Replication gaps underlie BRCA-deficiency and therapy response

Abstract

Cancers that are deficient in BRCA1 or BRCA2 are hypersensitive to genotoxic agents, including platinums and other first-line chemotherapeutics. The established models propose that these cancers are hypersensitive because the chemotherapies block or degrade DNA replication forks and thereby create DNA double strand breaks, both of which require functional BRCA proteins to prevent or resolve by mechanisms termed fork protection (FP) or homologous recombination (HR). However, recent findings challenge this dogma because genotoxic agents do not initially cause DNA double strand breaks or stall replication forks. Here, we propose a new model for genotoxic chemotherapy in which ssDNA replication gaps underlie the hypersensitivity of BRCA deficient cancer, and we propose that defects in HR or FP do not. Specifically, we observed that ssDNA gaps develop in BRCA deficient cells because DNA replication is not effectively restrained in response to genotoxic stress. Moreover, we observe gap suppression (GS) by either restored fork restraint or by gap filling, both of which conferred resistance to therapy in tissue culture and BRCA patient tumors. In contrast, restored HR and FP were not sufficient to prevent hypersensitivity if ssDNA gaps were not eliminated. Together, these data suggest that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose are fundamental to the mechanism of action of genotoxic chemotherapies. Introduction:Mutations in the hereditary breast cancer genes, BRCA1 and BRCA2, first demonstrated that cancer is a genetic disease in which susceptibility to cancer could be inherited (King et al., 1986). In addition to breast cancer, mutated BRCA1 or BRCA2 also cause a predisposition to other cancer types, including breast, ovarian, pancreatic, and colorectal cancers. Importantly, cancers with mutated BRCA genes are hypersensitive to cisplatin, a first-line anti-cancer chemotherapy that has been the standard of care for ovarian cancer for over 40 years (Munnell, 1968). BRCA deficient cancers are thought to be

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

“…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.…”

“…We envision that the toxicity threshold is more readily exceeded in BRCA deficient cells due to intrinsic defects in coordinating RAD51 replication restraint and gap avoidance functions ((Hashimoto et al, 2010; Kolinjivadi et al, 2017b; Zellweger et al, 2015)). Indeed, we demonstrate that in response to a range of drugs including cisplatin, BRCA deficiency interferes with replication restraint and gaps develop (Panzarino et al, 2019). Thus, we propose that HR deficiency is not the root cause of therapy response or for that matter “BRCAness”, but rather gaps are the key factor.…”

“…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 mechanism-of-action of genotoxic therapies (Panzarino et al, 2019).…”

Inhibition of the translesion synthesis polymerase REV1 exploits replication gaps as a cancer vulnerability

“…Our data also highlight the clinical importance of identifying TLS polymerase-dependent cancers, especially those that lack current treatment options such as ovarian that are sensitive to the TLSi in vitro. Leveraging this vulnerability by TLS inhibition alone or in conjunction with gap-inducing therapies that include inhibitors of ATR, Wee1 and PARP as well as cisplatin ideally will improve efficacy (20,21). Conceivably, targeting TLS factors in cancer will also reduce the ability of the cancer to mutate and, therefore, retain its vulnerability to other therapies.…”

“…While most appreciated in bypass of DNA adducts, emerging evidence reveals that TLS polymerases are activated in the absence of DNA damage and are required for replication of DNA structures enhanced by DNA replication stress such as G-quadruplexes (G4s), which limit replication progression and promote single-stranded DNA (ssDNA) gap formation (12,(17)(18)(19). Conceivably, ssDNA gaps underlie the mecha-nism of action of genotoxic therapies, and gap suppression (GS) is the key factor that confers resistance (20,21).…”