Abstract A038: Monitoring PARP trapping in live ovarian cancer cells using a CRISPR-engineered FRET biosensor

Abstract Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized treatment for high-grade serous ovarian carcinoma (HGSOC), particularly in homologous recombination-deficient (HRD) tumors. However, over half of patients relapse within four years, and acquired resistance remains a major barrier to durable clinical benefit. A key determinant of PARPi efficacy is their ability to trap PARP1 on DNA at sites of damage, leading to replication fork stalling and cytotoxicity. Reduced PARP trapping has been proposed as a resistance mechanism yet remains poorly understood due to the lack of tools that allow high-throughput, single-cell resolved measurements in live, physiologically relevant models. To address this, we developed a novel, endogenous Förster resonance energy transfer (FRET) based biosensor to monitor PARP1 conformational changes associated with DNA trapping. Using CRISPR-Cas9 genome editing, we inserted EGFP and mCherry at the N- and C-termini of the endogenous PARP1 gene in OVCAR4 cells. Successful dual tagging was confirmed using long-read nanopore DNA sequencing, verifying precise on-target integration of the fluorophores. This dual-labeled biosensor reports intramolecular FRET, reflecting conformational compaction associated with DNA engagement. When paired with fluorescence lifetime imaging microscopy (FLIM), this system enables high-content, single-cell analysis of real-time PARP1 trapping dynamics in live cells. We validated the biosensor by quantifying trapping responses to four clinically approved PARPi (veliparib, olaparib, rucaparib, and talazoparib) demonstrating dose-dependent decreases in fluorescence lifetime that reflect known differences in trapping potency. We then generated matched PARPi-resistant biosensor-expressing populations through continuous exposure to olaparib or rucaparib, both in vitro and in vivo using xenograft models. Interestingly, resistant populations exhibited significantly reduced PARP1 trapping upon re-exposure to PARPi. These populations also showed reduced cisplatin sensitivity, suggesting overlapping resistance pathways between platinum agents and PARPi. This study establishes a physiologically relevant, quantitative platform for monitoring PARP1 trapping in real time, at single-cell resolution. The biosensor enables direct comparison of PARPi activity and resistance dynamics and reveals mechanistic insight into therapy failure. Ongoing work includes performing BRCA1 KO to model HRD within this system and applying the biosensor in 3D spheroids and patient-derived organoids. Ultimately, this platform could support the development of more effective PARPi, refine biomarker strategies, and inform therapeutic combinations to overcome resistance in HGSOC. Citation Format: Dan Marks, Edwin Garcia, Sunil Kumar, Katherine Tyson, Caroline Koch, Hasan Mirza, William Flanagan, Christopher Dunsby, Paul MW. French, Iain A. McNeish. Monitoring PARP trapping in live ovarian cancer cells using a CRISPR-engineered FRET biosensor [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A038.