Relief of Inhibitory Autophosphorylation Activates RAF

Small-molecule BRAF inhibitors have shown clinical activity in BRAF-mutant melanomas but not in BRAF–wild-type tumors. Emerging evidence indicates that BRAF inhibitors can promote proliferation of cells with upstream RAS mutations and induce secondary cancers through paradoxical activation of wild-type RAF proteins, but the underlying mechanism is unclear. Holderfield and colleagues found that inhibitors of BRAF^V600E activated downstream mitogen-activated protein kinase (MAPK) signaling and stimulated proliferation in BRAF–wild-type cells and inhibited ERK signaling and proliferation in BRAF-mutant cells with the same potency, suggesting that activation of wild-type RAF proteins by BRAF inhibitors is kinase dependent. Purified wild-type RAF kinase domains were inhibited in the presence of ATP in association with an electromobility shift that was blocked by preincubation with a RAF inhibitor, indicating that inhibitory autophosphorylation regulates RAF activity and that RAF inhibitors relieve autoinhibition of wild-type RAF. Mass spectrometry identified phosphorylated residues in the phosphate-binding loop (P-loop) region, and mutation of these residues prevented both autoinhibition and activation by RAF inhibitors. Of note, P-loop mutations are among the most frequently occurring non-V600E BRAF mutations in human cancers, indicating that disruption of RAF autoinhibition may be an additional mechanism of oncogenic RAF activation. Furthermore, although previous studies have suggested that RAF inhibitors activate wild-type RAF by activating BRAF/CRAF heterodimers or promoting interactions with RAS, BRAF/CRAF and RAS/RAF coimmunoprecipitation did not correlate strongly with RAF inhibitor–induced MAPK activation in BRAF–wild-type cells, indicating that relief of inhibitory autophosphorylation may be largely responsible for paradoxical RAF activation. The development of noncatalytic RAF inhibitors that do not block RAF autoinhibition may therefore be less likely to result in acquired resistance or promote formation of secondary cancers.

Holderfield M, Merritt H, Chan J, Wallroth M, Tandeske L, Zhai H, et al. RAF inhibitors activate the MAPK pathway by relieving inhibitory autophosphorylation. Cancer Cell 2013;23:594–602.