Fig 2: Pyridostatin inhibits growth of BRCA1-deficient human PDTCs and PDTXs Dose-dependent viability assays of PDTCs derived from breast cancer samples (Bruna et al, 2016) treated with pyridostatin (PDS) at the indicated doses. Values from n = 2 independent experiments are shown. AB521: ER-negative tumour, no BRCA1 alteration; VHIO179: tumour with BRCA1 germline mutation and MAD2L2 inactivating mutation (olaparib-resistant).VHIO179 PDTXs were grafted into CB17-SCID female mice. Pyridostatin was administered intravenously (7.5 mg/kg/day) over the indicated periods of time. Vertical dotted line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 7 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 24, using an unpaired two-tailed t-test. ****P = 0.0001. Data information: Exact P values for (B) are provided in Appendix Table S8.

Fig 3: Antitumoral effect of pyridostatin, NU-7441 and paclitaxel combinations in BRCA2-deficient HCT116 xenograft modelsBRCA2-deficient HCT116 human cells were injected intramuscularly in SCID male mice. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and NU-7441 was administered intraperitoneally (i.p.; 10 mg/kg/day), over the indicated periods of time. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 6 mice. Error bars represent SEM. P values were calculated at day 24 using an unpaired two-tailed t-test NS, P > 0.05; **P = 0.01; ****P = 0.0001.Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) over the indicated periods of time, and paclitaxel (PACLI) was administered intravenously (i.v.; 20 mg/kg/day) on indicated single days. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 6 mice. Error bars represent SEM. P values were calculated at day 24 using an unpaired two-tailed t-test ****, P = 0.0001.Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day), NU-7441 was administered intraperitoneally (i.p.; 10 mg/kg/day), over the indicated periods of time, and paclitaxel (PACLI) was administered intravenously (i.v.; 20 mg/kg/day) on indicated single days. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 6 mice. Error bars represent SEM. P values were calculated at day 36 or 38 using an unpaired two-tailed t-test ****, P = 0.0001.Kaplan-Meier survival curve for mice treated as in (C), with each treatment group containing n = 6 mice. Statistical significance was assessed by Log-rank test (****P < 0.0001). Data information: Exact P values for (A-C) are provided in Appendix Table S8 and (D) in Appendix Table S6.

Fig 4: Pyridostatin inhibits growth of BRCA2-deficient DLD1 xenograft tumours and inflicts repairable DNA damage in BRCA2-deficient cells A, BCB17-SCID male mice were injected intramuscularly into the hind leg muscles with (A) BRCA2-proficient (BRCA2 +/+) or (B) BRCA2-deficient (BRCA2 -/-) DLD1 cells. Pyridostatin (PDS) was administered intravenously (i.v.; 7.5 mg/kg/day) and talazoparib was administered orally (p.o.; 0.33 mg/kg/day), over the indicated periods of time. Vertical dashed line indicates end of treatment. Tumour volume was measured at the timepoints shown on the graph and expressed relative to tumour volume at the beginning of treatment. Each experimental group included n = 5 mice. Error bars represent SEM. P values were calculated between treated and untreated tumours at day 17, using an unpaired two-tailed t-test. ***P = 0.001; NS, P > 0.05CDNA breaks were measured using alkaline comet assay in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with 2 µM of pyridostatin (PDS) for 16 h and released into fresh medium without pyridostatin. Representative images are shown. Scale bar represents 100 µm.DQuantification of DNA breaks shown in (C). Graph and error bars represent the mean and SEM of n = 3 independent experiments. A minimum of 50 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *P = 0.05; NS, P > 0.05.EQuantification of ?H2AX foci visualised using immunofluorescence staining in cells treated as in (C). A minimum of 200 cells were analysed per condition per experiment. Graph and error bars represent the mean and SEM of n = 3 independent experiments. P values were calculated using an unpaired two-tailed t-test. ***P = 0.001; NS, P > 0.05.FBRCA2-deficient (-BRCA2) human DLD1 cells were treated with 2 µM of pyridostatin (PDS) for 24 h. Next, pyridostatin was removed and cells were released in a medium containing 250 nM NU-7441 (NU). Whole-cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red.GDose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with pyridostatin (PDS) and NU-7441 at the indicated concentrations for 6 days. Graphs represent average values obtained from of n = 3 independent experiments, each performed in technical triplicates. Data information: Exact P values for (A-B, D-E) are provided in Appendix Table S8. Source data are available online for this figure.