Abstract A064: Chemical and structural mechanisms of RAS induced RAF:KSR dimerization

Abstract Background: Dimerization of RAF kinases (ARAF, BRAF, and CRAF) is a critical event in the activation of the RAS-MAPK pathway. GTP-bound RAS promotes the dimerization of RAF effectors through interaction with their RAS-binding domain (RBD). In addition to these active kinases, the RAF family includes two pseudokinases: Kinase Suppressor of RAS 1 (KSR1) and KSR2. Historically viewed as scaffolds that organize pathway components for efficient signaling, KSR proteins are now recognized to directly dimerize with RAF and promote pathway activation. Despite this functional significance, the molecular mechanisms that drive RAF:KSR dimerization remains unclear, as KSR proteins lack the canonical RAS binding domain. Our study seeks to unravel the structural and mechanistic basis underlying this interaction, offering new insights into KSR’s regulatory roles and therapeutic relevance in RAS–MAPK signaling. Methods: To investigate RAF:RAF/KSR dimerization dynamics, we employed live-cell reconstitution assays in HEK293T cells. These systems were used to study interactions across wild-type and oncogenic RAS isoforms. Site-directed mutagenesis was performed on key residues identified through preliminary data and structural predictions, including computational tools like Alphafold. Mutant constructs were evaluated using a combination of cellular, biochemical, and biophysical techniques to determine the functional roles of specific residues in dimerization. Additionally, we investigated the effects of current inhibitors of the RAS-MAPK pathway on dimerization, with a particular emphasis on RAF:KSR interactions using live cell target engagement and HTRF assays. Results: Quantitative live cell reconstitution assays to assess RAF:RAF/KSR interactions in the context of wild-type and mutant RAS variants revealed that oncogenic RAS mutants promote the formation of both homo and heterodimeric RAF and KSR complexes in a RAS-GTP dependent manner. Further, RAS-induced complex formation was found to be MEK-dependent, as both pharmacological inhibition using small molecules and MEK interface mutants, disrupted RAS-driven RAF and KSR dimerization. Complex formation of active dimers was also found to occur at the membrane, indicating that, similar to RAS-GTP, membrane anchoring of KSR is critical for enabling functional interactions within the pathway. Our preliminary experiments suggest a role for KSR1 in mediating resistance to RAS inhibitors, consistent with structural models for competition. These findings underscore the need for further investigation into the functional role of KSR-based RAS/RAF dimers in modulating sensitivity and resistance to RAS–MAPK inhibitors currently under clinical development. Conclusions: In summary, our findings elucidate the molecular basis of RAS-driven RAF:KSR dimerization and its relevance to resistance mechanisms, offering new avenues for therapeutic intervention. Ongoing studies aim to determine how these interactions influence the target engagement of diverse RAS-pathway inhibitors and their relevance to drug response in patients. Citation Format: Hiral U. Mistry, Arvin C. Dar. Chemical and structural mechanisms of RAS induced RAF:KSR dimerization [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A064.