The MAPK pathway is frequently hyperactivated in melanoma, driving aberrant proliferation and cell survival. Although oncogenic NRAS and BRAF mutations are well established, the functional and therapeutic relevance of RAF1 (CRAF) mutations remains unclear. Daraxonrasib (RMC-6236) is a clinical-stage RAS(ON) inhibitor that targets the active state of mutant and wild-type RAS. Here, we identify two recurrent RAF1 hotspot mutations in melanoma and examine how they modulate MAPK signalling and confer resistance to daraxonrasib in vitro.

RAF1 mutations were profiled in a hospital melanoma cohort by targeted sequencing and validated in TCGA-SKCM. Mouse melanocytes (Melan-a) expressing empty vector or MYC-tagged CRAF (WT, S257L, S259F) were used to assess CRAF autoinhibition, MAPK activation, and oncogenic properties; NRASG12V-transformed Melan-a cells served as a comparator for inhibitor response. Human melanoma cell lines (CHL1, C8161, and WM1361) expressing the same constructs were treated with daraxonrasib and/or cobimetinib (a MEK inhibitor) to evaluate MAPK activation and cell proliferation. IC₅₀ values and proliferation were quantified using IncuCyte live-cell imaging. MAPK signalling was assessed by immunoprecipitation and Western blotting, and RAF isoform dependency was tested by siRNA-mediated knockdown of BRAF or CRAF (endogenous mouse wild-type or ectopic human mutant).

By integrating our hospital cohort with TCGA-SKCM, we identified RAF1 S257L and S259F as recurrent melanoma hotspot mutations. In Melan-a cells, these mutations relieved CRAF autoinhibition and induced elevated, dimerization-dependent ERK activation. Both mutants transformed melanocytes in vitro and conferred intrinsic resistance to daraxonrasib in the resulting cells, with IC₅₀ not reached and sustained pS338-CRAF and p-ERK throughout the tested concentration range (0.1 nM–10 μM). By contrast, daraxonrasib inhibited NRASG12V cells with an IC₅₀ of 1.5 nM. RAF isoform knockdown further indicated that both basal and daraxonrasib-resistant ERK signalling were primarily sustained by mutant CRAF, rather than BRAF or wild-type CRAF. In human melanoma cell lines (CHL1, C8161, and WM1361), ectopic expression of both CRAF mutants maintained ERK activation despite daraxonrasib treatment, and this was suppressed by co-treatment with cobimetinib. Moreover, the combination exerted stronger anti-proliferative effects than either inhibitor alone in vitro.

Recurrent melanoma RAF1 hotspot mutations S257L and S259F are dimerization-dependent, gain-of-function oncogenic variants that confer intrinsic resistance to the RAS(ON) inhibitor daraxonrasib in vitro. Daraxonrasib-resistant ERK signalling is predominantly mediated by mutant CRAF and can be effectively suppressed by MEK co-inhibition, providing a rationale for genotype-guided combination strategies in RAF1-mutant melanoma.