Abstract 1477: Do MSH3 mutations really affect colorectal cells neoplastic transformation: an evolution of three African Americans novel mutations within the MSH3 ATPase domains

Abstract Background: DNA Mismatch Repair (MMR) genes’ (including MSH3) alterations through silencing and/or mutations plays a role in colorectal cancer (CRC) carcinogenesis. MSH3 interacts with MSH2 to form MutSβ, which recognizes and directs repair of insertion/deletion loops during in DNA replication; deficiencies lead to elevated mutation rates. Msh3 alterations are associated with instability at tetranucleotide repeats (termed Elevated Microsatellite Alterations at Selected Tetranucleotide repeats, EMAST), a phenotype elevated frequency among African American (AA). CRCs and associated with poor patient prognosis. Aim: To assess functional impact of 3 out of 4 Exomes (E) E20, E-21, E22, E23 novel deleterious AA variants within the ATPase domain of MSH3. Methods: We used CRISPR-Cas9 knock-in and successfully introduced 3 of these mutations (confirmed by Sanger sequencing) in Msh3 ATPase domain (c.G2926A, c.C3028T, c.G3241A) in SW620 colon cancer cell line (carry Msh3 gene wild-type). In Silico and wet-lab MSH3 functional analysis was performed. We used MTT for cell proliferation, tetranucleotide repeats instability assays, immunofluorescence for MSH3 localization, Western blot for protein expression, co-immunoprecipitation for MSH3 interaction with MSH2 MMR protein, and thin layer chromatography ATPase assays with purified MutSβ complexes. Results: In silico analysis showed deleterious effect of the 4 novel msh3 mutants. However, biological phenotypic assays revealed no observable changes in cell morphology or proliferation between wild-type and MSH3-variant knocked-in cells, with no EMAST after 7 to 10 passages. Subcellular localization of variant MSH3 was unaffected compared to wild type MSH3, and interaction between mutant MSH3 and MSH2 proteins was not impacted, However, the ATPase activity of two of the variant MutSβ complexes was affected. MSH3 c.G2759T (E20) exhibited overall reduced ATPase activity compared to wildtype and was not affected by the presence of DNA. MSH3 c.C3028T (E22) exhibited elevated ATPase activity compared to wildtype in the presence of specific (loop) or non-specific DNA (homoduplex). Conclusion: Despite computational deleterious predictions, novel MSH3 variants identified from AA CRCs appeared to show no biological or genetic evidence of pathogenicity, but did reveal altered in vitro biochemical properties. Altered MutSβ ATPase activity, both decreased and increased, has been shown to disrupt proper MMR activity. We propose that disruptions in the ATP hydrolytic cycle contribute to MMR deficiency, resulting in elevated mutation rates with contribution to colorectal cancer pathogenesis. This effect may be enhanced in distinct genetic backgrounds, and directly affect cancer cells’ proliferation and cell division. Citation Format: Mudasir Rashid, Hassan Brim, Katherine Casazza, Jennifer A. Surtees, Shaolei Teng, Adebiyi Sobitan, Adeyinka O. Laiyemo, Zaki Sherif, Koi Minoru, John Carethers, Hassan Ashktorab. Do MSH3 mutations really affect colorectal cells neoplastic transformation: an evolution of three African Americans novel mutations within the MSH3 ATPase domains. [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 1477.