Fig 2: Analysis of ATR signaling in primary resting CLL cells after treatment with fludarabine (F-Ara-A) or cladribine (CdA)(A) Activation of ATR by purine analogs. CLL cells were incubated with 3 µM F-Ara-A (F) or 1 µM CdA (C) for various times, or with or without 3 µM F-Ara-A for 48 h. Phospho-ATR(Thr-1989) and ATR protein level were analyzed by Western blot. (B) Effect of ATR inhibitors on fludarabine-induced ATR phosphorylation. CLL cells were incubated for 48 h with or without 3 µM F-Ara-A in the presence or absence of 10 µM VE-821 or 1 µM AZ20, followed by analysis of ATR phosphorylation by Western blot. (C) Effect of VE-821 on ?H2AX accumulation induced by F-Ara-A (left panel) or CdA (right panel). CLL cells were preincubated with or without 10 µM VE-821 before addition of 3 µM F-Ara-A or 1 µM CdA. At the indicated times, phosphorylation of H2AX (?H2AX) was analyzed by flow cytometry and expressed as fold-increase over untreated cells. Results are means ± SEM of 4 independent experiments. Significance relative to the absence of VE-821: ****P < 0.0001. (D) Effect of VE-821 on p53 activation induced by fludarabine or cladribine. CLL cells from three different patients were preincubated with 10 µM VE-821 before addition of 1 µM CdA or 3 µM F-Ara-A. Phospho-p53(Ser-15) and p53 protein level were analyzed by Western blot after 24 h of incubation. ß-Actin were used as a loading control.

Fig 3: BRD4 loss impairs the replication stress DNA damage checkpoint.a Overview of the ATR-TopBP1-Chk1 replication stress DNA damage checkpoint. TopBP1 is required for Chk1 activation and recruitment31,82. b Quantification of RT-qPCR of ATRIP, ATR, TopBP1, and Chk1 mRNA levels following treatment with 500 nM JQ1 for 24 and 48 h in HeLa cells. Data presented as mean ± SEM (n = 3 independent experiments). Significance assessed using ANOVA followed by Tukey’s test (24 h DMSOTopBP1 vs JQ1TopBP1 ****Adjusted P < 0.0001; 48 h DMSOTopBP1 vs JQ1TopBP1 ****Adjusted P < 0.0001). c WB of TopBP1, BRD2, BRD3, BRD4, and tubulin following transfection with control siRNA or siRNA to BRD2, BRD3, or BRD4. d, e WB of pATRthr1989, ATR, pTopBP1ser1138, TopBP1, and tubulin (d), and pChk1ser345, Chk1, and tubulin (e), following 24 h of treatment with JQ1. f IF images and quantification as in Fig. 1a of cells that stain triply positive for ?H2AX, CC3 and EdU (upper panel) or ?H2AX, CC3, and pHH3 (lower panel) following JQ1 treatment. Data presented as mean ± SEM (n = 3 independent experiments). Significance assessed using two-tailed unpaired t test (24 h DMSO?H2AX/CC3/EdU (+) vs JQ1?H2AX/CC3/EdU (+) ****P < 0.0001; 48 h DMSO?H2AX/CC3/EdU (+) vs JQ1?H2AX/CC3/EdU (+) ****P < 0.0001; 48 h DMSO?H2AX/CC3/pHH3 (+) vs JQ1?H2AX/CC3/pHH3 (+) **P = 0.0012). Scale bar = 5 µm. g Global model of how BRD4 loss results in DNA damage and cell death as a consequence of loss of productive RNAPII elongation, R-loop accumulation, transcription-replication conflicts, and failure to activate the ATR-TopBP1-Chk1 axis. Source data are provided as a Source data file.

Fig 4: Expression levels of proposed biomarkers of WEE1 and ATR inhibitor sensitivity in lung cancer and U2OS cell lines. (A) Immunoblot of lysates from exponentially growing cells. Bar charts (grey) show quantifications of protein levels relative to total protein and normalized to the levels in U2OS. Error bars: SEM (n = 3). (B) Flow cytometry analysis of ?H2AX levels in S-phase of exponentially growing cells normalized to U2OS. Error bars: SEM (n = 2).

Fig 5: Synergistic induction of S-phase DNA damage and cell killing in U2OS cells upon combined WEE1/ATR inhibition. (A) Immunoblotting of extracts from U2OS cells treated with MK1775 (200 nM) and VE822 (200 nM) as indicated. (B) Flow cytometry analysis of U2OS cells treated with MK1775 (200 nM) and/or VE822 (200 nM) for 3, 6, and 24 h or left untreated (mock). Scatter plots of ?H2AX versus DNA content (Hoechst) are shown. Numbers indicate percentage of cells with strong ?H2AX levels (indicated in red color). (C) Left: Histograms showing cell cycle profiles (counts versus DNA content) from the same experiment as in (B). Right: Quantification of cell cycle distribution from three independent experiments treated as in (B) for 24 h. All other results in (A–C) are representative of three or more independent similar experiments. (D) Flow cytometry analysis of phospho-B-MYB, phospho-MPM2, and chromatin-bound CDC45 in U2OS treated with MK1775 (500 nM) and/or VE822 (500 nM) for 1 h. Median levels were measured within a region of cells with S-phase DNA content as indicated in Figures S1D and S3. (E) Clonogenic survival of U2OS cells treated with MK1775 and/or VE822 at the indicated concentrations for 24 h. Clonal efficiency relative to that of untreated cells (control) is shown. Error bars: SEM (n = 3). In (C), p values were determined by the two-tailed two-sample Student’s t test (test criterion: treated sample ? mock), and in (D), p values were determined by the two-tailed Student’s one-sample t test (test criterion: fold change ? 1), * p = 0.05.