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Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor.
Daniel Marks1, Edwin Garcia1, Sunil Kumar2
1Ovarian Cancer Action Research Centre, Department of Surgery and Cancer, Imperial College London, London W12 0NN, UK; Francis Crick Institute, London NW1 1AT, UK; Department of Physics, Imperial College London, London SW7 2AZ, UK.
Cell Reports Methods
|December 31, 2025
Summary
We developed a novel biosensor to track Poly(ADP-ribose) polymerase inhibitors (PARPi) activity in real-time. This tool reveals how PARPi resistance develops, offering insights for improved ovarian cancer treatments.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Poly(ADP-ribose) polymerase inhibitors (PARPi) are vital in treating ovarian high-grade serous carcinoma (HGSC), especially in homologous recombination-deficient cancers.
- Tumor resistance to PARPi is a significant clinical challenge, leading to relapse in over 50% of patients within three years.
- Understanding PARP trapping mechanisms is crucial for overcoming PARPi resistance, but current methods lack the necessary resolution and throughput.
Purpose of the Study:
- To develop a high-resolution biosensor for real-time, single-cell monitoring of PARP trapping dynamics.
- To investigate the mechanisms of PARPi resistance by analyzing PARP trapping efficiency.
- To provide a tool for evaluating PARPi efficacy and informing personalized ovarian cancer therapy.
Main Methods:
- CRISPR-Cas9 dual labeling of endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells to create a FRET-based biosensor.
- Utilizing fluorescence lifetime imaging microscopy (FLIM) for quantitative, real-time analysis of PARP trapping.
- Assessing PARPi efficacy and resistance in vitro and in vivo models.
Main Results:
- The FRET biosensor successfully enabled real-time, single-cell analysis of PARP trapping dynamics.
- FLIM revealed dose-dependent PARP trapping and differentiated the efficiencies of four clinical PARPi.
- Reduced PARP trapping was observed in PARPi-resistant ovarian cancer models.
Conclusions:
- The developed FRET biosensor offers unprecedented insights into PARP trapping mechanisms and PARPi resistance.
- This technology can help elucidate resistance pathways and guide the development of more effective PARPi therapies.
- The biosensor has significant implications for advancing personalized treatment strategies for ovarian cancer patients.
