Abstract 2984: High throughput drug screening of patient-derived organoids reveals the ATR-mediated DNA damage response pathway as an esophageal cancer vulnerability

Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) is common worldwide and the deadliest of all squamous cell carcinomas (SCC). Standard-of-care chemotherapy and immune checkpoint inhibitors offer limited survival benefits, highlighting the need for novel therapeutic strategies to combat ESCC. Patient-derived organoids (PDO) recapitulate therapy response and are powerful tools to meet this need; however, current PDO drug screening protocols are limited to fewer than 100 drugs, restraining their use in high-throughput screening (HTS) campaigns. Methods: We developed a novel protocol enabling HTS of therapy-naïve ESCC (n=7) and normal esophageal mucosa (n=4) PDOs with >10, 000 bioactive compounds. We validated our results by preforming a dose response assay to determine the IC50 of select drugs using three representative ESCC PDO lines. We further validated our results in an orthogonal model of murine-derived organoids (MDO) generated from mice bearing normal mucosa, preneoplasia and ESCC, the latter two induced by esophageal carcinogen 4-nitroqunoline-1-oxide. We analyzed DNA damage response (DDR) genes in the Cancer Genome Atlas, ESCC human tissue microarrays, and MDO. We evaluated DNA damage in tissues and organoids by alkaline comet assay and γH2AX staining. We depleted ATR by CRISPR interference (CRISPRi) in MDO. Results: We identified 222 drugs (many FDA-approved) that killed (viability <80%) the majority of ESCC PDO (≥4 of 7) at 10 µM. We next selected drugs based on their pharmacodynamic properties. First, we selected drugs with IC50 values significantly lower than their Cmax values, the maximum plasma drug concentration documented in patients. We then selected 19 drugs that killed ESCC PDO significantly more (≥three-fold, P<0.05) than normal PDO, highlighting a potential therapeutic window for these strategies. Four of such ESCC-selective killers target the DDR regulators ATR, CHK1, and WEE1. We found that in DDR genes were significantly upregulated in ESCC patient tissue (n=88) relative to normal adjacent mucosa (n=11). Moreover, we determined that γH2Ax was significantly elevated in ESCC (n=215) relative to normal mucosa (n=93). We confirmed our results in MDO: γH2Ax labeling index, comet tail moments, and expression of DDR genes were significantly increased during ESCC pathogenesis. CRISPRi-mediated ATR depletion killed ESCC but not normal MDO. Conclusions: Through an innovative HTS campaign in PDOs, we determined that ESCC are addicted to DDR genes to combat endogenous DNA damage that would be lethal to cancer cells if left unrepaired. ATR and other DDR checkpoint kinases are therefore promising therapeutic targets for selectively killing ESCC cells, and possibly other SCC, underscoring the translational utility of PDO in precision cancer medicine. Grants: P01-CA098101, Torrey Coast Foundation, Mark Foundation Citation Format: Wataru Hirose, Samuel Flashner, Yasuto Tomita, Kento Kawasaki, Masaki Morimoto, Izabella Pacheco, Nour Kandil, Christian Lee, Cecilia Martin, Charles Karan, Joel T. Gabre, Anil K. Rustgi, Hiroshi Nakagawa. High throughput drug screening of patient-derived organoids reveals the ATR-mediated DNA damage response pathway as an esophageal cancer vulnerability [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 2984.