Abstract 4659: A genome-wide CRISPR screen identifies ARID1A as a potential resistance marker of IDH1 inhibitor in IDH1-mutant cholangiocarcinoma cell

Abstract Background: Isocitrate dehydrogenase 1 (IDH1) mutations have been identified in approximately 20% of intrahepatic cholangiocarcinoma cases. These predominant gain-of-function mutations at codon 132 lead to abnormal production of the oncometabolite D-2-hydroxyglutarate. Targeted pharmacological disruption of the IDH1 mutation demonstrates tumor growth inhibition in IDH1-mutated cholangiocarcinoma. However, the efficacy of an IDH1 inhibitor (AG-120) as monotherapy for IDH1-mutated cholangiocarcinoma has been proven clinically insufficient, suggesting a need for investigating predictive biomarkers. In this study, we employed a genome-wide CRISPR screen to pursue resistance mechanisms to pharmacological IDH1 inhibition. Methods: The screening utilized SNU1079 cells harboring the IDH1 R132C mutation and the Brunello genome-wide CRISPR knockout (KO) pooled library (Addgene #73178, Broad Institute GPP, 4 guides/gene). The read couns of sgRNAs were normalized to reads per million, and then log2 was transformed. The log fold change (LFC) of each sgRNA of the AG-120-treated group was determined relative to that of the DMSO-treated group. Hypergeometric distribution was used to determine statistical significance. P values per gene were calculated as the average -log10(P value) of individual sgRNAs. We validated the CRISPR screening outcomes in vitro using both IDH1R132C mutant (SNU1079) and IDH1R132S mutant (RBE) cells by WST-1 assays, clonogenic assay and competition assay. (Tsujino T et al. Nat Commun. 2023) In competition assay, GFP was constitutively expressed in ARID1A-knockout cells and cultured together with ARID1A intact cells, under treatment with 1 µM AG120 or DMSO for 21 days. Relative cell numbers of GFP-positive (ARID1A-KO) cells in comparison with GFP-negative (ARID1A intact) cells were quantified using a BD LSRFortessa Analyzer (BD Biosciences). Results: Based on a volcano plot analysis, we identified three genes (SMCHD1, ARID1A, and PGP) with LFC > 1.5. Following a recent report of frequent ARID1A alteration (20%; 84/412) in a clinical study (Boerner, et al. Hepatology. 2021), we focused our research on ARID1A (LFC = 1.531; −log10 P = 3.75). WST-1 assays and clonogenic assays as short-term (3-7 days) assays failed to validate the results of CRISPR screening; however, in competition assays under treatment with AG120, cells with ARID1A-KO exhibited significantly greater proliferation in comparison with ARID1A intact cells in two different cell lines (SNU1079, RBE). Conclusions: The results demonstrated that ARID1A disruption reduced the efficacy of the IDH1 inhibitor against IDH1-mutatedcholangiocarcinoma cells. Based on these findings, we are planning comprehensive analyses employing in vivo models and clinical data to substantiate these observations. Citation Format: Tomohiro Kondo, Osamu Kikuchi, Yafeng Wang, Yukie Nakai, Tomomi Ida, Yang Cao, Trang H. Vu, Yuki Kondo, Tomohiko Sunami, Yoshihiro Yamamoto, Tomoki Saito, Shigeki Kataoka, Atsushi Yamada, Masashi Kanai, Shinya Ohashi, Manabu Muto. A genome-wide CRISPR screen identifies ARID1A as a potential resistance marker of IDH1 inhibitor in IDH1-mutant cholangiocarcinoma cell [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(6_Suppl):Abstract nr 4659.