Abstract 401: Developing KRAS G12C inhibitor-resistant tumor models for efficacy evaluation of next-generation anticancer therapies

Abstract

Abstract Background: AMG510, a novel KRAS G12C mutation-specific inhibitor approved in 2021 by FDA, was the first therapy to directly target the KRAS oncoprotein in KRAS-mutant cancers with other direct KRAS G12C inhibitors currently being investigated in multiple clinical trials. However, the emergence of resistance in patients who initially were responsive is a challenge. Uncovering the underlying mechanism of resistance would support the identification and development of novel therapies to overcome drug resistance. In this study, we generated a panel of KRAS G12C inhibitor-resistant tumor models by introducing a secondary KRAS mutation on top of G12C for use as in vitro and in vivo tools to develop possible strategies to overcome such resistance. Methods: First, by using CRISPR/Cas9, we introduced a secondary KRAS mutation, including Y96C, Y96D and Y96S in MIA PaCa, which harbours a homozygous KRAS G12C mutation. Point mutation knock-in was validated by sanger sequencing and cell identity was confirmed by SNP. Then cell viability was measured by CellTiter-Glo after 5 days incubation with AMG510 and MRTX849. RAS-MAPK pathway activity was assessed in the parental and mutant cell lines Y96D by Western blot. Xenograft of MIA PaCa KRAS G12C/Y96D cells l was also established. Results: Successful homozygous point mutation knock-in was achieved as confirmed by sanger sequencing. The double mutant cells displayed the similar growth rate as well as the morphology. In cell viability assays, relative to KRAS G12C parental cells, cells expressing the double-mutant alleles showed marked resistance to AMG510 and MRTX849. Consistent with the effects on cell viability, persistent phosphorylated ERK (pERK) and pRSK levels was also observed in KRAS G12C/Y96D expressing cells even at high concentrations of MRTX849, indicating sustained RAS-MAPK activity. In addition, MIA PaCa-KRAS G12C/Y96D was able to grow in vivo and will be used for pharmacological evaluation of KRAS inhibitors. Conclusion: These double-mutant cells can be used to assess preclinical in vitro/in vivo efficacy of KRAS-targeted therapeutics. Crown Bioscience is in the process of developing a series of KRAS G12C inhibitor-resistant cell lines to better facilitate efficacy testing of KRAS-targeted therapeutics. Citation Format: Jun Zhou, Zi Ye, Ning Bo, Dan Zhang, Li Hua, Chenpan Nie, Jingjing Wang, Rajendra Kumari, Leo Price, Xiaoxi Xu. Developing KRAS G12C inhibitor-resistant tumor models for efficacy evaluation of next-generation anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 401.