Journal: Cell Death Discovery

Article Title: PARP inhibition-associated heterochromatin confers increased DNA replication stress and vulnerability to ATR inhibition in SMARCA4-deficient cells

doi: 10.1038/s41420-025-02306-1

Figure Lengend Snippet: A LUAD cell lines harboring deficiency of SMARCA4 (A549 and H1299), ARID1A (H2172 and SK-LU-1), and PBRM1 (ABC1 and H226) were constitutively expressed the corresponding wild-type proteins via lentiviral cDNA expression system. Expression of SMARCA4, ARID1A, and PBRM1 proteins was analyzed by western blotting. β-Actin was detected as loading control. B Cell viability screening for tuvusertib (0, 3.9–1000 nM) in combination with lartesertib (0, 39.1–10000 nM), peposertib (0, 39.1–10000 nM), or niraparib (0, 39.1-10000 nM). Cells were treated with these combinations for 4 days, then a cell viability assay was performed. Using SynergyFinder plus, dose response and Bliss synergy maps in A549 cells are shown in Fig. 1B, and other cells are shown in Fig. . These maps indicate the mean of three independent experiments. C Combination sensitivity score-Bliss synergy score plots of tuvusertib in combination with lartesertib, peposertib, or niraparib in the cell viability screening shown in Fig. 1B.

Article Snippet: ATRi (tuvusertib), ATMi (lartesertib), DNA-PKi (peposertib), and PARPi (niraparib) were provided by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945).

Techniques: Expressing, Western Blot, Control, Viability Assay

Journal: Cell Death Discovery

Article Title: PARP inhibition-associated heterochromatin confers increased DNA replication stress and vulnerability to ATR inhibition in SMARCA4-deficient cells

doi: 10.1038/s41420-025-02306-1

Figure Lengend Snippet: A Western blot analysis of SMARCA4 wild-type (WT) and knockout (KO) H1975 cells. α-Tubulin was detected as loading control. B Cell viability for tuvusertib or niraparib alone. Cells were treated with tuvusertib (0, 3.9–1000 nM) and niraparib (0, 39–10000 nM) combination for 6 days, then a cell viability assay was performed. These graphs indicate the mean ± SD of four independent experiments. C Cell viability at 62.5 nM tuvusertib and 625 nM niraparib in the experiment shown in Fig. 2B. The graph indicates the mean ± SD of four independent experiments and statistical significance by one-way ANOVA test with Tukey’s multiple comparisons test. P -value: * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, and **** ≤ 0.0001. D Representative images of each samples show each and merged images of DAPI (blue), PCNA (green), and γH2A.X (red). Cells were treated with 62.5 nM tuvusertib and 625 nM niraparib each alone and combination for 48 h, then carried on immunofluorescence analysis. Scale bars indicate 50 µm. E QIBC plots of DAPI sum intensity versus PCNA mean intensity, with γH2A.X mean intensity (color scale) per nuclei in the experiment shown in Fig. 2D. These plots indicate 2000 cells. F Dot plot of γH2A.X mean intensity per nuclei in S phase cells in the experiment shown in Fig. 2D. PCNA-positive cells were defined as S phase cells. The graph indicates the median of 500 cells and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: ** ≤ 0.01, *** ≤ 0.001, and **** ≤ 0.0001. G Representative images of each samples show merged images of DAPI (blue) and PLA foci of EdU and γH2A.X (red). Cells were treated with 62.5 nM tuvusertib and 625 nM niraparib each alone and combination for 48 h, then carried on PLA analysis. Scale bars indicate 10 µm. H Quantification of the average number of PLA foci per positive nuclei (foci ≥ 1) in S phase cells. The graph indicates the mean ± SD of four independent experiments and statistical significance by one-way ANOVA test with Tukey’s multiple comparisons test. P -value: not significant (ns) > 0.05 and * ≤ 0.05.

Article Snippet: ATRi (tuvusertib), ATMi (lartesertib), DNA-PKi (peposertib), and PARPi (niraparib) were provided by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945).

Techniques: Western Blot, Knock-Out, Control, Viability Assay, Immunofluorescence

Journal: Cell Death Discovery

Article Title: PARP inhibition-associated heterochromatin confers increased DNA replication stress and vulnerability to ATR inhibition in SMARCA4-deficient cells

doi: 10.1038/s41420-025-02306-1

Figure Lengend Snippet: A Dot plot of fork velocity in H1975 cells. Cells were treated with tuvusertib for 24 h prior to IdU and CldU labeling, then were carried on DNA fiber assay. The graph indicates the mean of 200 fibers and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05 and **** ≤ 0.0001. B Dot plot of fork velocity in H1975 cells. Cells were treated with niraparib for 24 h prior to IdU and CldU labeling, then were carried on DNA fiber assay. The graph indicates the mean of 200 fibers and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05 and **** ≤ 0.0001. C Representative immunofluorescent images of DNA fiber assay in H1975 cells. Cells were treated with 10 nM tuvusertib and 2.5 µM niraparib for 24 h prior to IdU (red) and CldU (green) labeling, then a DNA fiber assay was performed. Scale bars indicate 10 µm. D Dot plot of fork velocity in the experiment shown in Fig. 3C. The graph indicates the mean of 200 fibers and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05 and **** ≤ 0.0001. E Dot plot of fork velocity in H1975 cells. Cells were transfected with siRNA for 24 h, were treated with 10 nM tuvusertib for 24 h prior to IdU and CldU labeling, then were carried on DNA fiber assay. The graph indicates the mean of 200 fibers and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05 and **** ≤ 0.0001. F Dot plot of H3K9me2 mean intensity per nuclei in H1975 cells. Cells were treated with niraparib and 2 µM UNC0642 for 24 h, then were carried on immunofluorescence analysis. The graph indicates the median of 200 cells and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05, * ≤ 0.05, ** ≤ 0.01, and **** ≤ 0.0001. G Dot plot of fork velocity in H1975 cells. Cells were treated with 10 nM tuvusertib, 2.5 µM niraparib, and 2 µM UNC0642 for 24 h prior to IdU and CldU labeling, then were carried on DNA fiber assay. The graph indicates the mean of 200 fibers and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05 and **** ≤ 0.0001. H Dot plot of γH2A.X mean intensity per nuclei in S phase cells. Cells were treated with 100 nM tuvusertib, 625 nM niraparib, and 2 µM UNC0642 each for 48 h, then an immunofluorescence analysis was performed. PCNA-positive cells were defined as S phase cells. The graph indicates the median of 500 cells and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05, *** ≤ 0.001, and **** ≤ 0.0001. I Cell viability relative to untreated or UNC0642-treated cells in H1975 cells. Cells were treated with 100 nM tuvusertib, 625 nM niraparib, and 2 µM UNC0642 for 6 days, then were carried on cell viability assay. The graph indicates the mean ± SD of three independent experiments and statistical significance by one-way ANOVA test with Tukey’s multiple comparisons test. P -value: not significant (ns) > 0.05, ** ≤ 0.01, and **** ≤ 0.0001.

Article Snippet: ATRi (tuvusertib), ATMi (lartesertib), DNA-PKi (peposertib), and PARPi (niraparib) were provided by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945).

Techniques: Labeling, Transfection, Immunofluorescence, Viability Assay

Journal: Cell Death Discovery

Article Title: PARP inhibition-associated heterochromatin confers increased DNA replication stress and vulnerability to ATR inhibition in SMARCA4-deficient cells

doi: 10.1038/s41420-025-02306-1

Figure Lengend Snippet: A Dot plot of fork velocity in SMARCA4 WT and SMARCA4 KO H1975 cells. Cells were treated with 10 nM tuvusertib and 625 nM niraparib for 24 h prior to IdU and CldU labeling, then a DNA fiber assay was performed. The graph indicates the mean of 200 fibers and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05 and **** ≤ 0.0001. B Dot plot of H3K9me2 mean intensity per nuclei in SMARCA4 WT and SMARCA4 KO H1975 cells. Cells were treated with 2.5 µM niraparib for 24 h, then were carried on immunofluorescence analysis. The graph indicates the median of 200 cells and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. C Dot plot of fork velocity in SMARCA4 WT and SMARCA4 KO H1975 cells. Cells were treated with 10 nM tuvusertib, 625 nM niraparib, and 2 µM UNC0642 for 24 h prior to IdU and CldU labeling, then a DNA fiber assay was performed. The graph indicates the mean of 200 fibers and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: not significant (ns) > 0.05, * ≤ 0.05, and **** ≤ 0.0001. D Western blot analysis of SMARCA4 WT and SMARCA4 KO cells. Cells were treated with 62.5 nM tuvusertib and 625 nM niraparib for 48 h, then carried on western blot analysis. α-Tubulin was detected as loading control. E Dot plot of γH2A.X mean intensity per nuclei in S phase cells. Cells were transfected with siRNA for 24 h, then were treated with 62.5 nM tuvusertib and 625 nM niraparib for 48 h and 25 µM Mirin for 24 h prior to immunofluorescence analysis. PCNA-positive cells were defined as S phase cells. The graph indicates the median of 500 cells and statistical significance by Kruskal–Wallis test with Dunn’s multiple comparisons test. P -value: ** ≤ 0.01 and **** ≤ 0.0001.

Article Snippet: ATRi (tuvusertib), ATMi (lartesertib), DNA-PKi (peposertib), and PARPi (niraparib) were provided by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945).

Techniques: Labeling, Immunofluorescence, Western Blot, Control, Transfection

Journal: Cell Death Discovery

Article Title: PARP inhibition-associated heterochromatin confers increased DNA replication stress and vulnerability to ATR inhibition in SMARCA4-deficient cells

doi: 10.1038/s41420-025-02306-1

Figure Lengend Snippet: A, B Body weight (B.W.) changes relative to day 0 and tumor growth in each group. H1975:SMARCA4 WT and SMARCA4 KO xenograft mice were treated with niraparib (50 mg/kg, Daily), tuvusertib (20 mg/kg, 4 days on/3 days off), or combination. The graph indicates the mean ± SD of n = 10 mice/group and statistical significance by two-way ANOVA test with Tukey’s multiple comparisons test. P -value: * ≤ 0.05.

Article Snippet: ATRi (tuvusertib), ATMi (lartesertib), DNA-PKi (peposertib), and PARPi (niraparib) were provided by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945).

Techniques:

Journal: Cell Death Discovery

Article Title: PARP inhibition-associated heterochromatin confers increased DNA replication stress and vulnerability to ATR inhibition in SMARCA4-deficient cells

doi: 10.1038/s41420-025-02306-1

Figure Lengend Snippet: PARP inhibition elevates H3K9me2-associated heterochromatin that increases ATR dependence for fork progression and cell survival, resulting in moderate synergistic cytotoxicity with ATR inhibition in SMARCA4 wild-type (WT) cells. In SMARCA4-deficient (Def) cells, Mus81 and Mre11 nucleases cooperate to exacerbate ATRi/PARPi-induced replication catastrophe. Therefore, ATRi and PARPi combination causes potent cytotoxicity in SMARCA4-deficient cells.

Article Snippet: ATRi (tuvusertib), ATMi (lartesertib), DNA-PKi (peposertib), and PARPi (niraparib) were provided by the healthcare business of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID: 10.13039/100009945).

Techniques: Inhibition

Journal: Cell reports

Article Title: Thymidine rescues ATR kinase inhibitor-induced deoxyuridine contamination in genomic DNA, cell death, and interferon-α/β expression

doi: 10.1016/j.celrep.2022.111371

Figure Lengend Snippet: (A) Proliferation of Pmel-1 CD8 + T cells activated with gp100 ex vivo. CTV histograms of live, CD44 hi CD8 + T cells at indicated times post-activation are shown. Red histograms are un-activated CD8 + T cells that have largely died. The number of divisions is shown. (B) Immunoblots of CD8 + T whole-cell extracts prepared the same number of cells at 0 and 30 h post-activation. (C) CD8 + T cells were activated and treated with 5 μM AZD6738 (ATRi) from 0 to 24 h (blue) or 24 to 48 h (red). At 48 h, live CD8 + T cells (eFluor 780 − CD8 + TCRβ + ) were quantitated. (D) Unactivated CD8 + T cells were maintained in IL-7 and treated with ATRi from 0 to 24 or 24 to 48 h. At 48 h, live CD8 + T cells were quantitated. (E) CD8 + T cells were activated and treated with ATRi, 500 nM AZD0156 (ATMi), 5 μM olaparib (PARPi), or 500 nM AZD1775 (WEE1i) from 24 to 48 h. At 48 h, live CD8 + T cells were quantitated. (A and B) Two biological replicates. (C and D) Three biological replicates. (E) Two biological replicates; three technical replicates. (C–E) Mean and SD bars are shown. *p < 0.05, **p < 0.01, ****p < 0.0001, by ANOVA with Tukey’s multiple comparisons.

Article Snippet: Olaparib (PARPi) , AstraZeneca , N/A.

Techniques: Ex Vivo, Activation Assay, Western Blot

Journal: Cell reports

Article Title: Thymidine rescues ATR kinase inhibitor-induced deoxyuridine contamination in genomic DNA, cell death, and interferon-α/β expression

doi: 10.1016/j.celrep.2022.111371

Figure Lengend Snippet: (A) EdU versus DNA plots of CD8 + T cells and fibroblasts. The percentage of cells in each cell cycle phase and the doubling time were used to estimate the length of G1, S, and G2/M. (B) The circumference of the circle represents the doubling time, and the lengths of G1, S, and G2/M are drawn to scale. (C) Immunoblot of MCM4 in the chromatin fraction prepared from CD8 + T cells treated with 5 μM AZD6738 (ATRi), 500 nM AZD0156 (ATMi), 5 μM olaparib (PARPi), or 500 nM AZD1775 (WEE1i) for 1 h. (D) Repli-seq analyses of the replication timing program of CD8 + T cells treated with ATRi (red) or DMSO (blue) for 1 h. Upper: whole genome. Lower: chromosome 1. Data were deposited at GEO: GSE183412. (E) EdU incorporation in early (2N–3N) and late (3N–4N) S phase in CD8 + T cells treated with ATRi for 1 h. 10 μM EdU was added from 30 to 60 min. (F) DNA combing analyses of CD8 + T cells and fibroblasts treated with ATRi for 1 h. Cells were treated with IdU from 30 to 40 min and CldU from 40 to 60 min of the treatment. (G) Immunoblots of MCM4 in the chromatin fraction prepared from CD8 + T cells treated with increasing concentrations of Ro-3306 (CDK1i), CVT-313 (CDK2i), or PHA-767491 (CDC7i) for 75 min. Cells were treated with ATRi from 15 to 75 min. (H) Immunoblot of RIF1 phosphoserine-2153 in whole-cell extracts prepared from CD8 + T cells treated with Ro-3306 for 75 min. Cells were treated with five ATRi’s from 15 to 75 min. (I) Immunoblot of MCM4 in chromatin fraction and CDC25A in the soluble fraction prepared from CD8 + T cells treated with ATRi and/or 5 mM HU in the sequence indicated. (J) Cartoon of a model in which ATR limits origin firing across active replicons. Green circles indicate origins that fired, and red circles indicate origins that have not fired. (A) CD8 + T cells: 12 biological replicates, 1–5 technical replicates; fibroblasts: 7 biological replicates, 1–2 technical replicates. (E) 10 biological replicates. (F) Left panel, CD8 + T cells DMSO: n = 179 fibers from 4 biological replicates, CD8 + T cells ATRi: n = 203 fibers from 3 biological replicates, fibroblasts DMSO: n = 30 fibers from 2 biological replicates, fibroblasts ATRi: n = 28 fibers from 2 biological replicates. Right panel, CD8 + T cells DMSO: n = 675 fibers from 5 biological replicates, CD8 + T cells ATRi: n = 520 fibers from 4 biological replicates, fibroblasts DMSO: n = 226 fibers from 2 biological replicates, fibroblasts ATRi: n = 219 fibers from 2 biological replicates. (C and G–I) Two biological replicates. (E and F) Mean and SD bars are shown. **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-tailed, unpaired t test.

Article Snippet: Olaparib (PARPi) , AstraZeneca , N/A.

Techniques: Western Blot, Sequencing, Two Tailed Test

Journal: Cell reports

Article Title: Thymidine rescues ATR kinase inhibitor-induced deoxyuridine contamination in genomic DNA, cell death, and interferon-α/β expression

doi: 10.1016/j.celrep.2022.111371

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Olaparib (PARPi) , AstraZeneca , N/A.

Techniques: Recombinant, Protease Inhibitor, Saline, Staining, Membrane, Cell Isolation, Flow Cytometry, Purification, Fluorescence, Software