Abstract 3909: PARG inhibitor sensitivity is correlated with accumulation of single strand DNA gaps in preclinical models of ovarian cancer
Authors: Ravindranathan Ramya , Somuncu Ozge , Jiao Yuqing , da Costa Alexandre André B A , Lamarre Benjamin P , Martignetti David B , Zambrano Gabriella A , Mukkavalli Sirisha , Liu Joyce , Kochupurakkal Bose , Lazaro Jean-Bernard , Parmar Kalindi , Shapiro Geoffrey I , D'Andrea Alan D. 
Published: March 2024

Abstract
Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair in conjunction with PARP1 by removal of poly (ADP-ribose) (PAR). Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA damaging agents, suggesting it is a potential therapeutic target for cancer therapy. Indeed, PARG inhibitors are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as ovarian and breast cancer patients with BRCA1/2-mutation. In this study, we investigated PARG inhibitor (PARGi)-PDD00017273 activity in preclinical models of ovarian cancer cell lines and primary patient-derived organoids. PARGi exhibited sensitivity in a subset of ovarian cancer cell lines that was distinct from PARP inhibitor sensitivity. Initially, we evaluated a PARGi-sensitive cell line, RMUGS. By gradually increasing the concentration of PARGi, we generated drug resistant cells. PARGi treatment resulted in increased S phase specific PAR, DNA-PARylation, DNA damage, and replication stress in the sensitive cells but not in cells with de novo or acquired resistance. In the sensitive cells, PARGi exposure also increased accumulation of PAR at the replication fork resulting from unligated Okazaki fragment processing. More importantly, PARGi treatment or PARG loss caused accumulation of single strand DNA (ssDNA) gaps in sensitive cells, as detected by the DNA fiber assays combined with S1 nuclease treatment. Regardless of the BRCA-status, the induction of ssDNA gaps in ovarian cancer cells correlated with PARGi sensitivity. Consistent with an increase in ssDNA gaps, PARGi exposure increased p-RPA, a ssDNA surrogate, at the replication fork in sensitive cells. p-RPA sites also colocalized with PAR suggesting that accumulation of PAR occurs at the ssDNA sites, perhaps, leading to the ssDNA gaps. Collectively, these results suggested that toxic accumulation of PAR at the replication fork leads to accumulation of ssDNA gaps due to Okazaki fragment processing defects and ovarian cancer cell killing after PARG inhibition. We further assessed PARG inhibition in patient-derived organoids (PDOs) and patient-derived xenograft (PDX)-derived organoids (PDXOs) as model systems of ovarian cancer. PARGi exhibited monotherapy activity in specific HR-deficient PDOs and PDXOs with BRCA1 or RAD51C mutation and in a HR-proficient PDXO model. Importantly, PARGi exposure resulted in accumulation of ssDNA gaps in organoids of ovarian cancer and this phenotype directly correlated with drug sensitivity. PDOs can therefore be a useful model system for testing PARGi sensitivity. The detection of ssDNA gaps in PDOs may provide a functional biomarker to predict the response to PARG inhibitors in clinical trials.
Citation Format: Ramya Ravindranathan, Ozge Somuncu, Yuqing Jiao, Alexandre André B A da Costa, Benjamin P Lamarre, David B Martignetti, Gabriella A Zambrano, Sirisha Mukkavalli, Joyce Liu, Bose Kochupurakkal, Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I Shapiro, Alan D. D'Andrea. PARG inhibitor sensitivity is correlated with accumulation of single strand DNA gaps in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3909.