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The combination of 5\u2010Fluorouracil (5FU, a thymidylate synthase inhibitor) and irinotecan (a topoisomerase 1 inhibitor) is widely used in first\u2010line and subsequent treatments. This study aimed to identify novel therapeutic targets to enhance combinatorial therapy, improving treatment efficacy and durability of response.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We performed a loss\u2010of\u2010function 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\u00a0weeks. Genes enriched in resistant clones were identified through next\u2010generation sequencing. Amongst candidate genes, PARG was selected for functional validation.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>CRISPR\/Cas9\u2010mediated knockout (HT29 PARG\u2010KO) resulted in increased global poly(ADP\u2010ribosyl)ation after 5FU and SN38 treatment. PARG depletion led to reduced cell viability and increased apoptosis, particularly after 5FU exposure. Pharmacological PARG inhibition (PDD00017273) synergised with 5FU and SN38 across three CRC models (HT29, DLD1, HT115). In vivo, HT29 PARG\u2010KO xenografts were more sensitive to 5FU. Immunohistochemical analysis of 170 CRC patient tumours revealed that positive PARG expression correlated with poor response to 5FU + Irinotecan, increased liver metastases, and worse long\u2010term survival.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Our findings highlight PARG as a promising therapeutic target for CRC, where its inhibition enhances the efficacy of standard chemotherapy.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Key points<\/jats:title>\n                    <jats:p>\n                      <jats:list list-type=\"bullet\">\n                        <jats:list-item>\n                          <jats:p>LOF screening identified PARG as a modulator of CRC response to FUIRI treatment.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>HT29 PARG\u2010KO cells showed increased PAR, DNA damage, apoptosis, and 5FU sensitivity.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>Pharmacological PARG inhibition synergised with 5FU and SN38 in CRC cell lines.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>HT29 PARG\u2010KO tumours exhibited increased sensitivity to 5FU treatment in vivo.<\/jats:p>\n                        <\/jats:list-item>\n                        <jats:list-item>\n                          <jats:p>PARG expression in CRC tumours correlated with poorer responses and patient survival.<\/jats:p>\n                        <\/jats:list-item>\n                      <\/jats:list>\n                    <\/jats:p>\n                  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