The activity of poly(ADP-ribose) polymerase inhibitors (PARPi) in ovarian high-grade serous carcinoma (HGSC) depends upon inducing synthetic lethality in homologous recombination-deficient cancer cells, impairing DNA double-strand break repair. Despite initial success, over 50% of patients recur within four years. One proposed mechanism of resistance is reduced PARP trapping, where the PARP1-inhibitor complex fails to remain adequately trapped at DNA damage sites. Traditional methods for assessing PARP trapping rely on bulk biochemical assays with limited throughput, typically performed on fixed cells.
This thesis aimed to develop a novel tool to assay PARP trapping with single-cell resolution in a high-throughput manner. For this, an intramolecular FRET biosensor was developed using CRISPR-Cas9 knock-in technology to dual-label endogenous PARP1 in OVCAR4 cells with EGFP and mCherryFP. The FRET biosensor allows real-time analysis of PARP1-DNA binding dynamics in live cells and was assayed using automated high-content analysis (HCA) with wide-field time-gated fluorescence lifetime imaging microscopy (FLIM).
This approach enabled quantitative FRET measurements across 96-well plates, allowing statistical robustness for the comparison of perturbations due to the relatively high sampling rate. Significant intra-population heterogeneity was detected, which traditional pooled-sample assays, such as chromatin immunoprecipitation, could not resolve. The biosensor was first validated using clinically approved PARPi with varying trapping potencies and subsequently applied to novel models of PARPi resistance. A dose-dependent decrease in fluorescence lifetime was observed upon PARPi treatment, with reduced PARP trapping detected in cells resistant to olaparib and rucaparib.
The biosensor may provide critical insights into resistance mechanisms and enable screening of novel PARPi to enhance therapeutic options. Furthermore, it may be applied to clinical samples in the future, supporting personalized medicine approaches. The ability to assess PARP trapping in live cells with single-cell resolution offers an unprecedented capability for advancing therapeutic strategies and enhancing the efficacy of PARPi treatments for HGSC patients.