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, 153 (2), 320-34

Selective Inhibition of Tumor Oncogenes by Disruption of Super-Enhancers

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Selective Inhibition of Tumor Oncogenes by Disruption of Super-Enhancers

Jakob Lovén et al. Cell.

Abstract

Chromatin regulators have become attractive targets for cancer therapy, but it is unclear why inhibition of these ubiquitous regulators should have gene-specific effects in tumor cells. Here, we investigate how inhibition of the widely expressed transcriptional coactivator BRD4 leads to selective inhibition of the MYC oncogene in multiple myeloma (MM). BRD4 and Mediator were found to co-occupy thousands of enhancers associated with active genes. They also co-occupied a small set of exceptionally large super-enhancers associated with genes that feature prominently in MM biology, including the MYC oncogene. Treatment of MM tumor cells with the BET-bromodomain inhibitor JQ1 led to preferential loss of BRD4 at super-enhancers and consequent transcription elongation defects that preferentially impacted genes with super-enhancers, including MYC. Super-enhancers were found at key oncogenic drivers in many other tumor cells. These observations have implications for the discovery of cancer therapeutics directed at components of super-enhancers in diverse tumor types.

Figures

Figure 1
Figure 1. Mediator and BRD4 Co-occupy Promoters of Active Genes in Multiple Myeloma
(A) Gene tracks of MED1, BRD4, H3K27Ac, and H3K4Me3 ChIP-seq occupancy at the enhancer (left) and promoter (right) of SMARCA4 in MM1.S MM cells. The x axis shows genomic position, and enhancer-containing regions are depicted with a white box. The y axis shows signal of ChIP-seq occupancy in units of reads per million mapped reads per base pair (rpm/bp). (B) Metagene representation of global MED1, BRD4, H3K27Ac, and H3K4Me3 occupancy at enhancers and promoters. The x axis shows the ±2.5 kb region flanking either the center of enhancer regions (left) or the TSS of active genes (right). The y axis shows the average background subtracted ChIP-seq signal in units of rpm/bp. (C) Median MED1 and BRD4 levels in the ±1 kb region around the TSSs of actively transcribed genes ranked by increasing RNA Pol II occupancy in MM1.S cells. Levels are in units of rpm/bp, with the left y axis showing levels of MED1 and the right y axis showing levels of BRD4. Promoters were binned (50/bin), and a smoothing function was applied to median levels. See also Figure S1.
Figure 2
Figure 2. Super-Enhancers Identified in Multiple Myeloma
(A) Total MED1 ChIP-seq signal in units of reads per million in enhancer regions for all enhancers in MM1.S. Enhancers are ranked by increasing MED1 ChIP-seq signal. (B) Metagene representation of global MED1 (red line) and BRD4 (blue line) occupancy at typical enhancers and super-enhancers. The x axis shows the start and end of the enhancer (left) or super-enhancer (right) regions flanked by ±5 kb of adjacent sequence. Enhancer and super-enhancer regions on the x axis are relatively scaled. The y axis shows the average signal in units of rpm/bp. (C) Gene tracks of MED1 (top) and BRD4 (bottom) ChIP-seq occupancy at the typical enhancer upstream of TOP1, the super-enhancer downstream of IGLL5, the typical enhancer upstream of SMARCA4, and the super-enhancer overlapping the CCND2 gene TSS. The x axis shows genomic position, and super-enhancer-containing regions are depicted with a gray box. The y axis shows signal of ChIP-seq occupancy in units of rpm/bp. (D) Left: box plots of expression values for genes with proximal typical enhancers (white) or with proximal super-enhancers (pink). The y axis shows expression value in Log2 arbitrary units. Right: box plots of cell-type specificity values for genes with proximal typical enhancers (white) or with proximal super-enhancers (purple). The y axis shows the Z score of the Jensen-Shannon (JS) divergence statistic for genes, with higher values corresponding to a more cell-type-specific pattern of expression. Changes between expression levels are significant (two-tailed Welch's t test, p < 2 × 10−16), as are changes between cell-type-specificity levels (two-tailed Welch's t test, p = 1 × 10−14). (E) Bar graph depicting luciferase activity of reporter constructs containing cloned fragments of typical enhancers and super-enhancers in MM1.S cells. 2 kb fragments of three super-enhancers, IGLL5, DUSP5, and SUB1, and three typical enhancers, PDHX, SERPINB8, and TOP1, ranked 1, 129, 227, 2352, 4203, and 4794, respectively, in terms of MED1 occupancy, were cloned into reporter plasmids downstream of the luciferase gene, driven by a minimal MYC promoter. Luciferase activity is represented as fold over empty vector. Error bars represent SD of triplicate experiments. See also Figure S2 and Data S1.
Figure 3
Figure 3. Super-Enhancers Are Associated with Key Multiple Myeloma Genes
(A and B) Gene tracks of MED1 and BRD4 ChIP-seq occupancy at super-enhancers near genes with important roles in MM biology (A) or genes with important roles in cancer (B). Super-enhancers are depicted in gray boxes over the gene tracks. The x axis shows genomic position, and super-enhancer-containing regions are depicted with a gray box. The y axis shows signal of ChIP-seq occupancy in units of rpm/bp.
Figure 4
Figure 4. Inhibition of BRD4 Leads to Loss of BRD4 Genome Wide
(A) Tracks showing BRD4 ChIP-seq occupancy on the 35 Mb right arm of chromosome 21 after DMSO (top) or 500 nM JQ1 (bottom) treatment. The chromosome 21 ideogram is displayed above the gene tracks with the relevant region highlighted in blue. The x axis of the gene tracks shows genomic position, and the y axis shows BRD4 ChIP-seq signal in units of rpm/bp. (B) Box plot showing the distributions of BRD4 ChIP-seq signal at BRD4-enriched regions after DMSO (left) or 500 nM JQ1 (right) treatment. BRD4-enriched regions were defined in MM1.S cells treated with DMSO. The y axis shows BRD4 ChIP-seq signal in units of rpm/bp. The loss of BRD4 occupancy at BRD4-enriched regions after JQ1 is highly significant (p value < 1 × 10−16, Welch's t test). (C) Gene tracks of BRD4 ChIP-seq occupancy at the enhancer (left) and promoter (right) of SMARCA4 in MM1.S cells after DMSO (top) or 500 nM JQ1 (bottom) treatment for 6 hr. The x axis shows genomic position, and enhancer-containing regions are depicted with a white box. The y axis shows signal of ChIP-seq occupancy in units of rpm/bp. (D) Metagene representation of global BRD4 occupancy at enhancers and promoters after DMSO (solid line) or 500 nM JQ1 (dotted line) treatment. The x axis shows the ±2.5 kb region flanking either the center of enhancer regions (left) or the TSS of active genes. The y axis shows the average background subtracted ChIP-seq signal in units of rpm/bp. (E) Gene tracks of BRD4 binding at super-enhancers after DMSO (top) or 500 nM JQ1 (bottom) treatment. The x axis shows genomic position, and super-enhancer-containing regions are depicted with a gray box. The y axis shows signal of ChIP-seq occupancy in units of rpm/bp.
Figure 5
Figure 5. BRD4 Occupancy at Super-Enhancers Is Highly Sensitive to Bromodomain Inhibition
(A) Schematic example of how cooperative interactions of enhancer-associated factors at super-enhancers lead to both higher transcriptional output and increased sensitivity to factor concentration. (B) Measuring the effects of various concentrations of JQ1 genome wide on BRD4 occupancy. Schematic depicting the experimental procedure. (C) Short-term JQ1 treatment (6 hr) has little effect on MM1.S cell viability. JQ1 sensitivity of MM1.S cells by measurement of ATP levels (CellTiterGlo) after 6, 24, 48, and 72 hr of treatment with JQ1 (5, 50, 500, or 5,000 nM) or vehicle (DMSO, 0.05%). Error bars represent the SD of triplicate experiments. (D) Western blot of relative MYC levels after 6 hr of JQ1 or DMSO treatment. (E) Western blot of relative BRD4 levels after 6 hr of JQ1 or DMSO treatment. ChIP-western blot of the relative levels of immunoprecipitated BRD4 after 6 hr of JQ1 or DMSO treatment. (F) Line graph showing the percentage of BRD4 occupancy remaining after 6 hr treatment at various JQ1 concentrations for typical enhancers (gray line) or super-enhancers (red line). The y axis shows the fraction of BRD4 occupancy remaining versus DMSO. The x axis shows different JQ1 concentrations (DMSO [none], 5 nM, 50 nM, and 500 nM). Error bars represent 95% confidence intervals of the mean (95% CI). (G) Gene tracks of BRD4 ChIP-seq occupancy after various concentrations of JQ1 treatment at the IgH-MYC-associated super-enhancer (left) and the SMARCA4-associated typical enhancer (right). The x axis shows genomic position, and gray boxes depict super-enhancer regions. The y axis shows signal of ChIP-seq occupancy in units of rpm/bp. The percent of BRD4 remaining after each concentration of JQ1 treatment is annotated to the right of the gene tracks.
Figure 6
Figure 6. JQ1 Causes Disproportionate Loss of Transcription at Super-Enhancer Genes
(A) Box plots showing the Log2 change in gene expression for all actively transcribed genes in JQ1-treated versus control cells for a time course of cells treated with 500 nM JQ1 (left) or for a concentration course of cells treated for 6 hr with varying amounts of JQ1 (right). The y axis shows the Log2 change in gene expression versus untreated control cells (left graph) or control cells treated with DMSO for 6 hr (right graph). (B and C) Line graph showing the Log2 change in gene expression versus control cells after JQ1 treatment in a time- (B) or dose (C)-dependent manner for genes associated with typical enhancers (gray line) or genes associated with super-enhancers (red line). The y axis shows the Log2 change in gene expression of JQ1 treated versus untreated control cells. The x axis shows time of 500 nM JQ1 treatment (B) or JQ1 treatment concentration at 6 hr (C). Error bars represent 95% confidence intervals of the mean (95% CI). (D) Graph showing the Log2 change in gene expression after JQ1 treatment over time for genes ranked in the top 10% of expression in MM1.S cells. Each line represents a single gene, with the MYC and IRF4 genes drawn in red. The y axis shows the Log2 change in gene expression of JQ1-treated versus untreated control cells. The x axis shows time of 500 nM JQ1 treatment. (E) Line graph showing luciferase activity after JQ1 treatment at various concentrations for luciferase reporter constructs containing either a fragment from the IGLL5 super-enhancer (red line) or the PDHX typical enhancer (gray line). The y axis represents relative luciferase activity in arbitrary units. The x axis shows JQ1 concentrations. Error bars are SEM. (F) Bar graphs showing the percentage loss of either MED1 (top, red) or CDK9 (bottom, green) at promoters, typical enhancers, and super-enhancers. Error bars represent 95% CI. (G) Graph of loss of RNA Pol II density in the elongating gene body region for all transcriptionally active genes in MM1.S cells after 6 hr of 500 nM JQ1 treatment. Genes are ordered by decrease in elongating RNA Pol II in units of Log2 fold loss. Genes with a greater than 0.5 Log2 fold change in elongating RNA Pol II are shaded in green (loss) or red (gain). The amount of RNA Pol II loss is indicated for select genes. (H) Bar graph showing the Log2 fold change in RNA Pol II density in elongating gene body regions after 6 hr of 500 nM JQ1 treatment for genes with typical enhancers (left, gray) or genes with super-enhancers (red, right). Error bars represent 95% confidence intervals of the mean (95% CI). (I and J) Gene tracks of RNA Pol II ChIP-seq occupancy after DMSO (black) or 500 nM JQ1 treatment (red) at the super-enhancer proximal MYC gene (I) and IRF4 gene (J). The y axis shows signal of ChIP-seq occupancy in units of rpm/bp. See also Figure S3.
Figure 7
Figure 7. Super-Enhancers Are Associated with Key Genes in Other Cancers
(A and D) Total MED1 ChIP-seq signal in units of reads per million in enhancer regions for all enhancers in (A) the GBM cell line U-87 MG or (D) the SCLC cell line H2171. Enhancers are ranked by increasing MED1 ChIP-seq signal. (B and E) Metagene representation of global MED1 and BRD4 occupancy at (B) typical GMB enhancers and super-enhancers or (E) typical SCLC enhancers and super-enhancers. The x axis shows the start and end of the enhancer (left) or super-enhancer (right) regions flanked by ±5 kb of adjacent sequence. Enhancer and super-enhancer regions on the x axis are relatively scaled. The y axis shows the average signal in units of rpm/bp. (C and F) Gene tracks of MED1 and BRD4 ChIP-seq occupancy at (C) super-enhancers near BHLHE40 and BCL3, genes with important roles in GBM, or at (F) super-enhancers near INSM1 and ID2, genes with important roles in SCLC. Super-enhancers are depicted in gray boxes over the gene tracks. See also Figure S4.

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