Abstract B025: Potential limitation of oncogenic KRAS-targeted therapy in lung cancer due to stromal remodeling

Abstract RAS inhibitors have revolutionized personalized therapy for non-small cell lung cancer (NSCLC). However, acquired resistance is common, likely potentiated by alterations in the composition and architecture of the tumor microenvironment (TME). Interactions between tumor cells and stromal fibroblasts profoundly shape NSCLC cell behavior, impacting progression and outcome. Here, we sought to uncover mechanisms by which fibroblasts mediate resistance in NSCLC to inform the development of more effective therapeutic strategies. In our published L-iKRASG12D model of lung cancer, we identified oncogenic KRAS-dependent immune suppression that was partially reversed upon KRAS inhibition (Lasse-Opsahl & Barravecchia et al., JCI Insight 2025). To further investigate how TME-mediated resistance occurs in response to KRAS inhibition, we developed a novel orthotopic model of KRASG12D-driven NSCLC derived from the L-iKRASG12D model. Using single-cell RNA sequencing of lung tumor tissue from mice with active KRAS (KRAS ‘ON’) or with genetic or pharmacologic loss of KRAS (KRAS ‘OFF’ or RMC-7977), we observed major shifts in stromal cell composition, providing insight into the cellular landscape induced by KRASG12D. Specifically, we identified two distinct populations of cancer-associated fibroblasts (CAFs). Both were defined by smooth muscle alpha-2 actin (ACTA2 +), fibroblast activation protein (FAP +), and fibroblast-specific protein 1 (FSP1 +); however, while one cluster showed a less activated phenotype, with lower expression of CAF markers, the other was highly activated, expressing elevated levels of CAF markers, along with genes involved in tumor progression, such as extracellular matrix (ECM) proteins (COL1A1, COL1A2, FN1, TNC, DCN), ECM crosslinking proteins (LOX, LOXL2), proteins involved in TGF-β signaling (INHBA, TGFBR3, ACVR1), and cytokines (CCL2, IL6). These data suggest that CAF populations exhibit functional heterogeneity following KRAS inhibition, with the activated cluster sustaining a pro-tumorigenic phenotype. Genetic KRAS inhibition resulted in near-complete reversal of ECM gene expression (reduced expression of COL1A1, COL1A2, COL3A1, COL5A1, CXCL12 (SDF), FN1, HGF, MMP2, MMP14, LOX, LOXL2, TGFB1, TNC) in the activated CAF cluster. Yet a gene expression profile largely like the ‘ON’ group was retained in RMC-7977 treated tumors, suggesting that RMC-7977 fails to normalize these highly activated CAFs. Notably, EGFR remained upregulated in activated CAFs in RMC-7977-treated lungs compared to the ‘OFF’ group. This was unexpected, as EGFR-mediated resistance is typically attributed to cancer cells, not stromal cells. Our data suggest that the activated CAFs may contribute to persistent therapeutic resistance, introducing a stromal-specific mechanism of drug escape. Our findings reveal major changes in the stroma following KRASG12D inhibition and highlight potential TME-dependent mechanisms of resistance to KRAS inhibitors, which may inform next-generation therapeutic strategies in NSCLC. Citation Format: Brock Humphries, Niloofar Khairkhah, Ali Namvar, Mostafa M H. Ibrahim, Emily L. Lasse-Opsahl, Carlos E. Espinoza, Megan Faunce, Lily Rober, Marina Pasca di Magliano, Stefanie Galban. Potential limitation of oncogenic KRAS-targeted therapy in lung cancer due to stromal remodeling [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 B025.