaptive phenotype induced by BRAFi. Combination treatment with either an IGF1R or AKT inhibitor was sufficient to overcome IGF1R-mediated survival. Enforced expression of SPROUTY2 reversed the resistance mediated by EGFR or FGFR1 activation. SPROUTY2 expression also conferred sensitivity to BRAFi in LOX-IMV1 cells that were inherently resistant to BRAFi and expressed activated EGFR and FGFR1. Together, our data suggests that RTKs vary in the signalling pathways that are induced upon ligand-activation, particularly in the context of BRAFi resistance. Our findings have clinical relevance for the rational design of drug combinations to target specific RTK signalling and overcome RTK-mediated resistance. Citation Format: Frederic Zhentao Li, Rachel Ramsdale, Amardeep Dhillon, Grant McArthur, Petranel Ferrao. Differences in signalling and phenotypic effects in melanoma between receptor tyrosine kinases that confer resistance to BRAF inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C70.

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 the drug-naive state. In contrast, activated IGFR did not alter the drug-induced effects in A375. Our study reveals that RTKs differ in their ability to confer proliferation signaling and the EMT-like phenotype-switching, both having strong implications in the development of resistance to BRAFi in melanoma. Citation Format: Zhentao Li, Rachel Ramsdale, Robert Jorissen, Karen Sheppard, Amardeep Dhillon, Grant McArthur, Petranel Ferrao. Receptor tyrosine kinases can mediate compensatory signaling and phenotype-switching associated with resistance to BRAF inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2687. doi:10.1158/1538-7445.AM2015-2687