Abstract 6211: Development and validation of a novel sSTRIDE-MMR functional assay to study the efficiency of MMR inhibitors

Abstract

Abstract Mismatch repair (MMR) is considered to be one of the fundamental pathways that guards genome stability and mutations in genes encoding for crucial MMR proteins have been shown to promote cancer initiation. On the other hand, inactivation of MMR components in cancer cells leads to increased neoantigen formation and in consequence, exposes the tumor to the immune system. Therapeutic strategies aiming at inhibiting MMR are thus under development, yet methods reporting on the status of MMR are lacking. We report here the development, optimization and validation of a first MMR-specific functional assay. The assay, which is based on the STRIDE platform technology, detects DNA nicks localized in close proximity to PMS2 protein and can thus be utilized as a direct reporter of inhibition of PSM2 itself or upstream acting proteins. The optimization phase of assay development was performed in U2OS cells. First, in untreated cells we have shown that the sSTRIDE-MMR signals constitute ca. 10-15% of total single-strand DNA breaks foci detected by the classic sSTRIDE assay variant. In a series of technical negative controls we have then shown that the number of false-positive foci never exceeds 10% of the total number of signals, with as little as 2% for the isotype controls. The assay was further validated in HAP1 wild-type and PMS2-KO isogenic cell line pair. As expected, the number of sSTRIDE-MMR foci in the PMS2-KO cell line was significantly lower than in wild-type cells and additionally, no response to the treatment with 6TG, a known inducer of MMR, was detected in PMS2 null cells. Finally, the assay was used to verify the efficiency of first-in-class small-molecule MMR inhibitors. After treatment with the compounds, the number of detected sSTRIDE-MMR foci was significantly lower when compared to the number of foci in untreated cells. In conclusion, we show here that sSTRIDE-MMR is an assay characterized by high specificity, low false positives rate and a high-dynamic range. We believe that sSTRIDE-MMR can prove to be a very useful cell-based solution to study the efficiency of MMR inhibitors. Citation Format: Kamil Solarczyk, Karolina Uznańska, Olga Wójcikowska, Maja Białecka, Agnieszka Waligórska, Szymon Koman, Magdalena Kordon-Kiszala. Development and validation of a novel sSTRIDE-MMR functional assay to study the efficiency of MMR inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6211.