Abstract 4190: CRISPR-CAS9 library screening identifies novel vulnerabilities in CRC drug-tolerant persister cells

Abstract Introduction: Efficacy of targeted therapies in metastatic colorectal cancer (mCRC) is hindered by onset of resistance, therefore novel strategies are needed. A subpopulation of drug-tolerant persister cells (DTPs) survives to targeted therapies and initiates adaptive mutability response. DTPs experience an increased DNA damage and a shift to error-prone DNA damage repair, thus promoting mutability and resistance. Aim: The purpose of this project is to explore key players modulating adaptive mutability to prevent DTPs’ evolution to resistance, thus preventing tumor recurrence. Methods: CAS9-expressing CRC cells were infected with a custom CRISPR library targeting over 500 DNA damage response (DDR) genes, divided in 12 subpopulations, and grown either for 4 weeks in normal cell culture conditions (untreated) or treated with specific oncogenic inhibitors until the development of resistance in a Time-To-Progression (TTP) assay. CAS9-expressing clones infected with empty library were grown in parallel, in absence or presence of targeted therapies, as control. Next Generation Sequencing-based analysis of sgRNA (single guides RNA) was then performed on resistant subpopulations grown in the presence of targeted therapies. Results: We initially isolated CAS9-expressing clones from a microsatellite stable (MSS) CRC cell line and selected clones with growth rate and drug-sensitivity comparable to that of parental population. The selected CAS9-expressing clone was then infected with the CRISPR DDR library or empty library and, after the selection with puromycin, the selected populations were divided in 12 subpopulations, to exclude the possibility that pre-existing resistant cells might outgrow and interfere with the effect of DDR genes knock-outs (KO) on the development of drug-resistance. The subpopulations were then grown in absence or presence of oncogenic inhibitors for several weeks until the onset of resistance. Notably, while the 12 subpopulations infected with the empty library simultaneously developed resistance, the development of resistance in the DDR library-infected cells was spread over multiple weeks, thus highlighting that KO of DDR genes might either accelerate or delay the onset of resistance. sgRNA of resistant population were then analyzed by next-generation sequencing. A negative selection analysis will be applied to identify sgRNA that are lost in the resistant populations, thus targeting DDR genes whose depletion is potentially synthetical lethal with targeted therapies in mCRC. Conclusion: A DDR CRISPR screening identified novel vulnerabilities of CRC DTPs able to improve efficacy of targeted therapies and prevent tumor relapse in mCRC. Citation Format: Martina Miotto, Simona Lamba, Gaia Grasso, Giorgio Corti, Julie Bonetto, Alberto Sogari, Alberto Bardelli, Mariangela Russo. CRISPR-CAS9 library screening identifies novel vulnerabilities in CRC drug-tolerant persister cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4190.