Abstract Objectives: The ability to sensitize high-grade serous ovarian cancer (HGSOC) to poly (ADP-ribose) polymerase inhibitors (PARPi) remains an unmet clinical need. Our prior work showed that depletion of COP9 signalosome subunit 5 (COPS5), a deubiquitinating enzyme, increases DNA damage in ovarian cancer cells. Here, we evaluate the therapeutic potential of combining COPS5 inhibition with olaparib. Methods: COPS5 expression was assessed by immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded samples. Synergy between COPS5 inhibition and PARPi was evaluated in viability assays using the Loewe model. COPS5 was depleted using CRISPR/Cas9, and CSN5i-3, a selective COPS5 inhibitor, was used for pharmacologic inhibition. Cell viability was assessed via MTS and clonogenic assays. Two murine models were used to evaluate in vivo synergy. DNA damage was assessed by immunofluorescence for γH2AX foci. Homologous recombination (HR) efficiency was measured using a DR-GFP reporter assay and by immunofluorescence for RAD51 foci. DNA fiber assays with S1 endonuclease digestion were used to detect single-stranded DNA gaps. DNA: RNA hybrids were assessed by immunofluorescence with S9. 6 antibody. Transcription-replication conflicts (TRC) were evaluated by proximity ligation assay (PLA) for PCNA and RNA Polymerase II. Western blotting confirmed protein-level changes. Results: High COPS5 expression in patient-derived tumors was associated with reduced overall survival (HR 2. 4, 95% CI 1. 1–5. 4) and lower DNA damage (P0. 001). COPS5 depletion reduced cell viability by 60–70% (P0. 001) following olaparib treatment. CSN5i-3 was synergistic with olaparib in 12 established cancer cell lines (synergy score: 10. 73–48. 2), including five novel PARPi-resistant lines. In vivo, coadministration of CSN5i-3 and olaparib significantly reduced tumor mass in both syngeneic and human-derived PARPi-resistant models. Overall survival was assessed in the syngeneic model which also demonstrated a survival benefit with coadministration. To investigate synergy mechanisms, we examined DNA repair pathways. Combination treatment increased γH2AX foci in replicating cells marked by geminin. Neither genetic nor pharmacologic depletion reduced RAD51 foci, and DR-GFP assays showed no change in HR efficiency. DNA fiber analysis revealed shortened fibers post-S1 digestion, indicating increased DNA gaps. S9. 6 staining was elevated with CSN5i-3 alone and in combination, suggesting increased R-loop presence. PLA revealed increased PCNA-RNA Pol II colocalization, implicating TRCs in the observed DNA damage. Conclusions: COPS5 inhibition sensitizes ovarian cancer cells to PARPi, in part through enhanced R-loop accumulation and transcription-replication conflicts. This effect is observed in both HR-proficient and -deficient cell lines, including PARPi-resistant models, highlighting broad clinical potential. Validation of 12 additional COPS5 inhibitor analogues is ongoing to optimize therapeutic potential. Citation Format: Joshua Brill, Kevin Rodriguez, Megan Loeb, Maggie Minett, Elena Lomonosova, Eden Gallup, Prasanth Thuthika, Angela Schab, Lillian Van Biljon, Rikki Drexler, Brooke E. Sanders, Lindsay M. Kuroki, Carolyn K. McCourt, Andrea R. Hagemann, Ian S. Hagemann, Premal H. Thaker, David G. Mutch, Matthew A. Powell, Nima Mosammaparast, Priyanka Verma, Katherine C. Fuh, Dineo Khabele, Mary Mullen. COPS5 Inhibition sensitizes high-grade serous ovarian cancer to PARP inhibition via R-loop–mediated DNA damage 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 (18Suppl): Abstract nr A020.
Brill et al. (Fri,) studied this question.