UEG Week 2025 DDM Summit Presentations

Introduction: RNaseH2 plays a critical role in ribonucleotide excision repair, and its deficiency leads to spontaneous double-stranded DNA breaks and cytosolic release of dsDNA 1 .We have previously shown that epithelial deficiency of RNaseH2b (H2b IEC ) leads to a p53-dependent stem cell suppression, and additional deficiency of p53 results in spontaneous intestinal carcinogenesis 2 .Due to the central role of cGAS/STING in sensing dsD-NA 3 , we aim to investigate the role of epithelial STING in a mouse model of chronic epithelial DNA damage and tumor prevention in this study.Aims & Methods: To understand the role of epithelial STING in the DNA damage response, DNA damage was assessed using the comet assay in the ModeK cell line with cGAS and STING knock-out.Intestinal organoids were generated from H2b IEC and H2b IEC /Tmem173 -/-mice.Colony forming assay and EdU staining were performed in all genotypes to assess the impact of STING on epithelial stem cell function, and proliferation was assessed by CellTiter Glo assay.Strand-sequencing and RNA sequencing were performed in intestinal organoids to study genomic and transcriptomic changes upon DNA damage.Spontaneous carcinogenesis was assessed in 1-year-old mice.Protein kinase activity was assessed by the Pamgene Kinase Assay.Results: Loss of H2b results in cGAS/STING dependent IFN-I activation, while additional STING deficiency protects mice from DSS compared with H2b single deficiency.IFN-I independent DNA damage repair (DDR) was disrupted under cGAS/STING deficiency evidenced by WB and comet assay.Impaired DDR caused by STING deficiency was further confirmed by increased DNA breakpoints and chromosomal instability observed through Strand-sequencing in organoids.RNA sequencing from H2b IEC / Tmem173 -/-organoid further shows a downregulated G2/M DNA damage checkpoint and P53 signature compared to H2b IEC and highlights a pro-tumorigenesis signature.In vivo, we confirmed that H2b IEC /Tmem173 -/-mice have insufficient DNA damage repair and developed spontaneous small bowel adenocarcinoma after 53 weeks.Organoids isolated from tumors also exhibited invasiveness and high metastatic potential as shown by the transplantation model.Using protein kinase assay, we observed an unbalanced CDK1-P21 axis upon impaired DDR as the driver of tumorigenesis and a potential therapeutic target in the clinic. Conclusion:We provide evidence that the non-canonical role of epithelial STING in maintaining genome homeostasis and tumor prevention by mediating DNA damage repair and directing the P21-CDK1 axis.Our study provides new insights into the role of STING in small bowel carcinogenesis, with potential for clinical application.