Abstract Emerging data from clinical trials suggests that mechanisms that reactivate the MAPK pathway via gene amplification events can drive resistance to RAS inhibitors. This includes amplification of KRAS itself, as well as RAF1, RICTOR, and MYC. How amplification events affect signaling networks and the implications for therapeutic opportunities to overcome resistance remain unclear. To model this, we employed RMC-7977, a RAS (ON) multi-selective inhibitor structurally related to the clinical candidate daraxonrasib (RMC-6236), and established a RMC-7977–resistant (RMC-7977-R) H358 isogenic model that exhibit RAF1 amplification, as confirmed by amplicon-level analysis of whole-exome sequencing (WES), western blotting, and RNA sequencing. We evaluated this resistant cell line responses to a panel of RAS pathway–targeting agents, including RAF inhibitors (LXH-254 and exarafenib), MEK1/2 PROTAC degrader (MS934), pan-RAF-MEK non-degrading molecular glue (NST-628), RAF/MEK clamp (avutometinib) and MEK1/2 inhibitor (trametinib). Cell viability analysis demonstrated increased sensitivity of RMC-7977 resistant cells to RAF inhibitors compared to the parent H358. Combinations of MEK-targeting agents with either RAF inhibitors or RMC-7977 more effectively suppressed ERK phosphorylation in resistant cells compared to RAF inhibitors combinations with RMC-7977. However, combining RAF inhibitors with RMC-7977 or MEK-targeting agents (MS934, NST-628, avutometinib, or trametinib) more potently inhibited cell proliferation in resistant cells compared with combinations of RMC-7977 with MEK-targeting agents. In the context of siRNA-mediated RAF1 knockdown in RMC-7977 resistant cell line, trametinib treatment reduced ERK phosphorylation and inhibited cell proliferation more deeply than RMC-7977. This suggested that RAF1 may have additional signaling mechanisms beyond activation of MEK/ERK. To elucidate the mechanistic basis of LXH-254 activity in RAF1-amplified cells, we performed RNA sequencing and transcriptional analyses in cells treated with LXH-254, which revealed enrichment of G2/M checkpoint, MYC target, mitotic spindle, E2F target, and DNA repair signatures. Consistent with these observations, RAF inhibitors induced G2/M cell cycle arrest and apoptosis in RAF1-amplified cells, and the apoptotic response was enhanced when combined with RMC-7977 or MEK-targeting agents. Resistant cells exhibited increased nuclear RAF1 expression compared with parental H358. Co-immunoprecipitation experiments reveal enhanced interactions between RAF1 and the mitotic kinases PLK1 and AURKA in resistant cells. Our data indicates that RAF1 amplification rewires signaling states by affecting nuclear G2/M signaling networks in addition to the MEK/ERK pathway. More broadly, our results suggest that RAF1 gene amplification associated with RAS inhibitor resistance may produce neomorphic signaling states that affect therapeutic sensitivity. Citation Format: Hitendra S. Solanki, Denis Imbody, Bina Desai, Andriy Marusyk, Eric B. Haura. RAF1 gene amplification drives RAS inhibitor resistance via nuclear translocation and interactions with PLK1 and Aurora A kinases abstract. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₁): Abstract nr B026.
Solanki et al. (Thu,) studied this question.