Super-enhancers define a proliferative PGC-1α-expressing melanoma subgroup sensitive to BET inhibition

Abstract

Metabolic changes are linked to epigenetic reprogramming and play important roles in several tumor types. PGC-1α is a transcriptional coactivator controlling mitochondrial biogenesis and is linked to oxidative phosphorylation. We provide evidence that melanoma models with elevated PGC-1α levels are characteristic of the proliferative phenotype and are sensitive to bromodomain and extra-terminal domain (BET) inhibitor treatment. A super-enhancer region highly occupied by the BET family member BRD4 was identified for the PGC-1α gene. BET inhibitor treatment prevented this interaction, leading to a dramatic reduction of PGC-1α expression. Accordingly, BET inhibition diminished respiration and mitochondrial function in cells. In vivo, melanoma models with high PGC-1α expression strongly responded to BET inhibition by reduction of PGC-1α and impaired tumor growth. Altogether, our findings identify epigenetic regulatory elements that define a subset of melanomas with high sensitivity to BET inhibition, which opens up the opportunity to define melanoma patients most likely to respond to this treatment, depending on their tumor characteristics.

Figure 1

Differential in vitro impact of BET inhibitors on melanoma models. (a) Chemical structure of BAY 1238097. (b) Color-coded ranking of GI₅₀ values of BAY 1238097 and JQ1. (c) Cell cycle distribution following JQ1 treatment for 24 h. (d) Comparison of proliferative and invasive transcriptional signatures of CHL-1 and COLO-792 cells. Data are from GSE36133. Whiskers denote min to max. (e) Enrichment plot of proliferative signatures comparing the phenotypes of DMSO-treated control (n=4) and BAY 1238097-treated (n=4) CHL-1 cells. (f) Heatmap of differential gene expression following BAY 1238097 GI₉₀ treatment of CHL-1 cells for 4 h. (g) Comparison of PGC-1α gene expression between sensitive (n=15) and insensitive cells (n=5) classified according to the data shown in b. Levels were normalized to expression in normal human epidermal melanocytes. Whiskers denote min to max. FDR, false discovery rate; NES, normalized enrichment score.

Figure 2

BRD4-bound super-enhancers define gene regulatory regions of PGC-1α and SOX10 phenotype master regulators. (a) Dot plot showing putative enhancers ranked by normalized BRD4 signal (BRD4 signal—input signal in r.p.m./bp). (b) Venn diagram showing the overlap of downregulated, super-enhancer and proliferative signature genes. (c) ChIP-seq tracks of H3K27ac and BRD4 of DMSO- or BET inhibitor-treated CHL-1 cells (4 h, 1 μM) at the PGC-1α super-enhancer. Enrichment of signal is presented as log likelihood ratio (logLR) over background signal. (d) H3K27ac ChIP-seq tracks of proliferative or invasive primary melanoma cells at the PGC-1α super-enhancer (data from GSE60666).

Figure 3

BET inhibitor-dependent downregulation of PGC-1α expression affects mitochondrial function, oxygen consumption rate and proliferation of OXPHOS melanoma cells. (a) Time-dependent response of PGC-1α gene expression to BET inhibition. After normalization to housekeeping genes, the control DMSO treatment group of each cell line was set to a value of 1. Gene expression at each time point was normalized to the control. Data are shown as the mean+s.e.m. of three replicates. (b) Time-dependent reduction of PGC-1α, as determined by western blot analysis. Neither protein was detectable at any time point in SK-MEL-2 or in RPMI-7951 cells, despite long exposure times (data not shown). Results are representative of triplicate experiments. (c) Overexpression rescue experiments. Transfected CHL-1 cells were treated with JQ1 for 24 h and subjected to 10 μM EdU 4–6 h prior to staining. The EdU-positive cell population was analyzed by flow cytometry. Representative results from a single experiment are shown as well as the mean values obtained in three independent experiments. *P<0.05% in t-test. Numerical results were normalized to the DMSO-treated control sample. (d) Determination of OCR and ECAR (an indirect measure of glycolysis) using a Seahorse analyzer to visualize the energy phenotype profile following BET inhibitor treatment (1 μM for 20 h). Data are represented as mean±s.d. of three separate experiments, each measured six times. (e) Determination of OCR using a Seahorse analyzer following consecutive injections of oligomycin (1 μM), FCCP (0.5 μM) and antimycin A (1 μM)/rotenone (1 μM). The plots are representative of two independent experiments, each measured six times. (f) Microscopy analysis and quantification of mitochondrial function. Red signal indicates Mitotracker Deep Red dye staining of functional mitochondria, while nuclei (blue) are visualized following DAPI staining. Images shown are representative of two independent replicates. The scale bar indicates 20 μm. Data are represented as the mean Mitotracker signal compared to DMSO-treated cells, +s.e.m. ECAR, extracellular acidification rate; OCR, oxygen consumption rate.

Figure 4

PDX and syngeneic mouse melanoma models exhibit sensitivity to BAY 1238097 related to downregulation of PGC-1α expression. (a) Mean tumor volume (±s.e.m.) of PDX model MEXF 1792 during treatment with vehicle or BAY 1238097 (7.5 mg/kg p.o.). Vehicle-treated mice with high tumor burden were killed before the final day of the study, as shown by the ‘n’ value over the vehicle group curve. (b) PGC-1α gene expression in MEXF 1792 samples from a, reported as the mean+s.e.m. relative to the vehicle control group (treated samples n=9, control samples n=7). Tumor samples were harvested 6 h post treatment on day 56. (c) B16F10 mean tumor volume (±s.e.m.) during treatment with vehicle or BAY 1238097 (15 mg/kg p.o.). (d) Dose-dependent reduction of PGC-1α gene expression in BAY 1238097-treated B16F10 tumors, 3 h post treatment. Gene expression results are shown relative to the vehicle-treated control group as the mean+s.e.m. of three tumor samples per dose.

Figure 5

Model of BRD4-bound super-enhancer defining a ‘PGC-1α high’ subgroup of proliferative melanoma cells. PGC-1α suppresses the metastatic character of melanoma cells (PGC-1α high vs PGC-1α low), while promoting mitochondria biogenesis and protecting from oxidative stress. Pharmacological blockade of PGC-1α expression by BET inhibition leads to downstream loss of mitochondrial function and reduced OXPHOS, ultimately resulting in impaired cell proliferation. This does not induce WNT, TGF-β and integrin pathway activity, which could otherwise promote a switch from a proliferative to an invasive phenotype.