Novel mechanisms of resistance to vemurafenib in melanoma - V600E B-Raf reversion and switching VEGF-A splice isoform expression.

Targeting activating mutations in the proto-oncogene B-Raf, in melanoma, has led to increases in progression free survival. Treatment with vemurafenib, which inhibits the most common activating-mutated form of B-Raf (B-Raf(V600E)), eventually results in resistance to therapy. VEGF-A is the principal driver of angiogenesis in primary and metastatic lesions. The bioactivity of VEGF-A is dependent upon alternative RNA splicing and pro-angiogenic isoforms of VEGF-A are upregulated in many disease states dependent upon angiogenesis, including cancers. Using techniques including RT-PCR, Western blotting, ELISA and luciferase reporter assays, the effect of vemurafenib on proliferation, ERK1/2 phosphorylation and the levels of pro- and anti-angiogenic VEGF-A isoforms was investigated in melanoma cell types expressing either wild-type B-Raf or B-Raf(V600E), including a primary melanoma culture derived from a highly vascularised and active nodule taken from a patient with a V600E mutant melanoma. The primary melanoma culture was characterised and found to have reverted to wild-type B-Raf. In B-Raf(V600E) A375 cells ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA, total VEGF-A protein expression and VEGF-A 3'UTR activity were all decreased in a concentration-dependent manner by vemurafenib. Conversely vemurafenib treatment of wild-type B-Raf cells significantly increased ERK1/2 phosphorylation, pro-angiogenic VEGF-A mRNA and total VEGF-A expression in a concentration-dependent manner. A switch to pro-angiogenic VEGF-A isoforms, with a concomitant upregulation of expression by increasing VEGF-A mRNA stability, may be an additional oncogenic and pathological mechanism in B-Raf(V600E) melanomas, which promotes tumor-associated angiogenesis and melanoma-genesis. We have also identified the genetic reversal of B-Raf(V600E) to wild-type in an active melanoma nodule taken from a V600E-positive patient and continued vemurafenib treatment for this patient is likely to have had a detrimental effect by promoting B-Raf(WT) activity.