Targetable BET proteins- and E2F1-dependent transcriptional program maintains the malignancy of glioblastoma

Abstract

Competitive BET bromodomain inhibitors (BBIs) targeting BET proteins (BRD2, BRD3, BRD4, and BRDT) show promising preclinical activities against brain cancers. However, the BET protein-dependent glioblastoma (GBM)-promoting transcriptional network remains elusive. Here, with mechanistic exploration of a next-generation chemical degrader of BET proteins (dBET6), we reveal a profound and consistent impact of BET proteins on E2F1- dependent transcriptional program in both differentiated GBM cells and brain tumor-initiating cells. dBET6 treatment drastically reduces BET protein genomic occupancy, RNA-Pol2 activity, and permissive chromatin marks. Subsequently, dBET6 represses the proliferation, self-renewal, and tumorigenic ability of GBM cells. Moreover, dBET6-induced degradation of BET proteins exerts superior antiproliferation effects compared to conventional BBIs and overcomes both intrinsic and acquired resistance to BBIs in GBM cells. Our study reveals crucial functions of BET proteins and provides the rationale and therapeutic merits of targeted degradation of BET proteins in GBM.

Keywords: BRD2; BRD3; BRD4; E2F; glioma.

Fig. 1.

dBET6 induces efficient degradation of BET proteins and inhibits the proliferation of GBM cells. (A) Chemical structures of JQ1, dBET1, thalidomide, and dBET6. dBET6 was developed based on dBET1 by extending the linker. (B) dBET6 selectively depleted BET proteins. U87 GBM cells were treated with eqimolar concentrations (0.5 µM) of thalidomide, BBIs, and dBET6 for 24 h. (C) dBET6 depleted BET proteins within 2 h after treatment. (D) siRNA-mediated silencing of CRBN abolished dBET6-induced degradation of BET proteins. U87 cells were transfected with either si-NT or si-CRBN for 72 h before dBET6 treatment (0.5 µM, 2 h). (E) Proteasome inhibitor MG132 restored the expression of BET proteins in dBET6-treated cells. MG132 (10 µM) and dBET6 (0.5 µM) were added into culture medium for 2 h before cell harvest. (F) Effects of BBIs and BET protein degraders (dBET1 and dBET6) on the expression and chromatin loading of BRD2, BRD3, and BRD4. U87 cells were treated with indicated compounds (1 µM, 2 h) before chromatin fractionation. (G) Genome-wide depletion of BET protein occupancy as determined by time-course ChIP-seq of BRD2/3/4 in U87 cells treated with dBET6 (0.5 µM). Each point on the plot represents an individual peak of indicated BET protein. ChIP-seq peak intensity of BRD2/3/4 in mock-treated U87 cells was set as baseline control for comparison. (H) Hierarchical clustering of mean IC₅₀ values of growth inhibition of four BBIs (JQ1, CPI203, OTX015, and I-BET151) and two BET degraders (dBET1 and dBET6) in 11 GBM cell lines. BR163, DA66, LC84, and YK163 were primary GBM explants. IC₅₀ values were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (96-h treatment), repeated independently at least three times.

Fig. 2.

dBET6 exerts superior anti-GBM activities over BBIs. (A) dBET6 showed a stronger inhibitory effect on BrdU incorporation of GBM cells than BBIs; mean ± SD, n = 3. (B) Cell cycle analysis of GBM cells in response to dBET6 and JQ1 treatment; mean ± SD; n = 3. (C) Levels of cell cycle-related proteins in U87 (0.5 µM) and U251 (1 µM) GBM cells after dBET6 treatment (24 h). (D) Short-term dBET6 pretreatment impaired tumor formation of U87 cells in an intracranial model and prolonged nude mice survival postimplantation. U87 cells were treated with indicated concentration of dBET6 for 24 h before stereotaxic implantation. Log-rank test was applied for survival analysis; n = 6 or 8. (E) Effects of DMSO, JQ1, and dBET6 exposure (24 h) on GBM cell tumorigenicity in NOD/SCID gamma mice. Tumor-initiating cell (TIC) frequency was estimated by in vivo limiting tumor-formation assay. P values in E are shown to indicate the pair-wise differences in active cell frequency between groups; χ² likelihood ratio test was applied. *P < 0.05; **P < 0.01; ***P < 0.001. n.s., not significant.

Fig. 3.

dBET6 inhibits the propagation and stemness of patient-derived gliomaspheres. (A) Relative cell viability of patient-derived gliomaspheres treated with the indicated concentrations of dBET6 for 5 d. IC₅₀ values (mean, n = 3) of dBET6 are shown. (B) Levels of cell cycle-related proteins in gliomaspheres after dBET6 treatment (50 nM, 24 h). (C) In vitro limiting-dilution assay showing the effect of dBET6 treatment (14 d) on gliomasphere formation. Sphere-forming cell (SFC) frequency was estimated. P values in C (Lower) were shown to indicate the pair-wise differences in active cell frequency between groups; χ² likelihood ratio test was applied. (D–G) Short-term dBET6 pretreatment impaired tumor formation of NNI-21 cells (D–F) and NNI-31 cells (G) in an intracranial model and prolonged the survival of recipient NOD/SCID gamma mice. GBM propagating cells were treated with indicated concentration of dBET6 for 24 h before stereotaxic implantation. Log-rank test was applied for survival analysis; n = 8. (D) H-score and (E) representative images showing immunohistochemistry staining of Ki67 signals in end point tumors harvested from individual animals in F. (Scale bar, 100 μm.) Data of D represent mean ± SD; n = 3. **P < 0.01; ***P < 0.001. n.s., not significant.

Fig. 4.

dBET6 and BET protein depletion overcome acquired resistance to BBIs in GBM cells. (A) Schematic diagram showing the establishment of JQ1-resistant U87 and U251 GBM cells (designated U87R and U251R, respectively) by chronic exposure to increasing doses of JQ1. (B) Resistance of U87R and U251R to JQ1, OTX015, and I-BET151. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (96-h treatment); mean ± SD, n ≥ 3. (C) Transcriptome responses of parental U87 cells and U87R cells to JQ1 treatment (1 µM, 24 h). JQ1-responsive genes (FDR < 0.1; absolute log₂ fold change > 1.2) were identified based on parental cells. Their amplitudes of differential expression in parental and resistant cells were then compared using paired t test. (D) Responses of U87R, U251R, and their parental cells to shRNA-mediated silencing of BRD2/3/4. Cell viability was determined by MTT assay (96 h postseeding); mean ± SD, n = 3. All sh-BRD2/3/4 groups were significantly different from sh-NT control group (P < 0.001, Student’s t test). (E and F) Effects of dBET6 on cell viability (E) and BrdU incorporation (F) of U87R, U251R, and their parental cells; mean ± SD, n = 3. (G) Principle component (PC) analysis of parental U87 and U87R cells in response to DMSO, JQ1, and dBET6 treatment. After JQ1 withdrawal for 48 h, U87R cells were treated with indicated compounds (1 µM, 24 h). (H) Heat map showing the transcriptome responses of parental U87 cells and U87R cells to dBET6 treatment (1 µM, 24 h). Genes that were sensitive to dBET6 treatment (FDR < 0.1; absolute log₂ fold change > 1.2) were identified in parental cells and used for plotting. *P < 0.05; ***P < 0.001. n.s., not significant.

Fig. 5.

dBET6 impairs RNA-Pol2 activity and BET protein-dependent transcription. (A) Effect of dBET6 treatment on RNA-Pol2 phosphorylation. U87 and U251 GBM cells were incubated with dBET6 (0.5 µM and 1 µM, respectively) for indicated durations. (B) Heat map for the ChIP-seq signal of RNA-Pol2 across the gene body in mock-treated and dBET6-treated U87 cells. The x axis was extended to 2 kb upstream of the TSS and 2 kb downstream of the transcription end site (TES). Color density reflects enrichment of ChIP signal. (C) Metagenes showing average RNA-Pol2 ChIP-seq signals across active RefSeq genes with basal RNA-Pol2 peaks in their promoter/TSS regions. Blue, mock treatment; red, dBET6 treatment. Units are mean tags per 20-bp bin per million reads (RPM) across the transcribed region of each gene with 2-kb upstream and downstream flanking regions. (D) Effect of dBET6 treatment on RNA-Pol2 pausing index. RefSeq genes showing no or moderate reduction of RNA-Pol2 signal in TSS region after dBET6 treatment were included in this analysis. Mann–Whitney U test was applied for statistical comparison of RNA-Pol2 pausing indexes under mock condition (shown in blue) and dBET6 treatment (shown in red). (E) Heat map showing the time-course transcriptomic response of U87 cells to dBET6 treatment (0.5 µM). RNA-seq was performed in triplicate, and data were subjected to model-based clustering analysis by MCLUST. (F) Enrichment analyses of genes with RNA-Pol2, BRD2, BRD3, and BRD4 peaks in their promoter/TSS regions in each cluster. Average frequency of each protein’s occupancy at the promoter/TSS of all clusters was used as reference. Clusters with P values smaller than 0.05 are highlighted in red; diameter of the circle represents the P value of Fisher’s exact test (one-sided).

Fig. 6.

dBET6 inhibits E2F protein-dependent transcriptional networks. (A) Hyperenrichment of down-regulated genes with E2F binding motifs in their promoters [Molecular Signature Database (MSigDB) C3]. U87 and NNI-24 cells were treated with 0.5 µM and 50 nM of dBET6, respectively. (B) Heat maps for the ChIP-seq signals of indicated antibodies ±2 kb from TSS in U87 cells. (C) Enrichment analysis of genes with E2F1 peaks in their promoter/TSS regions in each cluster. Average frequency of E2F1 occupancy at the promoter/TSS of all clusters was used as reference. Clusters with P values smaller than 0.05 are highlighted in red; diameter of the circle represents the P value of Fisher’s exact test (one-sided). (D) GSEA plots of a ChIP-seq–defined E2F1 target gene set in the U87 and NNI-24 GBM cells treated with DMSO versus dBET6. (E) Heat map showing the differential expression of genes within dBET6-responsive gene signature in response to dBET6 treatment in U87 and NNI-24 cells. (F) The dBET6-responsive gene signature stratified glioma molecular subtypes, pathological grades, and patient survival in three clinical glioma databases (TCGA, REMBRANDT, and Gravendeel). Log-rank test was applied to compare the Kaplan–Meier survival curves. Hazard ratio (HR) from the Cox proportional hazards model was reported. (G) Effects of JQ1 and dBET6 treatment (1 µM, 24 h) on BET proteins, phosphorylation of RNA-Pol2, and the expression of mesenchymal master regulators in U87 cells.