Fig 2: WRNi kills BRCA2-deficient cancer cells and retards BRCA2-/- xenograft tumor growth in mice.a Colony survival of WT and BRCA2-/- DLD1 cells exposed to WRNi (left panel) or olaparib (right panel). WT and BRCA2-/- DLD1 cells were plated on a six-well plate at 1,500/well and exposed to the indicated doses of WRNi or olaparib 4 h after cell seeding. b Line graphs showing relative colony survival of PEO1 and PEO4 cells exposed to increasing doses of WRNi (left panel) or olaparib (right panel). PEO1 and PEO4 cells were plated on a six-well plate at 1,000 cells/well and treated with the indicated doses of WRNi or olaparib 4 h after cell seeding. In a and b, cells were grown in presence of WRNi or olaparib for 12 days before colony staining. Data represent mean ± SD of three independent experiments. c Line graph showing a relative growth rate of BRCA2-/- (upper panel) and WT (lower panel) xenograft tumors in athymic nude mice treated with either drug vehicle or NSC61745. After the tumors reached the palpable size, mice were injected intraperitoneally with either drug vehicle or NSC617145 (25 mg/kg body weight) every alternate day for a total of 14 days. Mean tumor volume ± SD (n = 6) at each time point is shown. Tumor growth inhibition (TGI) is indicated. p-values were determined using two-tailed unpaired Welch’s t-test. d and e Immunohistochemical (IHC) staining for ?H2AX (d) and 53BP1 (e) expression in tissue sections obtained from vehicle or NSC617145 treated wild type and BRCA2-/- xenograft tumors. Representative images (×40 magnification) of DAB-stained tissue sections are shown. Scale bar represents 50 µM. Box-whisker plots showing the median integrated density of DAB staining analyzed from n = 5 tumor tissue samples under each condition. Bounds of the box represent 25th–75th percentile, middle horizontal lines show the median, whiskers indicate maximum and minimum values of the dataset; p values were calculated using two-tailed unpaired t-test. Middle horizontal lines of the boxes represent the median. Source data are provided as a Source Data file.

Fig 3: WRNi triggers MUS81-dependent double-strand break formation in BRCA2-mutated cancer cells.a Immunofluorescence staining of ?H2AX following treatment of PEO4 and PEO1 cells with WRNi (5 µM) for 6 h. Cells were co-stained with PCNA to identify replicating cells. Nuclei were stained with DAPI. DAPI, PCNA, and ?H2AX are shown in blue, red, and green, respectively. Scale bars are shown. Bar graph represents quantification of % PCNA-positive cells with =10 ?H2AX foci under untreated (UT) and WRNi-treated conditions. Data represent mean ± SD of three independent experiments; two-tailed unpaired t-test; p values are indicated. b Analysis of DSB formation using neutral Comet assay. Control or MUS81-depleted PEO4 and PEO1 cells were subjected to neutral comet assay following treatment with WRNi (5 µM) for 6 h. Representative images of comets obtained from individual experimental conditions are shown. Scale bars are shown. Scatter dot plot showing tail moment in individual samples. Data represent mean ± SD of n = 120 comets analyzed under each condition over n = 2 independent experiments. Mean tail moments (horizontal red bars) ± SD are shown. =120 comets were analyzed for each condition using the OpenComet plugin in ImageJ. Two-tailed Mann–Whitney test; p values (p < 0.0001, p < 0.0001, p = 0.0030, p < 0.0001, p < 0.0001) are indicated. Source data are provided as a Source Data file.

Fig 4: WRN helicase inhibition causes increased fork degradation and replication restart defects in BRCA2-mutated cancer cells.a (Upper panel) Schematic of the fork stability assay performed in PEO1 and PEO4 cells upon WRN knockdown. (Lower panel) Scatter dot plot showing quantification of IdU/CldU ratios in individual experimental conditions. Representative of n = 3 independent experiments; p-values (p = 0.0255, p < 0.0001, p = 0.0080) were derived from n = 200 DNA fibers using two-tailed Mann–Whitney test. Knockdown of WRN was verified by immunoblotting. b (Upper panel) Schematic of the fork degradation experiment in PEO1 and PEO4 cells upon WRNi treatment. (Lower panel) Graphical quantification of the results obtained from the fork stability experiments as described above. Representative of n = 2 independent experiments; p-values (p < 0.0001, p < 0.0001) were derived from n = 125 DNA fibers using two-tailed Mann–Whitney test. c (Upper panel) Scheme of the fork restart assay performed in PEO1 and PEO4 cells upon siRNA-mediated knockdown of WRN. (Lower panel) Scatter plot showing IdU/CldU ratios in individual experimental conditions. Representative of n = 2 independent experiments; p-values (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001) were derived from n = 195 DNA fibers using two-tailed Mann–Whitney test. Immunoblots showing relative knockdown levels of WRN as assessed 72 h post-transfection. d, (Upper panel) Schematic of the fork restart assay in HU-treated PEO1 and PEO4 cells upon WRN helicase inhibition. (Lower panel) Scatter plot showing IdU/CldU tract length ratios in individual experimental conditions. Representative of n = 2 independent experiments; p-values (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001) were derived from n = 125 DNA fibers using two-tailed Mann–Whitney test. Horizontal red bars in scatter plots indicate the median of IdU/CldU ratios; median IdU/CldU values are indicated; purple and green colors indicate CldU and IdU labeling, respectively; data points within the specified axis limits are shown. ß-actin was used as a loading control in immunoblots in (a) and (c). Western blots a, c were repeated independently at least two times with similar results. Source data are provided as a Source Data file.

Fig 5: Comparison of BRCA1/BRCA2 mRNA levels in CD34-positive cells of primary and leukemia cell lines. (A) CD34+CD38−cells accounted for 86% of Kasumi-1 and 93.6% of KG1a cells. (B) CD34+ cells consisted of up to 99% after being sorted from bone marrow of healthy individuals. (C) Diagram of DSB-related signaling pathways. (D) Expression of BRCA1 mRNA in the Kasumi-1 cell line was 0.2040-fold (±0.07139; n=6; bone marrow fluid; ****P<0.0001) of normal BM-HSCs. Expression level of BRCA1 mRNA in the KG1a cell line was 0.3820-fold (±0.1132; n=6; bone marrow fluid; ***P<0.001) of normal BM-HSCs. The expression of BRCA2 mRNA in the Kasumi-1 cell line was 0.3775-fold (±0.1500; n=6; bone marrow fluid; **P<0.01) of normal BM-HSCs. There was no significant difference in the level of BRCA2 mRNA expression in KG1a cell line compared with normal BM-HSCs (bone marrow fluid). (E) Expression of BRCA1 and BRCA2 proteins. The protein expression level of BRCA1 in KG1a and Kasumi-1 was lower than that in the BM-HSCs (n=3; KG1a: **P<0.01; Kasumi-1: **P<0.01). The protein expression level of BRCA2 in KG1a and Kasumi-1 was lower than that in the BM-HSCs (n=3; KG1a: ***P<0.001; Kasumi-1: **P<0.01). BRCA1, breast cancer type 1 susceptibility protein; BRCA2, breast cancer type 2 susceptibility protein; DSB, DNA double-strand breaks; BM-HSCs, bone marrow-hematopoietic stem cells.