PARG inhibition sequesters nuclear PAR-binding proteins, including XRCC1 and its partners, into nuclear condensates to elicit cytotoxicity

DNA breaks activate PARP1/2 to synthesize poly(ADP-ribose) (PAR), which relaxes chromatin and recruits DNA repair factors. Normally, PAR is short-lived, rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG). While PARP1/2 inhibitors are established therapies for homologous recombination (HR)-deficient cancers, predictive biomarkers for PARG inhibition (PARGi) remain undefined. Using parallel genome-wide CRISPR screens with PARP and PARG inhibitors, we show that PARGi is synthetically lethal with loss of several PAR-binding factors, including XRCC1-LIG3, POLB, ALC1/CHD1L, ARH3, and PARG itself, but notably not with HR deficiency. Conversely, loss of PARP1, NMNAT1 (required for nuclear NAD synthesis), or UNG (upstream of APE1 cleavage and PARP1 activation), confers PARGi resistance. Mechanistically, PARGi induces time- and dose-dependent formation of PARP1-and PAR-dependent nuclear condensates containing XRCC1 and associated repair factors in otherwise undamaged cells. These condensates do not harbor active DNA breaks but instead sequester PAR-binding repair proteins, depleting their available nuclear pool and impairing their recruitment to genuine DNA breaks. While our analysis focused on XRCC1, PARG inhibition likely sequesters additional PAR- and PARP1-binding proteins. Thus, we propose that PARGi sequesters PAR-binding proteins to elicit toxicity, explaining the essentiality of PARG (but not PARP1) and identifying the loss of PAR-binding factors as candidate predictive biomarkers for PARG-targeted therapy.