Investigating the roles of receptor tyrosine kinases in Vemurafenib resistance and phenotype switching in melanoma

Abstract

Despite the initial efficacy of Vemurafenib treatment in BRAF-mutant metastatic melanoma, the majority of patients eventually develop progressive disease due to drug resistance. The main mechanisms of resistance reported include reactivation of the RAF-MEK-ERK signalling pathway, commonly through increased activity of upstream receptor tyrosine kinases (RTKs). This study assesses the role of feedback regulation in RTK-mediated Vemurafenib resistance, the influence of RTKs on Vemurafenib-induced phenotype switching of melanoma cells, and the assessment of combination therapies targeting signalling pathways induced by RTK activation. The thesis is mainly focused on in vitro analysis using the A375 cell line model overexpressing EGFR, FGFR1, MET, IGFR and KIT. Prior to this thesis, data generated in our laboratory (Ramsdale and Ferrao et al., unpublished) revealed that ligand activation of EGFR, FGFR1 and cMET in the A375 lines were each able to confer Vemurafenib resistance, sustain cell proliferation during Vemurafenib treatment and enhance activation of ERK and JNK signalling pathways, whereas IGFR activated by IGF was not able to sustain proliferation and enhanced activation of AKT signalling.

This thesis first confirmed the reported findings that Vemurafenib treatment resulted in reduced levels of feedback regulator SPROUTY2 (SPRY2). Ligand activation of EGFR, FGFR1 and MET prevented Vemurafenib-induced reduction of SPRY2, whereas IGFR and KIT did not. Using an inducible expression system, enforced expression of SPRY2 to maintain levels during Vemurafenib treatment was demonstrated to reverse the resistance mediated by EGFR, FGFR1 and MET in A375 cells. In multiple BRAF-mutant melanoma cell lines adaptation to longer-term Vemurafenib treatment induced resistance that was associated with reduction of SPRY2 and SPRY4, which was reversible by withdrawal of Vemurafenib. Adaptation to Vemurafenib treatment over 4 weeks for A375 cells also induced a switch to a more mesenchymal-like phenotype associated with elevated expression of EMT-inducing transcription factors (EMT-TFs) and increased cell migration. These Vemurafenib-induced effects were also reversible by drug withdrawal. Through overexpression and knockdown studies, cJUN increased by Vemurafenib treatment was identified as critical mediator of Vemurafenib-induced phenotype switching and cell survival. Activation of Rho-ROCK signalling was demonstrated to contribute to Vemurafenib-induced cJUN. Activation of EGFR in A375 prevented Vemurafenib-induced changes in the expression of cJUN and other EMT-TFs, whereas IGFR did not. Consequently, activation of EGFR but not IGFR prevented the Vemurafenibinduced change to a mesenchymal-like morphology associated with increased cell migration in A375.

Immuno-blotting and reverse phase protein array (RPPA) approaches were utilised to assess RTK-mediated signalling. Activated EGFR and IGFR were confirmed to induce different intracellular signalling pathways. EGFR could enhance JNK, Src/STAT3, PI3K/AKT and ERK signalling, whereas IGFR was confirmed to strongly increase PI3K/AKT signalling. The effects of various drug combinations with Vemurafenib including inhibitors to specific RTKs, AKT, JNK, Src and ERK on the A375 cells expressing EGFR, FGFR1, MET, IGFR and KIT were assessed using high throughput cell number assays and high-content imaging analysis. Combination with specific RTK or JNK inhibitors overcame the drug resistance mediated by the specific RTKs, confirming previous dose response assay data (Ramsdale and Ferrao et al, unpublished). Combination with an AKT inhibitor with Vemurafenib was able to reduce the viable cell numbers surviving at the treatment endpoint. Noticeably, immuno-blotting revealed that combination treatment with an AKT inhibitor prevented Vemurafenib-induced reduction of SPRY2 and reduced levels of Vemurafenib-induced cJUN in the RTK-expressing A375 cell lines.

Together, findings from this thesis provide direct evidence that disabled feedback regulation by Vemurafenib-induced reduction of SPRY2 contributes to RTKmediated drug resistance. Activation of RTKs can alter Vemurafenib-induced cJUN and EMT-like phenotype switching during Vemurafenib adaptation. Signalling analyses and assessment of combination drug treatments have implications for developing appropriate therapeutic strategies for overcoming RTK-mediated Vemurafenib resistance associated with melanoma progression.