Analyses of the oncogenic BRAFD594G variant reveal a kinase-independent function of BRAF in activating MAPK signaling.

Class 3 mutations in B-Raf proto-oncogene, Ser/Thr kinase (BRAF), that result in kinase-impaired or kinase-dead BRAF have the highest mutation frequency in BRAF gene in lung adenocarcinoma. Several studies have reported that kinase-dead BRAF variants amplify mitogen-activated protein kinase (MAPK) signaling by dimerizing with and activating WT C-Raf proto-oncogene, Ser/Thr kinase (CRAF). However, the structural and functional principles underlying their activation remain elusive. Herein, using cell biology and various biochemical approaches, we established that variant BRAFD594G, a kinase-dead representative of class 3 mutation-derived BRAF variants, has a higher dimerization potential as compared with WT BRAF. Molecular dynamics simulations uncovered that the D594G substitution orients the αC-helix toward the IN position and extends the activation loop within the kinase domain, shifting the equilibrium toward the active, dimeric conformation, thus priming BRAFD594G as an effective allosteric activator of CRAF. We found that B/CRAF heterodimers are the most thermodynamically stable RAF dimers, suggesting that RAF heterodimers, and not homodimers, are the major players in determining the amplitude of MAPK signaling in cells. Additionally, we show that BRAFD594G:CRAF heterodimers bypass autoinhibitory P-loop phosphorylation, which might contribute to longer duration of MAPK pathway signaling in cancer cells. Last, we propose that the dimer interface of the BRAFD594G:CRAF heterodimer may represent a promising target in the design of novel anticancer therapeutics.

Development of Allosteric BRAF Peptide Inhibitors Targeting the Dimer Interface of BRAF.

BRAF is the most frequently mutated kinase in human cancers and is one of the major effectors of oncogenic RAS, making BRAF a target of considerable interest for anticancer drug development. Wild-type BRAF and a variety of oncogenic BRAF mutants are dependent on dimerization of the kinase domain, which also emerges as a culprit of drug resistance and side effects of current BRAF therapies. Thus, allosteric BRAF inhibitors capable of disrupting BRAF dimers could abrogate hyperactivated MAPK (mitogen-activated protein kinase) signaling driven by oncogenic BRAF or RAS and overcome the major limitations of current BRAF inhibitors. To establish this, we applied an in silico approach to design a series of peptide inhibitors targeting the dimer interface of BRAF. One resulting inhibitor was found to potently inhibit the kinase activity of BRAF homo- and heterodimers, including oncogenic BRAFG469A mutant. Moreover, this inhibitor synergizes with FDA-approved, ATP-competitive BRAF inhibitors against dimeric BRAF, suggesting that allosteric BRAF inhibitors have great potential to extend the application of current BRAF therapies. Additionally, targeting the dimer interface of BRAF kinase leads to protein degradation of both RAF and MEK, uncovering a novel scaffolding function of RAF in protecting large MAPK complexes from protein degradation. In conclusion, we have developed a potent lead peptide inhibitor for targeting the dimer interface of BRAF in cancer cells. The dual function of this peptide inhibitor validates the strategy for developing allosteric BRAF inhibitors that specifically dissociate RAF dimers and destabilize the MAPK signaling complex.