Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo. Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance.

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Accurately identifying patients with high-grade serous ovarian carcinoma (HGSOC) who respond to poly(ADP-ribose) polymerase inhibitor (PARPi) therapy is of great clinical importance. Here we show that quantitative BRCA1 methylation analysis provides new insight into PARPi response in preclinical models and ovarian cancer patients. The response of 12 HGSOC patient-derived xenografts (PDX) to the PARPi rucaparib was assessed, with variable dose-dependent responses observed in chemo-naive BRCA1/2-mutated PDX, and no responses in PDX lacking DNA repair pathway defects. Among BRCA1-methylated PDX, silencing of all BRCA1 copies predicts rucaparib response, whilst heterozygous methylation is associated with resistance. Analysis of 21 BRCA1-methylated platinum-sensitive recurrent HGSOC (ARIEL2 Part 1 trial) confirmed that homozygous or hemizygous BRCA1 methylation predicts rucaparib clinical response, and that methylation loss can occur after exposure to chemotherapy. Accordingly, quantitative BRCA1 methylation analysis in a pre-treatment biopsy could allow identification of patients most likely to benefit, and facilitate tailoring of PARPi therapy.

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

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