Loss-of-Function genetic Screen Unveils Synergistic Efficacy of PARG Inhibition with Combined 5-Fluorouracil and Irinotecan Treatment in Colorectal Cancer

Abstract Colorectal cancer (CRC) remains a major global health concern, partly due to resistance to therapy and the lack of new effective treatments for advanced disease. The combination of 5-Fluorouracil (5FU, a thymidylate synthase inhibitor) and irinotecan (a topoisomerase 1 inhibitor) is widely used in first-line and subsequent treatments. This study aimed to identify novel therapeutic targets to enhance combinatorial therapy, improving treatment efficacy and durability of response. We performed a loss-of-function screen using HT29 CRC cell line and a retroviral library containing 7296 shRNAs targeting 912 chromatin genes. Cells were then treated with 5FU and SN38 (the active metabolite of irinotecan) or left untreated for 4 weeks. Genes enriched in resistant clones were identified through next-generation sequencing. Among candidate genes, PARG was selected for functional validation. CRISPR/Cas9-mediated knockout (HT29 PARG-KO) resulted in increased global poly(ADP-ribosyl)ation after 5FU and SN38 treatment. PARG depletion led to reduced cell viability and increased apoptosis, particularly after 5FU exposure. Pharmacological PARG inhibition (PDD00017273) synergized with 5FU and SN38 across three CRC models (HT29, DLD1, HT115). In vivo , HT29 PARG-KO xenografts were more sensitive to 5FU. Immunohistochemical analysis of 170 CRC patient tumors revealed that positive PARG expression correlated with poor response to 5FU + Irinotecan, increased liver metastases, and worse long-term survival. Our findings highlight PARG as a promising therapeutic target for CRC, where its inhibition enhances the efficacy of standard chemotherapy.