Abstract LB304: NTHL1-D239Y variant enhances tumorigenesis in colorectal cancer through cancer initiation and chronic inflammation

Abstract Introduction: Genomic DNA is continually exposed to endogenous and exogenous stressors, leading to DNA damage and genomic instability. The base excision repair (BER) mechanism is crucial for maintaining genetic integrity by repairing damaged nucleobases. BER begins when DNA glycosylases cleave the N-glycosidic bond to remove oxidized pyrimidines. DNA glycosylases can be monofunctional or bifunctional. In this study, we explore the bifunctional NTHL1 variant in inflammation-associated colorectal cancer (CRC). NTHL1 tumor syndrome is linked to increased lifetime risks of CRC, breast cancer, and colorectal polyposis. The rare polymorphism rs3087468 (D239Y) results in a loss of glycosylase activity and acts as a dominant-negative mutation. Hypothesis: We hypothesize that the NTHL1-D239Y variant increases CRC susceptibility under chronic inflammation in the AOM/DSS cancer initiation model. Methods: Male and female C57BL/6 mice (6-8 weeks old) with wild-type (NTHL1WT/WT), heterozygous (NTHL1WT/D239Y), or homozygous (NTHL1D239Y/D239Y) NTHL1 variants were used. In the first experiment, mice were injected with 7.5 mg/kg azoxymethane (AOM), followed by three cycles of 2.5% dextran sulfate sodium (DSS) (5 days DSS alternating with 14 days of normal water). The second experiment used DSS alone. We monitored body weight, collected fecal samples for 16S rRNA sequencing, and measured colon length and polyp number/size at euthanasia. Histology was assessed for inflammation and tumor development. Blood samples were analyzed for micronuclei formation, and immunohistochemistry (IHC) staining was used for tissue proliferation. Results: In the AOM/DSS model, male NTHL1WT/D239Y and NTHL1D239Y/D239Y mice had significantly more polyps compared to NTHL1WT/WT controls. Female NTHL1D239Y/D239Y mice had more polyps than NTHL1WT/D239Y females. In the DSS-only experiment, male NTHL1WT/D239Y mice developed a significant number of polyps compared to wild-type males, while female NTHL1D239Y/D239Y mice developed more polyps than NTHL1WT/WT females. No significant differences in polyp size were observed in the DSS-only experiment. Tumors extending into the proximal colon were seen only in female NTHL1D239Y/D239Y mice. Male NTHL1WT/D239Y mice developed high-grade adenomas in the distal colon compared to wild-type males.Micronucleated erythrocytes were significantly higher in male NTHL1D239Y/D239Y mice compared to controls, but this was not observed in the DSS-only group. No changes in colitis score or colon length were observed. Genotype or AOM/DSS treatment did not affect microbial alpha diversity, but treatment-dependent changes in microbial composition were noted based on sex and genotype. IHC data indicated elevated KI67 expression in NTHL1D239Y/D239Y and NTHL1WT/D239Y mice following AOM/DSS treatment. Conclusion: The NTHL1-D239Y variant promotes malignancy only under chronic inflammation and AOM/DSS treatment. This variant confers a complex, sex- and zygosity-dependent susceptibility to inflammation-associated CRC, with females showing increased tumor development in the proximal colon and males displaying enhanced tumor size and progression. Citation Format: Tarek Michael Masannat, Fayez K. Ghishan, Pawel R. Kiela, Joann B. Sweasy. NTHL1-D239Y variant enhances tumorigenesis in colorectal cancer through cancer initiation and chronic inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB304.