Fig. 5: Combination PARPi–ATRi increases replication stress and apoptosis.

a–f Experimental design for replication fork analysis for parental BRCA^MUT (PEO1), acquired PARPi and platinum-resistant (PEO1-PR, PEO1-CR, and JHOS4-PR), and platinum-resistant CCNE1^AMP (OVCAR3) cells were pretreated with PARPi (1 μM), ATRi (1 μM), or combination for 30 min, subsequently pulse-labeled with CldU (red) followed by IdU (green) for 15 min each, in the continuous presence of inhibitors. b, c Replication fork speed as calculated by length of track/duration both pulses and >100 intact unidirectional tracks were counted. ATRi exposure attenuated fork speed compared with control (P < 0.0001, all lines). PARPi–ATRi further reduced fork speed compared with ATRi alone in all lines (PEO1-PR and JHOS4-PR, P < 0.0001; PEO1-CR, P = 0.02; OVCAR3, P = 0.04; PEO1, P = 0.03). d, e Fork asymmetry as calculated by long green/short green length of replication initiation tracks and >100 intact initiation tracks were counted for each condition. PARPi treatment produced asymmetric forks in parental cells (PEO1, P < 0.0001). ATRi caused an increase in fork asymmetry in all lines (PEO1, PEO1-PR, PEO1-CR, and OVCAR3, P < 0.0001; JHOS4-PR, P = 0.0102). ATRi with PARPi further increased asymmetric fork ratios in all models (PEO1-PR, JHOS4-PR, and OVCAR3, P < 0.0001; PEO1-CR, P = 0.005; PEO1, P = 0.01). Data shown are median ± 95% CI; n = 2 biological independent samples and two slides per condition counted for DNA combing. One-way ANOVA followed by nonparametric Kruskal–Wallis test for (c) and (e). f The coefficient of drug interaction (CDI) was calculated to determine drug interaction effects. CDI < 1 indicated synergism, CDI = 1 additivity, CDI > 1 antagonism (g, h). Cells treated with control, PARPi (1 μM), ATRi (1 μM), or combination for 3 days (PEO1, PEO1-PR, PEO1-CR, and OVCAR3) and 5 days (JHOS4 and JHOS4-PR). Apoptosis was evaluated by Annexin V-APC and propidium iodide staining (g). Apoptosis was higher in PARPi–ATRi group compared with ATRi monotherapy in all cell lines (PEO1-PR, P = 0.0022; all remaining lines P < 0.001). Immunoblot detection of cleaved-caspase-3 in cells treated as indicated (h). Data shown mean ± SD (n = 3 biologically independent samples), individual samples are presented as data points. One-way ANOVA followed by Tukey’s multiple-comparisons test for (g). ***P < 0.001, **P < 0.01, *P < 0.05. Source data are provided as a source data file.