Fig 2: FTY720-mediated reduction of S70pBcl2 is associated with enhanced DNA damage and cell death of CLL patient-derived primary cells upon treatment combination with Fludarabine. (A) Flow chart indicating the work sequence of obtaining peripheral blood mononuclear cells (PBMC) from CLL patients (refer to methods) to drug treatments at various timepoints prior to western blot or cell viability analysis. Primary cells were co-cultured with stroma NK.Tert cells for cell viability analysis (refer to methods). (B) Western blot showing S345pChk1, γH2AX, S70pBcl2, Chk1, Bcl-2, β-actin of CLL patient-derived primary cells following pre-treatment with FTY720 (2.5μM) for 2 h and treatment with increasing doses of fludarabine for 10 h. (C–E) Fold change of quantified and normalized S70pBcl2/Bcl-2, S345pChk1/Chk1 and γH2AX/β-actin of six CLL patient-derived primary cells following pre-treatment with FTY720 (2.5 μM) for 2 h and treatment with increasing doses of fludarabine for 10 h. N = 6 patient samples with enough cells were used for western blot analysis. FTY: FTY720, Flu10: Fludarabine 10 μM, Flu50: Fludarabine 50 μM. (F) Fold change in cell viability of N = 9 CLL patient-derived primary cells following pre-treatment with FTY720 (2.5 μM) for 2 h and treatment with increasing doses of fludarabine for 48 h. Cell viability measured by annexin V staining and gated for CLL patient-derived primary cells but not stroma NK.Tert cells. Graphs showing mean and SD. One-way ANOVA and Sidak's multiple comparison were performed for C-E. Two-way ANOVA and Sidak's multiple comparison were performed for F. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. (G) Diagram depicting S70pBcl2 as a sensor of oxidative stress and modulator of intracellular and mitochondrial ROS production via its manipulation of the mitochondrial machinery, activity and redox metabolism. Reduction in S70pBcl2 could be a potential sensitizing option for ROS-based chemotherapy.

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: TRIM21 ubiquitinated CLASPIN and regulated CLASPIN dependent CHK1 activation. (A) HEK293T cells were lysed and incubated with protein A agarose conjugated with normal rabbit IgG or anti-CLASPIN antibody for immunoprecipitation, followed by immunoblotting with the indicated antibodies. *, non-specific signal. (B) HEK293T cells co-transfected with HA-CLASPIN and FLAG-TRIM21 were lysed and incubated with anti-FLAG M2 agarose, the immunoprecipitates were then examined by immunoblotting with antibodies against HA and FLAG. (C) Schematic of the CLASPIN deletion mutants: F1:1–330aa, F2:301–630aa, F3:601–930aa, F4:901–1230aa, F5:1201–1339aa. (D) Whole cell lysates extracted from HEK293T cells co-transfected with HA-TRIM21 and wildtype or mutant FLAG-CLASPIN were incubated with anti-FLAG M2 agarose for immunoprecipitation, followed by immunoblotting with the indicated antibodies. (E) Bacterially-purified HIS-TRIM21 and GST-CLASPIN301-630aa/GST peptides immobilized on Glutathione Sepharose 4B beads were mixed and incubated for 4 h. The HIS-TRIM21 complex was examined by immunoblotting. GST-CLASPIN301-630aa/GST were directly visualized by Ponceau S staining. (F) In vitro ubiquitination assays were performed by incubating HIS-TRIM21 or its E3 ligase inactive mutant and GST-CLASPIN301-630aa in the presence of E1, E2 and HA-ubiquitin at 30°C for 1 h, followed by GST-pulldown and analysis by immunoblotting with the indicated antibodies. (G) HeLa cells were transfected with a negative control siRNA or an siRNA targeting TRIM21 or CLASPIN as indicated for 48 hours. Then, whole cell lysates were collected after being treated with 2 mM HU or a mock treatment for the indicated times. CHK1 activation was examined by immunoblotting with an antibody specifically recognizing phosphorylated CHK1 at Ser345. The expression levels of CLASPIN, TRIM21 and CHK1 were also examined. *Non-specific signal. Actin: loading control.

Fig 5: Effects of AZD6738 monotherapy or combined with CDC7 inhibitor in vivo. a Tumor volumes of Huh7 and MHCC97H tumor xenografts in BALB/c nude mice following vehicle or AZD6738 (50 mg/kg) treatment for 12 days (Huh7 xenograft) and 18 days (MHCC97H xenograft), respectively. b γH2AX staining was performed on formalin-fixed paraffin-embedded Huh7 and MHCC97H xenografts after 12 days (Huh7 xenograft) and 18 days (MHCC97H xenograft) of treatment with vehicle or AZD6738. c Tumor volumes of PLC/PRF/5 tumor xenografts in BALB/c nude mice following vehicle, AZD6738 (50 mg/kg), XL413 (50 mg/kg) or combination treatment for 12 days. d γH2AX staining was performed on formalin-fixed paraffin-embedded PLC/PRF/5 xenografts after 12 days treatment with vehicle, AZD6738, XL413 or combination. e Potential classification and treatment strategies for HCC patients. Upregulation of genes implicated in response to replication stress could serve as a biomarker for the classification of HCC patients. The heatmap shows expression level of genes implicated in replication stress of tumor and adjacent tissues. Among 159 patients with both tumor and paired adjacent tissues available, 69 patients have HCC tissues showing high replication stress, while the remaining 90 patients have HCC tissues with low replication stress according to K-means clustering. Kaplan-Meier curves depicting that high replication stress in tumor tissues correlates with poor prognosis of the patients. Patients with HCC tumors showing high replication stress would potentially benefit from ATR or CHK1 inhibition only, whereas patients with tumors that show low replication stress would need CDC7 inhibition to induce replication stress and sensitize tumor cells to ATR or CHK1 inhibition. *P < 0.05, **P < 0.01 and ***P < 0.001A