PARG inhibitor sensitivity correlates with accumulation of single strand DNA gaps in preclinical models of ovarian cancer

Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair by removal of poly (ADP-ribose) (PAR) from PARP1-parylated proteins. Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA damaging agents, suggesting that PARG inhibitors may provide a therapeutic option for cancer therapy. Indeed, PARG inhibitors (PARGi) are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as ovarian and breast cancer patients with germline or somatic BRCA1/2-mutations. Biomarkers of PARGi response will be required for the optimal development of these important new drugs. PARP inhibitors kill BRCA-deficient cancer cells by increasing ssDNA gaps during replication. Here, we report that, similar to PARP inhibitors, PARGi treatment can also cause an accumulation of ssGAPs in sensitive cells. PARGi exposure increased accumulation of S-Phase specific PAR, a marker for Okazaki fragment processing (OFP) defects on lagging strands, in sensitive cells but not in resistant cells. PARGi also caused accumulation of PAR at the replication fork and increased pRPA, a marker for replication stress, at the replication fork in sensitive cells. Additionally, PARGi exhibited monotherapy activity in some HR-deficient, as well as HR-proficient, patient-derived organoids of ovarian cancer, and drug sensitivity directly correlated with the accumulation of ssDNA gaps. Taken together, PARGi treatment results in toxic accumulation of PAR at replication forks resulting in ssDNA gaps due to OFP defects during replication. Regardless of the BRCA-status, the induction of ssDNA gaps in preclinical models of ovarian cancer cells correlates with PARGi sensitivity. Patient-derived organoids will be a useful model system for testing PARGi sensitivity and functional biomarkers Overall design: RMUGS, the most PARGi sensitive cell line, was exposed to a gradually increasing concentration of PARGi until the cells started to grow in the presence of PARGi(4 months) at a similar rate to the parental line without PARGi . The cells with acquired resistance to PARGi, namely RMUGS-R cells, exhibited an approximately 10 fold higher IC50 for growth inhibition by PARGi compared to the sensitive parental cells.For RNA seq, the resistant cells were maintained in a medium without the drug for 2 weeks. The experiment was performed in triplicates. Total RNAs were extracted and sequenced . Differential gene expression analysis was performed by DESeq2 and limma, comparing the mRNA expression levels of PARGi-resistant cells to those of the parent cells.