Volume 21, Issue 12, Pages 3398-3405 (December 2017) BET Bromodomain Inhibition Synergizes with PARP Inhibitor in Epithelial Ovarian Cancer  Sergey Karakashev, Hengrui Zhu, Yuhki Yokoyama, Bo Zhao, Nail Fatkhutdinov, Andrew V. Kossenkov, Andrew J. Wilson, Fiona Simpkins, David Speicher, Dineo Khabele, Benjamin G. Bitler, Rugang Zhang  Cell Reports  Volume 21, Issue 12, Pages 3398-3405 (December 2017) DOI: 10.1016/j.celrep.2017.11.095 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 3398-3405DOI: (10.1016/j.celrep.2017.11.095) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 BET Inhibitor JQ1 Suppresses TOPBP1 and WEE1 Expression (A) Venn diagram and significance of overlap for genes occupied by BRD4, decreased BRD4 binding and downregulated by JQ1, and implicated in DNA damage signaling and repair or cell-cycle checkpoint. The overlap was used to identify JQ1-regulated direct BRD4 target genes. (B) List of direct BRD4 target genes implicated in DNA damage signaling and repair or cell-cycle checkpoint regulated by JQ1. (C) BRD4 ChIP-seq and nascent RNA-seq normalized reads from control and JQ1-treated OVCAR3 cells were aligned for WEE1 and TOPBP1 genomic loci. (D) Validation of TOPBP1 and WEE1 downregulation by JQ1. Relative primary transcript expression level of the indicated genes was measured by qRT-PCR with or without 250 nM JQ1 treatment for 30 min. n = 3. ∗p < 0.001. (E) OVCAR3 cells were treated with the indicated concentrations of JQ1 or vehicle control for 48 hr; expression levels for TOPBP1, WEE1, and a loading control, GAPDH, were examined by immunoblot. (F) OVCAR3 cells were infected with lentivirus encoding the indicated shBRD4 or control. Drug-selected cells were examined for the expression of BRD4, WEE1, TOPBP1, and a loading control, GAPDH, by immunoblot. (G and H) JQ1 downregulates TOPBP1 (G) and WEE1 (H) expression in vivo in a xenograft model using OVCAR3 cells. (I and J) JQ1 reduces the association of BRD4 and RNA polymerase II (Pol II) with the promoters of TOPBP1 (I) and WEE1 (J) genes. OVCAR3 cells were treated with either control or JQ1 (250 nM) for 24 hr, and ChIP analysis was performed using antibodies against BRD4 or RNA Pol II for the human TOPBP1 and WEE1 gene promoter. An isotype-matched IgG was used as a control (n = 3). Data are represented as mean ± SEM. Cell Reports 2017 21, 3398-3405DOI: (10.1016/j.celrep.2017.11.095) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 BET Inhibitor JQ1 Synergizes with PARP Inhibitor Olaparib in BRCA1/2 Wild-Type Ovarian Cancer Cells (A) Synergy analysis for JQ1 and Olaparib in BRCA1/2 wild-type OVCAR3 cells. Cells were treated with the indicated concentration of JQ1 and Olaparib for 72 hr. The combination index (CI) value was determined. CI value indicates: < 1 synergism = 1 additive effect > 1 antagonism. n = 3. (B) Logarithmic combination index plot of JQ1 is generated in combination with Olaparib in OVCAR3 cells. (C) OVCAR3 cells were plated to 24-well plates and treated with 250 nM JQ1, 125 nM Olaparib, or a combination of both for 12 days in a colony formation assay. (D) Dose-response curves for OVCAR3 cells treated with the indicated concentration of Olaparib with or without 250 nM JQ1 in colony formation assay. n = 4. Arrow indicates a ∼50-fold reduction in Olaparib IC50 in the presence of JQ1 at a dose that does not significantly affect the growth of OVCAR3 cells. (E and F) OVCAR3 cells were treated with 250 nM JQ1, 5 μM Olaparib, or a combination for 72 hr and quantified for apoptosis by fluorescence-activated cell sorting (FACS) based on annexin V staining (E) or examined for expression of apoptosis marker cleaved PARP p85 and a loading control GAPDH by immunoblot (F). n = 3. Data are represented as mean ± SEM. See also Figure S1. Cell Reports 2017 21, 3398-3405DOI: (10.1016/j.celrep.2017.11.095) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 BET Inhibitor JQ1 Suppresses DNA Damage Signaling and G2-M Cell-Cycle Checkpoint Induced by PARP Inhibitor Olaparib to Promote Mitotic Catastrophe (A) OVCAR3 cells were treated with 250 nM JQ1, 5 μM Olaparib, or a combination of both for 48 hr. Expression of the indicated DNA damage signaling and cell-cycle checkpoint proteins and a loading control GAPDH was examined by immunoblot. (B and C) Same as in (A) but stained for RAD51 foci formation (B) and quantified for RAD51 foci formation (C). n = 3. Scale bars, 5 μm. (D and E) OVCAR3 cells treated with or without 250 nM JQ1 and with or without 10 Gy ionizing irradiation were stained for RAD51 foci formation (D) and quantified for RAD51 foci formation (E). n = 3. Scale bars, 5 μm. (F) Relative homologous recombination efficacy in OVCAR3 cells treated with or without 250 nM JQ1 was determined by the DR-GFP reporter assay. n = 3. (G and H) OVCAR3 cells expressing the indicated shRNAs or shControl treated with or without 5 μM Olaparib were stained for RAD51 foci formation (G) and quantified for RAD51 foci formation (H). n = 3. Scale bars, 5 μm. (I and J) Same as in (A) and (G), respectively, but imaged for mitosis using time-lapse video microscopy. Cell nuclei were visualized by staining for DNA using SiR-DNA in red, and tubulin was visualized by CellLight GFP-Tubulin in green. Scale bars, 30 μm. Time is expressed as minutes:seconds. Data are represented as mean ± SEM. See also Figure S2. Cell Reports 2017 21, 3398-3405DOI: (10.1016/j.celrep.2017.11.095) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 BET Inhibitor JQ1 Synergizes with PARP Inhibitor Olaparib in Suppressing the Growth of BRCA1/2 Wild-Type Xenograft Ovarian Tumors In Vivo (A) OVCAR3 cells were injected subcutaneously into the flank of NSG mice. The mice were randomized into 4 indicated treatment groups (n = 5) after 1 week and treated daily with vehicle control, 20 mg/kg JQ1, 50 mg/kg Olaparib, or in combination for 3 weeks. The tumor sizes were measured at the indicated time points. ∗p < 0.0003. (B) At the end of treatment, tumor weight was measured as a surrogate for tumor burden. ∗p < 0.05. (C) Same as in (A) and (B). Serial sections were subjected to immunohistochemcial staining using antibodies against Ki67, cleaved caspase-3, WEE1, TOPBP1, and γH2AX. Scale bars, 50 μm. (D–H). Quantification of histological scores from the indicated treatment group for Ki67 (D), cleaved caspase-3 (E), WEE1 (F), TOPBP1 (G), and γH2AX (H). n = 5; ∗p < 0.03. Data are represented as mean ± SEM. See also Figure S3. Cell Reports 2017 21, 3398-3405DOI: (10.1016/j.celrep.2017.11.095) Copyright © 2017 The Author(s) Terms and Conditions