BET Bromodomain Inhibition as a Therapeutic Strategy in Ovarian Cancer by Downregulating FoxM1

Abstract

Ovarian cancer is responsible for the highest mortality among all gynecologic malignancies, and novel therapies are urgently needed to improve patient outcome. Here we performed an integrative genomic analysis and identified the bromodomain and extraterminal domain (BET) protein BRD4 as a potential therapeutic target in ovarian cancer. Suppression of BRD4 using small-molecule BET inhibitors JQ1 and I-BET151, or dual kinase-bromodomain inhibitor volasertib, led to robust and broad antitumor effects across all subclasses of ovarian cancer. In contrast to many other cancers which are susceptible to BET inhibition due to downregulation of super-enhancer-dependent MYC transcript, we discovered that JQ1-sensitive ovarian cancer cells exhibited marked disruption of Forkhead box protein M1 (FoxM1) pathway, a key driver of ovarian carcinoma. These in vitro findings were further supported by in vivo efficacies of JQ1 targeting both cell line-based and patient-derived xenograft models. Our data establish a new treatment strategy against ovarian cancer by employing epigenetic vulnerabilities, and provide a mechanistic rationale for the clinical investigation of BET bromodomain inhibitors in this deadly disease.

Keywords: BET inhibitors; BRD4; FoxM1; ovarian cancer.

Figure 1

Integrated analyses identify BRD4 as a therapeutic target in ovarian cancer. A. Copy number analysis of BRD4 in TCGA ovarian cancer samples. Color scale: amplification in red and deletion in blue. B. BRD4 gene expression in ovarian cancer with different BRD4 copy number alterations. C. Quantitative PCR of BRD4 in an ovarian cancer tissue cDNA array containing 192 clinical samples. D. Results of shRNA lentiviral screen in SNU840 cells were presented in rank order of ascending shRNA scores. The effect of shRNAs targeting BET proteins was highlighted by colored dots. Gray dots represent results for non-BET shRNAs. E. Kaplan-Meier plot of disease free survival in ovarian cancer patients with or without BRD4 gene amplification. F. Kaplan-Meier plot of overall survival in ovarian cancer patients with or without BRD4 gene amplification.

Figure 2

BET bromodomain inhibition induces pan-subtype cell-cycle arrest in ovarian cancer. A. Western blot analysis of BRD2, BRD3, BRD4 and c-MYC in a panel of ovarian cancer cell lines. B. Heatmap of IC50s of the BET inhibitors (7-day treatment) in the indicated panel of ovarian cancer cell lines. Color scale: sensitive in red and resistant in black. C. Cell viability was assayed in cells treated with (-)-JQ1, JQ1 or I-BET151 (n = 6 biological replicates). D. Cells were treated with (-)-JQ1, JQ1 (1 μM) or I-BET151 (1 μM) for 10 days. The remaining cells were stained with crystal violet. E. Cell cycle analysis following 24-hour treatment with (-)-JQ1, JQ1 (1 μM) or I-BET151 (1 μM).

Figure 3

BET inhibition represses FoxM1 transcriptional program. A. Enriched gene sets of differentially expressed genes in OVTOKO cells following 24-hour of treatment with JQ1 (1 μM) or DMSO. FoxM1 pathway was highlighted in red. B. GSEA plots of indicated functionally defined gene sets in DMSO versus JQ1 treated OVTOKO cells at 24 hour. C. Heatmap of FoxM1 pathway components in DMSO versus JQ1 treated OVTOKO cells. D. Gene expression of c-MYC and FoxM1 in DMSO versus JQ1 treated OVTOKO cells. E. OVTOKO cells were treated with DMSO, JQ1 (1 μM) or I-BET151 (1 μM). FoxM1 and c-MYC were detected by Western blot. F. OVTOKO cells were treated with JQ1 and fractionated. Cell lysates were immunoblotted with indicated antibodies. G. HEK293T cells were transfected with the indicated expression plasmids, and subjected to co-immunoprecipitation using a BRD4 antibody.

Figure 4

FoxM1 is a functional target of BET bromodomain inhibitors. A. Quantitative PCR of FoxM1 pathway genes in OVTOKO and OVCA420 cell lines treated with DMSO, JQ1 (1 μM) or I-BET151 (1 μM). Each group had three biological replicates. P<0.05, ANOVA followed by Tukey's post-test. B. OVTOKO and OVCA420 cells were treated with DMSO, JQ1 (1 μM) or I-BET151 (1 μM). Cell lysates were immunoblotted with indicated antibodies. C. FoxM1 was knocked down in OVTOKO cells. Western blot demonstrated FoxM1 knockdown. D. OVTOKO cell growth upon FoxM1 knockdown or JQ1 treatment. *P<0.05, ANOVA followed by Tukey's post-test. E. FoxM1 was overexpressed in ES-2 cells. Western blot demonstrated FoxM1 overexpression. F. ES-2 cells were treated with DMSO or JQ1 (1 μM) for 10 days. The remaining cells were stained with crystal violet. G. Relative cell viability of ES-2 cells treated with DMSO or JQ1 (1 μM) in the presence or absence of FoxM1 overexpression.

Figure 5

BET bromodomain inhibition attenuates tumor growth and downregulates FoxM1 in vivo. A. OVCAR.x1 cells were treated with DMSO, JQ1 (1 μM) or I-BET151 (1 μM) for 10 days. The remaining cells were stained with crystal violet. B. Quantitative PCR of FoxM1 pathway genes in OVCAR.x1 cells treated with DMSO, JQ1 (1 μM) or I-BET151 (1 μM). Each group had three biological replicates. P<0.05, ANOVA followed by Tukey's post-test. C. OVCAR.x1 cells were treated with DMSO, JQ1 (1 μM) or I-BET151 (1 μM). Cell lysates were immunoblotted with indicated antibodies. D. Tumor growth of OVCAR.x1 cells treated with JQ1 (50mg/kg/day) or vehicle control, ten mice per group. *P<0.05, Student's t-test. E. OVCAR.x1 tumor lysates were immunoblotted with indicated antibodies. F. Tumor growth of ovarian PDX model OVA9 treated with JQ1 (50mg/kg/day) or vehicle control, eight mice per group. Each line represents an individual tumor growth curve, and the thick blue/red lines indicate mean tumor volume of the treatment group. Tumor volumes of the JQ1 group were statistically significantly lower than those of the vehicle control after 29 days of treatment (*P<0.05, unpaired Student's t-test). G. Tumor growth of ES-2 xenografts in the presence or absence of FoxM1 overexpression and treated with JQ1 (50mg/kg/day) or vehicle control. *P<0.05, ANOVA followed by Tukey's post-test.