Abstract B028: Adaptive Signaling Rewiring Enables Rapid, Sequential Resistance to KRAS and Pan-RAS Inhibitors

Abstract The purpose of this study was to define lineage-specific and pathway-level mechanisms of acquired resistance to allele-specific KRAS inhibitors and to determine how early adaptive rewiring influences subsequent responses to pan-RAS inhibition. We generated resistant derivatives of KRASG12C (H358, H1373, MIAPaCa-2) and KRASG12D (MIAPaCa-2, HCT116) cell lines through stepwise dose escalation to sotorasib, adagrasib, or MRTX1133. Resistant clones were assessed by viability assays, RAS-GTP measurements, phospho-signaling analyses, and RTK expression profiling. Additional resistance models were generated using the tri-complex RAS inhibitor daraxonrasib in both parental and pre-adapted backgrounds. Across models, resistant cells did not acquire secondary KRAS mutations but instead upregulated distinct repertoires of RTKs in a lineage-dependent manner and maintained proliferation despite reduced RAS-GTP levels. KRASG12D cells that became resistant to MRTX1133 also showed strong activation of alternative RTK pathways, with EGFR family members being the most prominent. Combination treatment with either afatinib or PI3K inhibitors did not restore drug sensitivity, indicating that resistant cells rely on compensatory signaling routes that are not blocked by these agents. Tri-complex inhibition partially suppressed growth in all KRAS inhibitor–resistant lines; however, each ultimately developed resistance. Notably, cells previously adapted to allele-specific inhibitors acquired daraxonrasib resistance more rapidly than naïve parental cells, suggesting that early adaptive rewiring accelerates failure of broader RAS inhibition. MIAPaCa-2 KRASG12C models exemplified this effect. Although daraxonrasib was more potent than adagrasib in parental cells, adagrasib-resistant (A-R) derivatives showed diminished sensitivity and cross-resistance to both drugs. Daraxonrasib resistance developed substantially faster in A-R cells than in parental cells. In A-R cells, daraxonrasib reduced pERK more effectively than adagrasib, consistent with partial retention of KRAS–MAPK dependence. In contrast, MIAPaCa-2 daraxonrasib-resistant (D-R) cells exhibited pERK suppression yet remained proliferative, suggesting reduced dependence on the KRAS–MAPK axis. Doubly resistant MIAPaCa-2A-R/D-Rcells displayed markedly elevated basal pERK despite reduced total KRAS, indicating a distinct mode of MAPK pathway reactivation. In conclusion, KRAS inhibitor resistance arises through extensive non-genetic plasticity, enabling reduced KRAS dependency and rapid adaptation to next-generation RAS inhibitors. These findings highlight the need to target lineage-specific bypass circuits and to consider resistance trajectories when designing treatment-sequencing strategies. Citation Format: Ines Pulido, Frida Pantoja, Julian Carretero, Agustin Lahoz, Khadel Abdelhady, Malek Massad, Takeshi Shimamura. Adaptive Signaling Rewiring Enables Rapid, Sequential Resistance to KRAS and Pan-RAS Inhibitors [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(5_Suppl_1):Abstract nr B028.