Abstract 1942: Characterization of a panel of CRISPR/Cas9 engineered KRAS G12C inhibitor-resistant tumor models

Abstract Introduction: KRAS is one of the most frequent mutated oncogenes and has been recognized as undruggable for many years. AMG510, the first therapy to directly target KRAS, was approved by the FDA to treat non-small cell lung cancer (NSCLC) bearing KRAS G12C mutation. However, the emergence of resistance in patients remains a challenge and limits its clinical benefits, which calls for next-generation targeted therapy or combination strategies to overcome the resistance to KRAS G12C inhibitors. A variety of secondary mutations in KRAS attributing to the resistance have been identified, which requires robust in vitro and in vivo preclinical models to validate potential therapeutics targeting these mutations. Therefore, we generated a panel of KRAS G12C inhibitor-resistant tumor models to facilitate the development of possible strategies to overcome such resistance. Methods: First, a secondary KRAS mutation, including H95D, H95Q, H95R, Q61H and R68S, was introduced by using CRISPR/Cas9 technology in MIA PaCa cell line, which harbors a homozygous KRAS G12C mutation in addition to Y96D, Y96C and Y96S previously published by us. Point mutation knock-in was validated by sanger sequencing, and cell identity was confirmed by SNP assay. In vitro, the parental and mutated cells were treated with either AMG510 and MRTX849 and cell viability was measured by CellTiter-Glo. In vivo efficacy of KRAS G12C inhibitors, AMG510 and MRTX849, SOS1 inhibitor, BI-3406 and MEK inhibitor, Trametinib were evaluated in the Y96D mutated MIA PaCa subcutaneous xenograft. Results: Homozygous secondary point mutation knock-in in MIA PaCa cells was confirmed by sanger sequencing. Similar growth rate and morphology was observed in selected clones compared to the parental line. Similar to Y96D mutation previously published, R68S mutation was highly resistant to both KRAS G12C inhibitors, with IC50 increased more than 100 fold for both MRTX849 and AMG510, whereas H95D, H95Q and H95R mutation was more resistant to MRTX849, while Q61H had a minimal effect on either one of the inhibitors. In addition, cells expressing KRAS G12C/Y96D were also resistant to AMG510 and MRTX849 in in vivo study, whereas combination of BI-3406 (single treatment: TGI of 27%) and Trametinib (single treatment: TGI of 76%) suppressed tumor growth significantly with TGI of 93% on day 17 after randomization compared to control group (p0. 001). Also, the combination showed significant improvement compared to BI-3406 single treatment (p0. 001) whereas no significant improvement was observed compared to Trametinib single treatment (p0. 05). Conclusion: CRISPR/Cas9 engineered second site KRAS mutations in cells harboring KRAS G12C mutation displayed a differentially resistant profile to KRAS G12C inhibitors. Thus, H95D/Q/R, R68S and Y96D can be used as preclinical inhibitor-resistant models to evaluate clinical strategies to overcome resistance to KRAS-targeted therapies. Citation Format: Jun Zhou, Li Hua, Jian Feng, Ning Bao, Dan Zhang, Jingjing Wang, Marrit Putker, Ludovic Bourre. Characterization of a panel of CRISPR/Cas9 engineered KRAS G12C inhibitor-resistant tumor models abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1942.