Abstract 5524: Developing KRASG12C inhibitor-resistant tumor models for efficacy evaluation of next-generation anticancer therapies

Background: For decades, KRAS was considered undruggable due to the lack of suitable binding sites. However, advancements in bioengineering and chemistry have enabled the approval of targeted therapies. Success was first seen with allele-specific targeting of KRASG12C in non-small cell lung cancer (NSCLC), leading to the approval of sotorasib (AMG510, LumakrasTM). Despite its clinical benefits, resistance emerged in some patients due to secondary KRAS mutations, which necessitates next-generation or combination therapy development. In this study, we outline the development of KRASG12C inhibitor-resistant models overcome this hurdle. Methods: Secondary KRAS mutations (Y96D/C/S, H95D/Q/R, R68S, Q61H, A59T/S, and Q99L) were introduced by CRISPR/Cas9 in MIA PaCa-2 with a homozygous KRASG12C mutation. Knock-in of point mutation was validated by Sanger sequencing. Cell viability was assessed by CellTiter-Glo (CTG) with AMG510 and MRTX849 (Adagrasib, KrazatiTM). RAS-MAPK pathway activity was evaluated by western blot. Xenograft models of MIA PaCa-2 cells with Y96D/C, H95D/Q/R, R68S, Q61H and A59T were established. Additionally, in vitro chronic dosing of AMG510 generated AMG510-resistant MIA PaCa-2 and NCI-H358 cell lines were validated by CellTiter-Glo and western blot. RNA-seq identified potential resistance mechanisms. Xenograft models were also established. Results: A successful homozygous point mutation knock-in was confirmed by Sanger sequencing. Cells expressing double-mutant alleles KRAS G12C Y96D/C/S, A59T/S and R68S showed resistance to both AMG510 and MRTX849, while KRAS G12C H95D/Q/R was more resistant to MRTX849, and KRAS G12C Q61H, Q99L didn’t show significant resistance. Persistent phosphorylated ERK (pERK) and pRSK levels indicated sustained RAS-MAPK activity in cells expressing KRAS G12C Y96D, H95D, A59T/S, and R68S, even at high KRAS inhibitor concentrations. Furthermore, a KRAS G12C Y96D/C, A59T, Q61H, R68S and H95D/Q/R double mutant cell-derived xenograft was established in vivo. Additionally, MIA PaCa-2 AMG510-resistant and NCI-H358 AMG510-resistant cells showed resistance to AMG510 and MRTX849 in cell viability assays. RNA-seq data identified c-MET amplification in AMG510-resistant MIA PaCa-2 cell, while FGFR1/3/4 amplification was found in AMG510-resistant NCI-H358 cells. Conclusion: CRISPR/Cas9-engineered KRAS secondary mutations cell lines displayed differentially resistant profile to KRASG12C inhibitors, and drug-induced resistant cell models developed in vitro displayed KRAS-independent mechanisms of resistance. These novel cell models offer a valuable preclinical platform to evaluate therapeutic strategies to overcome resistance to KRAS-targeted therapies. Citation Format: Jian Feng, Dan Zhang, Aaron Hua, Chenpan Nie, Jessie(Jingjing) Wang, Ludovic Bourre, Jun Zhou, Peng Wang. Developing KRASG12C inhibitor-resistant tumor models for efficacy evaluation of next-generation anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular s); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1): nr 5524.