Abstract 3295: Pi3k autoinhibition dictates rras2 dependency across HER2-amplified and PI3K-mutant cancers

Abstract Aberrant PI3K signaling drives cancer growth, yet tumors activate p110α through distinct mechanisms. In normal cells, the regulatory subunit p85 restrains p110α until activated receptors recruit the complex to membranes. HER2-amplified tumors intensify this route through phosphorylated HER3, which provides multiple phospho-sites that both recruit PI3K and relieve autoinhibition. PIK3CA mutations found in various cancer types, including those with HER2 amplification, can weaken the restraint of p85 and bypass receptor control while preserving the requirement for membrane localization. Given that p110α still requires membrane localization even after autoinhibition is relieved, tumors often use oncogenic KRAS to provide this input. However, most HER2-amplified and many PIK3CA-mutant cancers lack KRAS mutations, suggesting alternative RAS-family proteins may fill this role.To define the contribution of RAS to PI3K signaling in a HER2-amplified context with wild-type p110α, we used KRAS G12C KYSE-410 cells. Through targeted siRNA knockdown, we identified RRAS2, not KRAS G12C, as the dominant PI3K driver, resulting in a ∼60% reduction in pAKT. Replacing endogenous p110α with a RAS-binding-defective mutant produced the same effect, demonstrating that RRAS2 exclusively engages this site. Exogenous expression of oncogenic RRAS2 failed to restore pAKT in the presence of the HER2 kinase inhibitor tucatinib, even though RRAS2 remained GTP-loaded, membrane-localized, and bound to p110α. These results show that RRAS2 alone is insufficient to activate PI3K in the absence of phospho-HER3.To test whether p85 restraint prevents RRAS2 from activating PI3K, we expressed p110α mutants that destabilize the helical (E545K) or C2 (C420R) interface with p85. Both mutants restored RRAS2-driven signaling after tucatinib treatment, demonstrating that RRAS2 activates PI3K only when autoinhibition is relieved. To test the model in a physiological context, we used JIMT1 cells, which endogenously co-amplify HER2 and RRAS2 and carry the C420R mutation in p110α. Tucatininb treatment did not affect pAKT, indicating that RRAS2 can sustain PI3K activity when p110α autoinhibition is relieved. RRAS2 knockdown or re-expression of wild-type p110α restored tucatinib sensitivity, confirming that RRAS2 engagement and p110α–p85 regulation form key regulatory nodes.Further, DepMap analyses reveal increased RRAS2 dependency in cells with destabilizing p110α–p85 mutations in the absence of RTK amplification, supporting a model in which RRAS2-driven PI3K signaling requires both relief of autoinhibition and membrane localization.Together, our findings demonstrate that RRAS2 can drive PI3K signaling in contexts previously seen as RAS-independent and where recruitment mechanisms were unclear. Thus, highlighting an additional regulatory node of PI3K activation in HER2-amplified and PI3K-destabilized contexts. Citation Format: Miranda R. Cabanski-Dunning, Matthew J. Sale, Lucy C. Young, Maria Tarazona-Guzman, Dylan Aguinaldo, Madeleine Sitton, Rony Andre Francois, Frank McCormick. Pi3k autoinhibition dictates rras2 dependency across HER2-amplified and PI3K-mutant cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3295.