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.

Figure 1:. BRCA2 deficient cancer cells fail to restrain replication in the presence of stress, generating regions of ssDNA gaps that are destroyed after continued exposure.

A) Left, Western blot detects truncated BRCA2 protein in BRCA2 deficient PEO1 cells and detects full-length BRCA2 protein in BRCA2 proficient C4–2 cells that are derived from PEO1 cells. Right, cell survival assay confirms PEO1 cells are hypersensitive to cisplatin compared to C4–2 cells. B) Schematic and quantification of CldU track length shows (white panel) that PEO1 cells fail to arrest replication in the presence of stress. These regions are degraded by S1 nuclease (light grey panel), and are also destroyed after continued exposure to replication stress (dark grey panel). Each dot represents one fiber. Experiments were performed in biological triplicate with at least 100 fibers per replicate. Statistical analysis according to two-tailed Mann-Whitney test; *** p< 0.001. Mean and 95% confidence intervals are shown. C) Schematic and quantification of nuclear imaging identifies a greater percentage of EdU positive cells in PEO1 as compared to C4–2. p< 0.05 (*) as determined by t-test of biological triplicate experiments. D) Nondenaturing fiber assay identifies exposed ssDNA adjacent to newly replicating regions after stress in PEO1, but not C4–2 cells. Regions of active replication were detected with EdU-Click chemistry; p < 0.01 (***) as determined by t-test of biological triplicate experiments. E) Model of fiber assay interpretation.

Figure 2:. CHD4 depletion suppresses ssDNA gaps but does not restore fork restraint.

A) Left, Western blot confirms CHD4 is depleted by shRNA compared to non-silencing control (NSC) in BRCA2 deficient PEO1. Right, cell survival assay confirms PEO1 with depleted CHD4 are resistant to cisplatin compared to PEO1 NSC. B) Schematic and quantification of CldU track length shows that PEO1 with depleted CHD4 increase replication in the presence of stress (white panel). These regions are protected from S1 nuclease (light grey panel), and are also protected after continued exposure to replication stress (dark grey panel). Each dot represents one fiber. Experiments were performed in biological triplicate with at least 100 fibers per replicate. Statistical analysis according to two-tailed Mann-Whitney test; p < 0.001 (***). Mean and 95% confidence intervals shown. C) Schematic and quantification of nuclear imaging identifies a greater percentage of EdU positive cells in CHD4 depleted PEO1 as compared to NSC. p < 0.01 (**) as determined by t-test of biological triplicate experiments. D) Nondenaturing fiber assay identifies that ssDNA adjacent to newly replicating regions after stress is reduced when CHD4 is depleted in PEO1 cells. Regions of active replication were detected with EdU-Click chemistry; p < 0.05 (*) as determined by t-test of biological duplicate experiments. E) Model of fiber assay interpretation.

Figure 3:. Suppression of ssDNA gaps accurately predicts poor therapy response in both cell culture and patient xenografts.

A) Overview of the SILAC CHD4 immunoprecipitation experiment. B) SILAC immunoprecipitation reveals that CHD4 interacts with ZFHX3, FEN1, and EZH2 after cisplatin treatment. Red and blue circles are proteins significantly enriched in the CHD4 network of either PEO1 or C4–2 cells. Green (X) represents CHD4. Yellow circles are known CHD4 interacting partners from the NurD complex, including MTA1, HDAC1, MTA2, and HDAC2 (22); ZFHX4 was also identified and is a known CHD4 interacting partner (26). Black plus signs represent proteins not significantly enriched in the CHD4 network of either PEO1 or C4–2. Three biological replicates were performed; see methods section for statistical analysis. C) Western blot confirms ZFHX3 is depleted by shRNA in PEO1 as compared to NSC. Cell survival assay confirms PEO1 with depleted ZFHX3 are resistant to cisplatin compared to PEO1 NSC. D) Reduced ZFHX3 mRNA levels predict poor patient response to therapy (progression free survival) for ovarian cancer patients with germline BRCA2 deficiency from the TCGA database (p < 0.02). Shaded area represents the 95% confidence interval. E) Schematic and quantification of CldU track length shows that depletion of CHD4 (shRNA(B)), ZFHX3 or FEN1, or inhibition of EZH2, increase replication in the presence of stress (white panel) and protect nascent DNA from S1 nuclease (gray panel). F) Schematic and quantification of CldU track length shows S1 fiber sensitivity is suppressed in BRCA1 deficient patient derived xenografts that have acquired chemoresistance. Each dot represents one fiber. Experiments were performed in biological triplicate with at least 100 fibers per replicate; the xenograft fiber assay was performed in duplicate. Statistical analysis according to two-tailed Mann-Whitney test; p < 0.001 (***). Mean and 95% confidence intervals are shown.

Figure 4:. ssDNA replication gaps, and not FP or HR, determine patient response to chemotherapy.

A) Schematic and quantification of CldU track length in PEO1 cells shows that depleted SMARCAL1 or inhibited MRE11 does not increase replication in the presence of stress, and B) does not protect from S1 nuclease, unlike CHD4 depletion. C) Neither SMARCAL1 nor MRE11 mRNA levels predict response of ovarian cancer patients with germline BRCA2 deficiency in TCGA dataset (p > 0.8 and p > 0.5, respectively). In contrast, CHD4 mRNA levels do predict response in these patients (p = 0.03). Shaded area represents the 95% confidence interval. D) Top, Western blot confirms RADX is depleted by two shRNA reagents in T131P cells compared to non-silencing-control (NSC). Bottom, cell survival assay confirms RAD51 T131P cells remain hypersensitive to cisplatin even when RADX is depleted. E) Schematic and quantification of CldU track length shows (white panel) that fibroblasts from a Fanconi Anemia-like patient with a mutant allele of RAD51 (T131P; HR proficient cells, cisplatin hypersensitive) fail to arrest replication in the presence of stress even when RADX is depleted, and these regions are degraded by S1 nuclease (light grey panel). WT FA cells are corrected by CRISPR to delete the dominant-negative T131P RAD51 allele. Each dot represents one fiber. Experiments were performed in biological triplicate with at least 100 fibers per replicate. Statistical analysis according to two-tailed Mann-Whitney test; p < 0.001 (***). Mean and 95% confidence intervals are shown.

Figure 5:. DNA Double Strand Breaks are not Detected when Apoptosis is Inhibited.

A) Overview of model: therapy induces ssDNA gaps that trigger programmed cell death, and the nucleolytic machinery creates DNA DSBs. B) Left, flow cytometry with propidium iodide and annexin V shows that apoptosis is eliminated by 50uM Z-VAD-FMK in BRCA2 deficient PEO1 cells treated with 1uM CPT for 48h. Right, flow cytometry detects apoptosis in BRCA2 deficient PEO1 cells treated with 0.5uM cisplatin for 24h (see Figure S5A for matched untreated control) or 2.5uM cisplatin for 72h (see Figure S5F for matched untreated control). C) Overview of isolation procedure that maintains high molecular weight (megabase-scale) genomic DNA for pulsed field capillary electrophoresis (PFCE). D) PFCE of PEO1 genomic DNA reveals 50uM Z-VAD-FMK eliminates all detectable DNA DSBs for both 1uM CPT 48h and 2.5uM CDDP 24h. E) Model of BRCAness and chemoresponse. During stress, BRCA-deficient cells fail to effectively restrain replication, leading to ssDNA gaps that determine chemosensitivity: BRCAness. These cells acquire chemoresistance by eliminating the ssDNA gaps, either by gap filling, or by restoring fork slowing.