BET bromodomain proteins are required for glioblastoma cell proliferation

Abstract

Epigenetic proteins have recently emerged as novel anticancer targets. Among these, bromodomain and extra terminal domain (BET) proteins recognize lysine-acetylated histones, thereby regulating gene expression. Newly described small molecules that inhibit BET proteins BRD2, BRD3, and BRD4 reduce proliferation of NUT (nuclear protein in testis)-midline carcinoma, multiple myeloma, and leukemia cells in vitro and in vivo. These findings prompted us to determine whether BET proteins may be therapeutic targets in the most common primary adult brain tumor, glioblastoma (GBM). We performed NanoString analysis of GBM tumor samples and controls to identify novel therapeutic targets. Several cell proliferation assays of GBM cell lines and stem cells were used to analyze the efficacy of the drug I-BET151 relative to temozolomide (TMZ) or cell cycle inhibitors. Lastly, we performed xenograft experiments to determine the efficacy of I-BET151 in vivo. We demonstrate that BRD2 and BRD4 RNA are significantly overexpressed in GBM, suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. I-BET151 treatment enriched cells at the G1/S cell cycle transition. Importantly, I-BET151 is as potent at inhibiting GBM cell proliferation as TMZ, the current chemotherapy treatment administered to GBM patients. Since I-BET151 inhibits GBM cell proliferation by arresting cell cycle progression, we propose that BET protein inhibition may be a viable therapeutic option for GBM patients suffering from TMZ resistant tumors.

Keywords: bromodomain; epigenetics; glioblastoma; histone acetylation mimics; histones; stem cells; temozolomide.

Figure 1.BRD2 and BRD4 are elevated in Glioblastoma. A. Heat maps of genes elevated in GBM tumors ranked by P values. Genes with P < 0.001 (Bonferroni correction) are shown in bold. In italics are genes that pass a Benjamini-Hochberg post-test with a false discovery threshold of 1%. (B) Heat map of genes elevated in GBM ranked by fold change. (C) Phylogenetic Tree of Bromodomain Proteins. A gene-based phylogenetic tree was constructed using ChromoHub (Structural Genomics Consortium³⁰). Genes on the tree were color-coded according to level of significance between glioblastoma and control expression levels. The most significant genes were those that passed a t test with Bonferroni multiple comparisons correction (P < 0.001, green). Genes that passed a less stringent Benjamini-Hochberg post-test (allowing for a false discovery rate, or FDR, of 1%) are highlighted in yellow.

Figure 2.BRD4 knockdown reduces U87MG cell proliferation. (A) BRD4 RNA levels are reduced after siRNA transfection of U87MG cells U87MG cells were transfected twice with 50 nM BRD4 siRNA or control. Five days after transfection, RNA was extracted and used to verify the knockdown efficiency with quantitative RT-PCR. BRD4 RNA levels were normalized relative to the Actin housekeeping gene. (B) Cells transfected with BRD4 siRNA have less cellular ATP than control-transfected cells as measured by a CellTiter-Glo assay. Half of the cells transfected with Brd4 siRNA in A were analyzed for ATP content using the CellTiter-Glo Assay. RLU, Relative Luminescence Levels (C) Cells transfected with BRD4 siRNA proliferate less than control siRNA transfected cells as measured by an EdU incorporation assay (***P < 0.001 by the Student’s t test; n = 3). Results shown are from three independent experiments.

Figure 3. I-BET151 treatment reduces U87MG cellular ATP and proliferation in vitro. (A) IC₅₀ of I-BET151 in a CellTiter-Glo assay. U87 cells treated for 48 h with the indicated doses of I-BET151. (B) IC₅₀ of 72-h treatment of U87MG cells with I-BET151. (C) EdU incorporation assay of U87MG cells after 48-h treatment with I-BET151. (D) EdU incorporation assay of 72-h I-BET151 treatment of U87MG cells. 48 and 72 h CellTiter Glo, ***P < 0.001 by Bonferroni Post hoc comparison, n = 3. 48 and 72 h EdU assay, *P < 0.05 by the Student’s t test, n = 3. Results shown are from three independent experiments.

Figure 4. I-BET151 treatment increases G1 population of U87MG cells. PI-FACS analysis of U87MG cells treated with I-BET151 or DMSO control for the indicated times. Cell cycle phase was assessed by Flow-Jo analysis and represented as % Total cells. ****P < 0.0001 by 2way ANOVA, n = 3. Results shown are from three independent experiments.

Figure 5. I-BET151 treatment increases proportion of cells in the G1/S transition. (A) Percentage of U87MG cells in G1, S/G2/M, or G1/S phases treated with DMSO. (B) Percentage of U87MG cells in G1, S/G2/M, or G1/S phases treated with I-BET151. FUCCI analysis was performed as described in Materials and Methods. Results are from three independent experiments. Statistical analysis described in Figure S3.

Figure 6. I-BET151 treatment reduces growth of U87MG cells in immunocompromised mice. I-BET151 (10 mg/kg, n = 8) or Saline (n = 9) was injected into NU-Foxn1nu mice that had been previously injected with U87MG cells. Eight mice were injected with I-BET151 and 9 mice were injected with saline. Tumor sizes were measured every two to three days using caliper measurements. Average and standard deviation per day are shown. Average and standard error of the mean per day are shown for mice independently injected and analyzed.