Abstract C70: Differences in signalling and phenotypic effects in melanoma between receptor tyrosine kinases that confer resistance to BRAF inhibitors
Authors: Li Frederic Zhentao , Ramsdale Rachel , Dhillon Amardeep , McArthur Grant , Ferrao Petranel 
Published: May 2016

Abstract
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.