Investigating the relationship between BRAF status and melanoma response to anti-angiogenic therapy

A large phase 3 trial (AVAST-M) assessing bevacizumab as adjuvant therapy for melanoma has shown that patients with BRAF V600E melanomas had improved disease-free interval compared to the wild-type (WT) BRAF population, suggesting a differential sensitivity of BRAF V600E to Bevacizumab. Bevacizumab is a monoclonal antibody that inhibits VEGF, targeting angiogenesis and other endothelial cell functions. The aim of this project was to investigate two hypotheses: 1) Bevacizumab differentially targets autocrine VEGF-VEGF receptor (VEGFR) signalling on BRAF V600E vs WT tumour cells; 2) Inhibitory effects of bevacizumab in BRAF V600E melanoma are mediated indirectly via effects on angiogenesis or other components of the tumour microenvironment. Initially, VEGFRs were detected in melanoma cell lines, and secreted and intracellular VEGF levels were significantly higher in BRAF V600E cell lines, but this difference was not seen in newly-derived LM cell lines from Ludwig Institute Melbourne. To remove BRAF-unrelated variation between cell lines, isogenic BRAF WT/V600E clones were generated by CRISPR/Cas9 genome editing in BRAF WT CHL-1 cells. The introduced mutant allele was shown to mediate increased MEK-ERK pathway activation and sensitivity to mutant BRAF inhibitor PLX4720. There was no difference between BRAF WT and V600E clones in 2D growth, however, the V600E clones increased in size more rapidly in 3D growth compared to WT, indicating a more aggressive phenotype. In the cell line panel and isogenic clones, bevacizumab did not inhibit 2D or 3D growth or migration (scratch assays), suggesting that direct anti-melanoma effects are unlikely. However, VEGF ELISA showed that secreted and intracellular VEGF levels were significantly higher in isogenic BRAF V600E clones, suggesting that BRAF V600E upregulated VEGF. In vivo, xenografts grown from BRAF V600E isogenic cells were more vascular with less hypoxia and necrosis than BRAF WT tumours. Furthermore, bevacizumab significantly reduced tumour size and prolonged survival in nude mice bearing BRAF V600E xenografts, but not in the WT group. Immunohistochemical analysis showed that bevacizumab induced significantly more hypoxia and reduced the vascularity of BRAF V600E but not WT tumours. There was an increase in mouse serum VEGF levels in mice bearing BRAF V600E xenografts treated with bevacizumab, suggesting a possible compensatory up-regulation of murine VEGF upon human VEGF inhibition. To identify differentially expressed genes that may contribute to the sensitivity of BRAF V600E melanomas to anti-angiogenic therapy, microarray analysis on the isogenic clones was combined with four datasets including RNA-seq on RNA extracted from frozen patient tumours, tumour samples from the AVAST-M trial, publicly-available data on gene expression in the LM melanoma cell line panel, and patient-derived melanomas from TCGA. Out of the genes validated, ROR2 was shown to upregulate VEGF in BRAF V600E clones, indicating a possible mechanism for the link between BRAF V600E mutation and VEGF upregulation. In summary, this project generated an isogenic BRAFWT/V600E melanoma model and showed that the introduced V600E mutation led to increased 3D growth, VEGF upregulation, and increased tumour vascularity and bevacizumab sensitivity in vivo, paralleling results of the AVAST-M trial. Transcriptional analysis identified candidate genes upregulated by BRAF V600E that may mediate increased dependence on tumour angiogenesis.

Advisor: Macaulay, V
Middleton, M