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, 21 (3), 1021-1034

General Mechanism of JQ1 in Inhibiting Various Types of Cancer

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General Mechanism of JQ1 in Inhibiting Various Types of Cancer

Guojuan Jiang et al. Mol Med Rep.

Abstract

Bromodomain‑containing 4 (BRD4) is a histone modification reader and transcriptional regulator that has been reported to interact with acetylated lysine histone motifs transcription factors (TFs), transcription co‑activators and RNA polymerase II. The selective small molecule inhibitor JQ1, which binds competitively to bromodomains, has been reported to exhibit anti‑proliferative effects in various types of cancer. Previous studies on the mechanism of action of JQ1 mostly focused on a specific tumor type or disease; however, the general mechanism through which JQ1 affects various tumors remains to be determined. In the present study, chromatin immunoprecipitation sequencing data for BRD4 and its expression profiles in six cancer cell lines were integrated and analyzed systematically. The results indicated that BRD4 binds to enhancers with histone H3 acetylated at lysine 27 (H3K27Ac) and mediator complex subunit 1 in a cell type‑specific manner, as well as binds to promoter regions with the oncogenic TFs MYC and E2F1 in a cell type‑common manner. The cell type‑common sites across the six cell types investigated were found to be functionally important for tumorigenesis, whereas the cell type‑specific sites were functionally enriched with the cell identity, all of which were sensitive to JQ1 treatment. Furthermore, a core set of JQ1‑regulated BRD4 binding genes were obtained, which were significantly inhibited by JQ1 in various cancer cell lines and contributed to hallmarks of cancer. These results implied a common mechanism underlying the therapeutic effects of JQ1 and suggested its potential suitability as an anti‑cancer drug targeting BRD4‑mediated transcriptional regulation.

Figures

Figure 1.
Figure 1.
Genome-wide analysis of BRD4 binding characteristics. (A) Correlation analysis of BRD4 and its associated factors, including histone modifications (H3K27Ac and H3K4me3), co-activators (MED1 and MYC/MAX) and RNAPII, in six cell types in the merged BRD4 binding dataset. (B) Heatmap of ChIP-seq signals for BRD4, H3K27Ac, H3K4me3 and RNAPII centered on a ±2.5 kb window of the BRD4 peak summit in the six cell types for seven groups of cell type-specific and cell type-common BRD4 binding sites. (C) Bar plots showing the genomic distribution associated with RefSeq genes of cell type-specific and cell type-common BRD4 binding sites. (D) Gene tracks of the ChIP-seq signals for BRD4, H3K27Ac and RNAPII at the XBP1 gene loci in all six cell types. The enhancer regions unique to the MM.1S cell line are highlighted with a blue shadow, while the promoter regions are highlighted with a yellow shadow.
Figure 2.
Figure 2.
Interaction between BRD4 and TFs in a cell type-specific and cell type-common manner. (A) Motif enrichment of cell type-specific and cell type-common top 2,000 BRD4 binding sites with high binding signal of each sub-group by HOMER software. The color bar represents-log10(P-value) of the motif enrichment level. (B) Bar graphs displaying the fraction of cell type-common and cell type-specific BRD4 binding sites overlapped with TFs in the corresponding cell type, including E2F1 in Ly1 cells, MYC in U-87, MM.1S and H2171 cells, and IRF4 in MM.1S and NFκB-P65 in HUVEC-Cs. (C) Average density profiling of TF binding signals across cell type-common and cell type-specific BRD4 binding sites in each cell line. (D) Bar graphs displaying the genomic distribution associated with RefSeq genes of TF binding sites that overlapped with BRD4 in each cell line. (E) Heatmap of the chromatin immunoprecipitation sequencing signals for BRD4, RNAPII and TFs in each cell line at transcriptionally active promoters. Regions are centered on a ±2.5 kb window around the transcription start sites and are ranked by RNAPII occupancy.
Figure 3.
Figure 3.
BRD4 super-loaded regions/genes determined in each cell line. (A) BRD4 ChIP-seq signal at BRD4 binding regions in each cell type. The regions are ranked by increasing BRD4 binding signals. BRD4 super-loaded regions are indicated in red, while other regions are indicated in grey. Cell type-specific genes are indicated in red and cell type-common genes in blue. (B) Gene traces of ChIP-seq signals for BRD4 and H3K27Ac at MYC and BCL2L1 gene loci in all six cell types. The BRD4 super-loaded regions are depicted with a red line, and the promoter regions are highlighted with a yellow shadow.
Figure 4.
Figure 4.
Functional annotation and effects of JQ1 on cell type-common and cell type-specific BRD4 super-loaded genes. (A) Heatmap of the log2-BRD4 ChIP-seq signal (rpm) at cell type-common and cell type-specific BRD4 super-loaded regions selected using a custom procedure. The numbers in the left panel indicate the BRD4 super-loaded target genes in each group. (B) Boxplots of cell type-specific BRD4 super-loaded target genes that are significantly actively transcribed in each cell line vs. other cell types. (C) Boxplot presenting cell type-common BRD4 super-loaded target genes that are significantly actively transcribed vs. all expressed genes in each cell type. (D) Heatmap displaying functional annotation enrichment of cell type-common and cell type-specific BRD4 super-loaded target genes generated by the DAVID website. The color bar represents -log10(P-value) of the functional enrichment level. Functional items with P-values of <1×10−3 in at least one cell type are included. (E) Boxplots of expression changes in MM.1S-specific and cell type-common BRD4 super-loaded target genes after JQ1 treatment at a concentration of 50 or 500 nM and a duration of 3 or 6 h vs. all expressed genes in MM.1S cells. (F) Boxplots of expression changes among Ly1-specific, cell type-common BRD4 super-loaded target genes and all genes after treatment with 500 nM JQ1 for 6, 12 and 24 h. **P<0.001 and ***P<0.0001 (determined by a two-tailed t-test).
Figure 5.
Figure 5.
Core set of common BRD4-regulated genes concordantly sensitive to JQ1 in various cancer cell types. (A) Heatmap of log2-expression changes in the core set of common BRD4-regulated genes in HUVEC-C, Ly1 and MM.1S cells treated with JQ1 under various conditions. HUVEC-CH UVEC-C common BRD4 super-loaded genes that were significantly regulated [absolute log2(fold change)>0.75 and FDR<0.01] by JQ1 in two of the three cell types were selected. (B) Gene set enrichment analysis plot of the core set of the common BRD4-regulated gene set from gene expression profiling of different types of cancer cell lines treated with JQ1, including multiple myeloma (GSE31365), lymphoma (GSE29449) and neuroblastoma (GSE43392). (C) Functional enrichment analysis (performed with the DAVID online tool) of the core set of common BRD4-regulated genes. A count of >5 and P-value of <0.01 were considered to indicate statistical significance. BRD4, bromodomain containing 4; HUVEC-C, human umbilical vein endothelial cell; FDR, false discovery rate; TNF-α, tumor necrosis factor α; Conc., concentration; GO, Gene Ontology; hsa, Homo sapiens.

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