Major finding: BRAF(V600E) splicing variants are found in >30% of vemurafenib-resistant patients.
Mechanism: The variant form of BRAF(V600E) dimerizes independently of RAS activation.
Impact: Tumors use multiple resistance mechanisms to circumvent RAF inhibitor therapy.
Vemurafenib is an ATP-competitive RAF inhibitor that is effective for the treatment of BRAF(V600E)-mutant melanomas, but tumors ultimately acquire resistance. RAS mutations have been observed in some refractory patients but only account for a small percentage of cases of clinical resistance to RAF inhibition. To identify new mechanisms of resistance to RAF inhibitors, Poulikakos and colleagues exposed a BRAF(V600E)-mutant cell line to a high dose of vemurafenib and generated 5 independent drug-resistant cell populations after 2 months of continuous exposure. All of the clones retained BRAF(V600E) expression, and none had acquired additional BRAF or RAS mutations, but 3 of the 5 clones expressed a shorter BRAF(V600E) splice variant lacking exons 4 to 8. This transcript encoded a functional 61-kDa protein whose ectopic expression rendered extracellular signal-regulated kinase (ERK) signaling resistant to vemurafenib and whose knockdown specifically inhibited ERK signaling and the growth of vemurafenib-resistant cells. The authors further showed that a key consequence of aberrant BRAF(V600E) splicing was loss of the RAS-binding domain (RBD), which normally regulates BRAF dimerization and activation. In the absence of activated RAS, p61BRAF(V600E) dimerization was significantly increased compared with full-length BRAF(V600E), and vemurafenib sensitivity could be restored by a second BRAF mutation abrogating dimerization. Strikingly, BRAF splice variants were identified in 6 of 19 melanoma patients with acquired vemurafenib resistance, but not in any melanomas resected from vemurafenib-naïve patients or the 4 vemurafenib-resistant patients with acquired NRAS mutations. Although the cause of RBD exon exclusion remains unclear, these findings suggest that aberrant splicing of BRAF(V600E) transcripts is a common mechanism of RAF inhibitor resistance that can be added to a growing list.
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