Abstract 65: A novel tool for functional characterization of mutations and their response to targeted therapy

Abstract

Abstract Genomic profiling of tumors has uncovered staggering amounts of novel alterations, termed variants of uncertain significance (VUS), even in well-studied oncogenes. Strikingly, as the number of sequenced samples increases so does the number of VUS. While the study of alterations identified as VUS has shown that some are inconsequential, it has also been shown that many can cause protein activation and are involved in oncogenicity and resistance to therapy or might serve as novel drug targets. We developed a high-throughput cell-based assay that allows functional annotation of VUS and their sensitivity to different inhibitors. The system quantifies signaling pathway activation using fluorescent imaging coupled with image analysis of cells expressing the mutated protein together with a fluorescently labeled signaling pathway reporter. We show that this method accurately identifies known driver mutations in 10 different oncogenes, including receptor tyrosine kinases and signaling proteins, spanning the four major signaling pathways involved in cancer. We then deployed this system to characterize over 100 different VUS in these genes. We also show that the method correctly recapitulates differential sensitivity of cKIT and EGFR mutations to different inhibitors. Finally, we highlight the importance of concurrent mutations in KRAS which abrogate the response to EGFR inhibitors. Altogether, this system provides significant information both for development of new targeted agents as well as tailoring of optimal treatment for patients. Citation Format: Zohar Barbash, DIkla Haham, Natalie Fillipov-Levy, Limor Cohen, Lea Birnbaum, Shay Rotkopf, Ilona Kifer, Gabi Tarcic. A novel tool for functional characterization of mutations and their response to targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 65.