◥Purpose: Although KRAS represents the most commonly mutated oncogene, it has long been considered an "undruggable" target. Novel covalent inhibitors selective for the KRAS G12C mutation offer the unprecedented opportunity to target KRAS directly. However, prior efforts to target the RAS-MAPK pathway have been hampered by adaptive feedback, which drives pathway reactivation and resistance.Experimental Design: A panel of KRAS G12C cell lines were treated with the KRAS G12C inhibitors ARS-1620 and AMG 510 to assess effects on signaling and viability. Isoform-specific pulldown of activated GTP-bound RAS was performed to evaluate effects on the activity of specific RAS isoforms over time following treatment. RTK inhibitors, SHP2 inhibitors, and MEK/ERK inhibitors were assessed in combination with KRAS G12C inhibitors in vitro and in vivo as potential strategies to overcome resistance and enhance efficacy. Results:We observed rapid adaptive RAS pathway feedback reactivation following KRAS G12C inhibition in the majority of KRAS G12C models, driven by RTK-mediated activation of wild-type RAS, which cannot be inhibited by G12C-specific inhibitors. Importantly, multiple RTKs can mediate feedback, with no single RTK appearing critical across all KRAS G12C models. However, coinhibition of SHP2, which mediates signaling from multiple RTKs to RAS, abrogated feedback reactivation more universally, and combined KRAS G12C /SHP2 inhibition drove sustained RAS pathway suppression and improved efficacy in vitro and in vivo.Conclusions: These data identify feedback reactivation of wildtype RAS as a key mechanism of adaptive resistance to KRAS G12C inhibitors and highlight the potential importance of vertical inhibition strategies to enhance the clinical efficacy of KRAS G12C inhibitors. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis):

Highlights d prkdc À/À , il2rga À/À zebrafish reared at 37 C engraft a wide array of human cancers d Growth and therapy responses can be dynamically visualized at single-cell resolution d Combination olaparib and temozolomide kill xenografted human rhabdomyosarcoma d Engrafting patient-derived cancers opens new avenues for personalized therapy Authors

Purpose: KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy. Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C-activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models. Results: Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K-AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT-mTOR pathway inhibitors. The G12CiþPI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models. Conclusions: Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.