RAF1 S257L/S259F hotspot mutations: functional characterization and intrinsic resistance to the RAS(ON) inhibitor daraxonrasib in melanoma.

Constitutive activation of the MAPK pathway is a hallmark of melanoma, leading to aberrant cell proliferation and survival. Although oncogenic mutations in NRAS and BRAF are well established, the functional and therapeutic significance of RAF1 mutations remains incompletely defined. Here, we identify RAF1 S257L and S259F as recurrent hotspot mutations in melanoma by integrating our hospital cohort with TCGA-SKCM. These conserved region 2 mutations relieve CRAF autoinhibition by reducing inhibitory Ser259 phosphorylation, resulting in elevated ERK signalling in a dimerization-dependent manner. Both mutants exhibit oncogenic activity and transform melanocytes in vitro, and the resulting transformed cells display intrinsic resistance to the RAS(ON) inhibitor daraxonrasib. Subsequent RAF-isoform knockdown and ectopic expression further demonstrate that daraxonrasib-resistant ERK activation is primarily driven by mutant CRAF. Importantly, in human melanoma cells, combined treatment with daraxonrasib and the MEK inhibitor cobimetinib effectively overcomes this mutant CRAF-driven resistance, producing greater suppression of p-ERK and cell proliferation than either agent alone and demonstrating marked synergy. Our findings establish RAF1 S257L/S259F as critical determinants of MAPK pathway activation and cellular response under daraxonrasib, provide mechanistic insights into intrinsic resistance to RAS(ON) inhibition, and underscore a therapeutic framework for genotype-guided combination strategies in RAF1-mutant melanoma.