bioRxiv : the preprint server for biology ·22 Oct 2026 ·Added 10 Nov 2026

DIRAS3 broadly suppresses signaling from diverse oncogenic KRAS hotspot variants through an inhibitory interface.

Gamze Bildik, Hailing Yang, Weiqun Mao, Ritchel B Gannaban, Viyaj K Bhardwaj, Md Nasir Uddin, Sung Y Jung, Mariaelena Pierobon, Alison Gomeiz, Alemayehu A Gorfe, John F Hancock, Christopher B Marshall, Frank McCormick, Steven W Millward, Junchen Liu, Robert C Bast, Zhen Lu

DOI ↗

Abstract

UNLABELLED: Despite major advances in KRAS-directed therapies, broadly suppressing oncogenic KRAS signaling across diverse hotspot variants remains an unmet challenge. Here, we identify an endogenous inhibitory mechanism that suppresses signaling across the major oncogenic KRAS hotspot classes while largely sparing basal wild-type KRAS, revealing activation-state-selective inhibition. We demonstrate that the imprinted tumor suppressor DIRAS3 directly engages activated KRAS through a functionally defined inhibitory interface that can be molecularly mimicked to broadly inhibit KRAS-driven signaling. Using genetically controlled RASless reconstitution models, complementary cancer cell validation, structure-function analyses, molecular dynamics-based membrane topology modeling, and ultrastructural imaging, we show that dual lipid modification constrains DIRAS3 membrane topology, enabling an α5-encoded inhibitory interface to engage activated KRAS, disrupt signaling-competent membrane assemblies, and suppress downstream signaling. Structure-function analyses establish this interface as a critical determinant of DIRAS3-mediated KRAS inhibition. A conformationally constrained α5 mimetic recapitulates full-length DIRAS3 activity, broadly suppressing MAPK and PI3K signaling across multiple oncogenic KRAS variants, including Q61 mutants with reduced sensitivity to current tri-complex RAS(ON) inhibitors. Mechanistically, immunoprecipitation shows that the α5 mimetic reduces KRAS association with both RAF1 and p110α similarly across G12 and Q61 mutants, whereas RMC-7977 reduces inhibition of p110α association with Q61 KRAS. The α5 mimetic also disrupts KRAS nanocluster organization and inhibits tumor growth in vivo. These findings identify a targetable inhibitory interface within DIRAS3 and establish mimicry of an endogenous KRAS suppressor as a mechanistically distinct strategy for broad KRAS inhibition. SIGNIFICANCE: These findings establish that DIRAS3-mediated endogenous regulation extends across diverse oncogenic KRAS variants, including Q61X mutants, revealing an unrecognized principle of pan-KRAS regulation that can be recapitulated by a DIRAS3-derived peptide.