Mechanisms Underlying RAF Inhibitor Resistance by BRAF Splice Variants in Melanoma

The serine/threonine kinase, BRAF, is mutated in 7% of cancers and 50% of melanomas. The majority of the mutations result in constitutive protein activation due to a valine to glutamic acid substitution (V600E). FDA approved RAF inhibitors prolong survival for patients with BRAF V600E melanoma; however, acquired resistance mechanisms limit the long-term efficacy of such therapies. One of the most common mechanisms of resistance to RAF inhibitors in melanoma is the expression of aberrantly spliced BRAF. We study the mechanisms underlying BRAF splice variant mediated resistance to RAF inhibitors in melanoma. Prior studies have focused on the observation that BRAF splice variants homodimerize to a greater degree than full-length BRAF V600E. We demonstrate that low-dose treatment with the RAF inhibitor, vemurafenib, disrupts BRAF dimers at concentrations that do not affect BRAF signaling. Instead, we observe enhanced association with BRAF’s substrate, MEK1/2, and measure association levels that correlate with the response to vemurafenib. Point mutagenesis of BRAF’s MEK-binding interface demonstrates that enhanced BRAF/MEK association is required for splice variant mediated resistance. Aberrant BRAF splicing removes one of the binding sites for the 14-3-3 family of scaffolding proteins at serine 365 (S365). The other binding, serine 729 (S729), is always retained. 14-3-3 binding sites regulate BRAF activity in the physiologic setting but their involvement in oncogenic signaling and response to RAF inhibitor are not fully appreciated. We demonstrate that loss of S365 in full-length BRAF is sufficient to increase dimerization and reduce sensitivity to vemurafenib. Mutation of S729 in BRAF splice variants abrogates protein dimerization and MEK association and increases the sensitivity to vemurafenib. These studies demonstrate that the 14-3-3 binding sites, and S729 in particular, play an integral role in the response to RAF inhibitors. In total, we expand the model of splice variant mediated resistance to incorporate enhanced BRAF/MEK association and 14-3-3 binding site regulation. These studies inform the characterization of next-generation RAF inhibitors that may have unappreciated effects on BRAF substrate association and dimerization. Finally, we suggest that the regulation of S729 may represent a future therapeutic target for patients with RAF driven malignancies