Abstract 4210: Replication stress detection using STRIDE technology platform

Abstract Replication stress, characterized by the slowing or stalling of replication forks, is a hallmark of many cancers and a key driver of genomic instability. Therapeutics targeting the ATR kinase, a central regulator of the replication stress response, have emerged as promising strategies for selectively exploiting the vulnerabilities of cancer cells. A reliable marker of replication stress is essential to better understand its dynamics and to optimize the development and application of DNA damage response (DDR) inhibitors. Such markers can provide critical insights into therapeutic mechanisms and enhance the precision of targeted treatments. Single-strand DNA breaks (SSBs) are both a cause and a consequence of replication stress, making them a potential biomarker. However, their occurrence during normal metabolism requires additional specificity, which can be provided by detecting replication stress-associated proteins. Here, utilizing our STRIDE technology platform, we combined direct in situ detection of SSBs with protein markers leading to the development of an assay aimed at measuring replication stress levels. The assay combined the detection of single-strand DNA breaks (SSBs) with replication stress-associated proteins: RPA, RAD17, and RAD9, evaluated separately in distinct assay variants. Each variant was assessed for steady-state readouts, negative control signal levels, and sensitivity to replication stress induced by known compounds. All variants effectively informed on replication stress induction, and baseline readouts of the assays correlated with the cellular response to replication stress-inducing agents. Importantly, the assays were also tested on FFPE tissue sections, demonstrating their feasibility for application to this type of material. The data collected thus far suggests that all assay variants hold potential as effective replication stress markers. Additional studies are required to further refine these assays, determine their optimal use in different contexts, and confirm their utility in identifying high replication stress backgrounds to predict responses to drugs targeting proteins involved in the replication stress response. Citation Format: Karolina Stępień, Kamila Schirmeisen, Jakub Lechowski, Magdalena Bartyńska, Magdalena Kordon-Kiszala, Kamil Solarczyk. Replication stress detection using STRIDE technology platform [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 4210.