Abstract 6108: Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells

Abstract

Abstract Since the early days of the synthetic lethality concept in DNA Damage Response the status of homologous recombination (HR) repair in cancer cells have been the focus of attention of researchers and clinicians. While different approaches exist, such as the RAD51 immunofluorescence (IF) or HRD genomic assays, functional biomarkers that can assess HR proficiency are missing. We report here the development, optimization and validation of two complementary, HR-specific functional assays. The assays, which are based on the STRIDE platform technology, detect double-strand DNA breaks localized in close proximity to RPA or RAD51 proteins. The optimization phase of assay development was performed in U2OS cells. First, repeatability (intra-run variation) and reproducibility (inter-run variation) of the assays were measured in untreated cells. Then, a series of technical negative controls was performed which have shown that the number of false-positive readouts is below 10% of the total number of signals. Finally, treatment of cells with compounds known to induce double-strand DNA breaks (etoposide and cisplatin) resulted in statistically significant increase in the number of detected dSTRIDE-RAD51 and dSTRIDE-RPA foci when compared to untreated controls. The assays were further validated in NCI-H661 (BRCA2 wild-type) and NCI-H169 (BRCA2 KO) cell line pair. The cells were treated with two concentrations of etoposide and the readouts from dSTRIDE, detecting the total pool of DSBs and dSTRIDE-HR assays were compared. In NCI-H661 cells, treatment with etoposide resulted in an increase in the number of double-strand breaks detected by dSTRIDE and as expected, more DSBs were formed after treatment with the higher concentration. dSTRIDE-HR assays confirmed that approximately 15% and 10% of these DSBs contain RPA and RAD51 proteins, respectively. In NCI-H169 cells etoposide produced a stronger reaction with even more DSBs detected by dSTRIDE, but importantly, no increase in the number of dSTRIDE-RAD51 foci was observed. dSTRIDE-RPA foci increased after treatment hinting that this step of HR remains unperturbed. Interestingly, the number of dSTRIDE-RAD51 foci in untreated cells was comparable between the two cell lines. In conclusion, we show here that two newly developed dSTRIDE-HR assays are well validated and can be successfully applied to report on the status of homologous recombination repair in different cell models. Citation Format: Kamil Solarczyk, Agnieszka Waligórska, Karolina Uznańska, Zsombor Prucsi, Olga Wójcikowska, Ewelina Matuszyk, Magdalena Bartyńska, Agata Kitlińska, Aleksandra Bober, Franek Sierpowski, Maja Białecka, Monika Jarosz, Malgorzata Szczygiel, Szymon Koman, Karolina Korpanty, Lukasz Beben, Lukasz Bandzarewicz, Przemyslaw Stachura, Magdalena Kordon-Kiszala. Novel functional dSTRIDE-HR assays to report on the status of homologous recombination repair in cancer cells. [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 6108.