Targeting BRAF Class II and III Mutations in NSCLC with the pan-RAF inhibitor Exarafenib Reveals ARAF-KSR1-Mediated Resistance and Rational Combination Strategies

The serine/threonine kinase BRAF is frequently mutated in several tumor types, including melanoma and non-small cell lung cancer (NSCLC). Oncogenic BRAF mutations can be classified as Class I, II, or III owing to differences in underlying oncogenic mechanisms. While there are approved targeted therapies for BRAF Class I mutants, there are no approved targeted therapy strategies for Class II and Class III BRAF mutated cancers. By leveraging analysis of a large, real world circulating tumor DNA (ctDNA) profiling database, we highlight a significant subset of NSCLC patients whose tumors harbored Class II and Class III BRAF mutations. These mutations comprise ~65% of BRAF-mutant NSCLC cases, with Class II patients showing significantly worse clinical outcomes compared to Class I patients. In several preclinical tumor systems with Class II and III BRAF mutations, exarafenib, a novel type 2 pan-RAF inhibitor that binds RAF proteins irrespective of isoform or dimerization state, demonstrates robust anti-tumor activity. Initial clinical evaluation also showed promising activity in Class II BRAF-mutant NSCLC patients. Mechanistic studies reveal that exarafenib resistance involves adaptive rewiring from conventional oncogenic BRAF-dependent signaling to an ARAF-mediated bypass pathway, characterized by drug-induced ARAF-KSR1 scaffolding complexes that maintain MAPK signaling despite pan-RAF inhibitor treatment. This resistance is driven by upstream RTK activation and RAS-GTP accumulation, which specifically promotes complex assembly under drug treatment. Based on these insights, we identified MEK inhibition as a rational combination strategy that overcomes resistance by targeting the convergence point of both signaling pathways. The exarafenib plus binimetinib combination demonstrated superior efficacy in diverse preclinical models. This study establishes ARAF-KSR1 complex formation as a novel resistance mechanism to pan-RAF inhibition and provides mechanistic rationale for combination strategies with potential to address the unmet clinical need for BRAF Class II and III-mutated NSCLC.