Abstract 2902: Systematic mapping of chemo-genomic interactions between DNA damaging agents and DNA damage repair genes with targeted CRISPR knockout screening

Abstract Genetic instability is a hallmark of cancer, often arising from mutations in DNA damage repair and response (DDR) proteins. It is our goal to map the repair dependencies associated with different DNA-damaging agents (DDA) and DDR inhibitors (DDRi). To this end, we developed a DDR-targeted, CRISPR knockout screening approach to assess relationships among 355 DNA repair genes and 30 DDA and DDRi in LN229 glioma cells. Systems-level analysis of over 11, 000 interactions revealed that DDA cluster into distinct classes. In contrast, DDRi including ATR, Pol-θ, DNA-PK, WRN, Wee1, USP1, ATM and PKMYT1 inhibitors exhibited a variety of unique dependency profiles. Pathway analysis revealed clear associations between the knockout of Fanconi anemia pathway genes and sensitivity to bifunctional alkylating agents (e.g. chlorambucil), nucleotide excision repair and monofunctional alkylating agents (e.g. illudin S), homologous recombination and PARP inhibitors (e.g. talazoparib), and non-homologous end-joining and radiomimetics (e.g. bleomycin). Furthermore, this platform identified differences in repair dependencies of compounds with subtle variations in chemical structure, highlighted by the imidazotetrazines temozolomide (TMZ) and KL50. In contrast to the methyl adducts deposited by TMZ, DNA modifications deposited by KL50 slowly evolve into DNA interstrand crosslinks (ICLs), circumventing mismatch repair (MMR)-driven resistance observed with TMZ. Indeed, screening revealed that resistance to TMZ, but not KL50, is driven by loss of MMR genes. Furthermore, we observed that BRIP1, a helicase known to play a key role in repair of ICLs, emerged as a unique sensitizing hit for KL50, but not TMZ, providing biological support for the formation of ICLs by KL50. Finally, we sought to identify the most clinically actionable chemo-genomic interactions from our screens. Bi-allelic inactivating mutations (BI) of DNA repair genes are often necessary for manifestation of a DDR deficiency phenotype. Thus, we subjected our gene list to a custom bioinformatic pipeline to quantify rates of BI in samples from The Cancer Genome Atlas (TCGA). Excluding genes listed as Level 1 biomarkers on OncoKB, we found that 4.73% of TCGA samples harbored putative BI of at least one of the 138 genes that emerged as a hit (FDR < 0.01) in our screens. Overall, our screens validated established chemo-genomic interactions while also identifying genetic dependencies associated with DDA of previously unknown mechanisms of action. Furthermore, we elucidated subtle differences in repair dependencies between structurally similar compounds. Finally, we identified novel genetic biomarkers with frequent BI in human tumor samples that sensitized to specific therapeutic agents upon loss, nominating chemo-genomic interactions with potential clinical relevance. Citation Format: Collin D. Heer, Spenser S. Johnson, James L. Elia, Sam Friedman, Xin Pei, Sofia R. Arbelaez, Ranjini Sundaram, Vijay Menon, Joseph Edmonds, Karlie Lucas, Nadeem Riaz, Susan E. Gueble, Ranjit S. Bindra. Systematic mapping of chemo-genomic interactions between DNA damaging agents and DNA damage repair genes with targeted CRISPR knockout screening [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 2902.