Replication Gaps Underlie BRCA Deficiency and Therapy Response

Abstract

Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes BRCA1 and BRCA2 (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. Significance: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype “BRCAness,” yielding promising biomarkers, targets, and opportunities to resensitize refractory disease. See related commentary by Canman, p. 1214

Shaping the BRCAness mutational landscape by alternative double-strand break repair, replication stress and mitotic aberrancies

“…As a result, the mutation profile of PRIMPOL differs from that of Y- and B-family TLS polymerases, as it generates insertions and deletions rather than base misincorporations ( 158 ). Recent data demonstrated the presence of large amounts of ssDNA gaps in BRCA-deficient cells, especially upon treatment with replication stress-inducing agents ( 119 , 135 ). The accumulation of ssDNA gaps appears to be an important characteristic of the BRCAness phenotype, as suppression of gap formation in BRCA1/2-deficient cells, rescued the sensitivity to chemotherapeutic agents ( 135 , 136 ).…”

“…Recent data demonstrated the presence of large amounts of ssDNA gaps in BRCA-deficient cells, especially upon treatment with replication stress-inducing agents ( 119 , 135 ). The accumulation of ssDNA gaps appears to be an important characteristic of the BRCAness phenotype, as suppression of gap formation in BRCA1/2-deficient cells, rescued the sensitivity to chemotherapeutic agents ( 135 , 136 ). PARP inhibitors also induce large amounts of ssDNA gaps ( 160 ), and PARP inhibitor response correlates well with ability of cells to suppress gap formation ( 160 ).…”

Targeting translesion synthesis (TLS) to expose replication gaps, a unique cancer vulnerability

“…In contrast, loss of genes such as MRE11 or SMARCAL1 that restore FP, do not lead to RGS, nor confer cisplatin resistance. Moreover, low expression of these genes does not predict poor survival for BRCA2-mutant patients as found for low expression of CHD4, EZH2, or FEN1 [71]. Together, these findings indicate that restored FP alone through inhibition of fork reversal and/or nuclease degradation is not sufficient to confer chemotherapy resistance.…”

“…cells [71]. In contrast, loss of genes such as MRE11 or SMARCAL1 that restore FP, do not lead to RGS, nor confer cisplatin resistance.…”

“…Indeed, many RS-inducing agents generate gaps in human cells as noted in response to UV as early as 1976 [68,69]. In addition to UV, gaps form in response to HU, cisplatin, or inhibitors of ataxia telangiectasia and Rad3-related (ATR), WEE1 or poly(ADP-ribose) polymerases (PARP) as well as following oncogene expression [67,70,71]. The toxicity of these agents is however reduced in pro-TLS cells as gaps are also suppressed [67].…”