PARPi synthetic lethality derives from replication-associated single-stranded DNA gaps

Abstract

BRCA1 or BRCA2 (BRCA)-deficient tumor cells have defects in DNA double strand break repair by homologous recombination (HR) and fork protection (FP) that are thought to underlie the sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi). Given the recent finding that PARPi accelerates DNA replication, it was proposed that high speed DNA replication leads to DNA double strand breaks (DSBs). Here, we tested the alternative hypothesis that PARPi sensitivity in BRCA deficient cells results from combined replication dysfunction that causes a lethal accumulation of replication-associated single-stranded DNA (ssDNA) gaps. In support of a gap toxicity threshold, PARPi-induced ssDNA gaps accumulate more excessively in BRCA deficient cells and are suppressed in de novo and genetic models of PARPi resistance while defects in HR or FP often lack this correlation.We also uncouple replication speed from lethality. The clear link between PARPi sensitivity and ssDNA gaps provides a new paradigm for understanding synthetic lethal interactions. Schlacher et al., 2012). Consistent with the DSB inducing model of therapy response in BRCA deficient cells, restoration of HR or FP are associated with chemoresistance (Bouwman et al., 2010; Bunting et al., 2010; Chaudhuri et al., 2016; Edwards et al., 2008; Sakai et al., 2008). However, this model of therapy response is challenged by recent reports indicating that chemotoxic agents do not initially induce DSBs or even pause DNA replication forks (Huang et al., 2013; Mutreja et al., 2018; Zellweger et al., 2015). Indeed, PARPi accelerates DNA replication (Maya-Mendoza et al., 2018). To square this finding with the framework that HR and/or FP defects drive PARPi therapy response, it was proposed that high speed DNA replication ultimately induces DSBs (Maya-Mendoza et al., 2018; Quinet and Vindigni, 2018). However, we recently proposed a competing model in which ssDNA gaps underlie the BRCA deficiency phenotype, and not DSBs and we propose are fundamental to the mechanismof-action of genotoxic therapies (Panzarino et al., 2019). 2011;Here, we provide evidence that the genotoxic lesion driving PARPi synthetic lethality in BRCA deficient cancer is wide-spread single-stranded DNA (ssDNA) gaps. Gap induction is associated with PARPi potentially due to its role in the repair of ssDNA breaks, processing Okazaki fragments, or regulating replication-fork reversal -a mechanism by which replication forks reverse direction when confronted with replication obstacles (Berti et al. Sogo et al., 2002). Critically, we demonstrate that PARPi gaps are compounded in BRCA-deficient cells, but suppressed in cell lines and tumors with intrinsic, genetic or de novo mechanisms of PARPi resistance. These findings highlight that a key function of the BRCA-RAD51 proteins is to limit replication gaps (Hashimoto et al., 2010; Kolinjivadi et al., 2017a; Kolinjivadi et al., 2017b; Zellweger et al., 2015, Panzarino et al., 2019 and that loss of this function confers therapy response. RESULTS PARPi generates s...

Replication Gaps Underlie BRCA Deficiency and Therapy Response

“…Not surprisingly, TLS confers chemotherapy resistance, is a cancer adaptation, and is actively being targeted for cancer therapy (53,54). Moreover, we find that replication gaps due to BRCA deficiency is the basis for synthetic lethality to PARP inhibitors (55). Understanding how gap suppression functions align with other BRCA roles in genome preservation, cell viability, and tumor suppression will also be critical future questions.…”

PARP Inhibitors: Clinical Relevance, Mechanisms of Action and Tumor Resistance

“…This includes the substantially increased prevalence of ssDNA gaps following PARPi treatment in BRCA-deficient tumor cell lines, in comparison to those that were BRCA-wild type. Furthermore, significantly less ssDNA gaps were observed in PARPi resistant cell models, demonstrating that PARPi sensitivity correlates with the level of ssDNA gaps induced by PARPi treatment (Cong et al, 2019).…”

“…This suggests an underlying mechanism of PARPi toxicity could be the result of DSBs occurring as a result of high-speed replication (Maya-Mendoza et al, 2018;Quinet and Vindigni, 2018). Based on these findings, it was also recently proposed that increased replication speed may result in the accumulation of replication-associated single-stranded DNA (ssDNA) gaps (Cong et al, 2019). It was hypothesized that these cytotoxic ssDNA gaps were attributed to PARP1's role in processing Okazaki fragments or the reversal of stalled replication forks.…”

ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination deficient cells

“…Persistent base lesions in ALC1 deficient cells may impede replication fork progression and increase the frequency of replication-coupled gaps that trap PARPi. Interestingly, several recent studies reported the accumulation of ss-DNA enriched postreplicative territories upon treatments with alkylating agents or PARPi 69,70 . Replication restart by re-priming events downstream of DNA lesion can leave ss-DNA gaps to be filled post replication 71,72 .…”