Super-Enhancers Promote Transcriptional Dysregulation in Nasopharyngeal Carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southeast Asia and Southern China. The pathogenic mechanisms of NPC, particularly those involving epigenetic dysregulation, remain largely elusive, hampering clinical management of this malignancy. To identify novel druggable targets, we carried out an unbiased high-throughput chemical screening and observed that NPC cells were highly sensitive to inhibitors of cyclin-dependent kinases (CDK), especially THZ1, a covalent inhibitor of CDK7. THZ1 demonstrated pronounced antineoplastic activities both in vitro and in vivo An integrative analysis using both whole-transcriptome sequencing and chromatin immunoprecipitation sequencing pinpointed oncogenic transcriptional amplification mediated by super-enhancers (SE) as a key mechanism underlying the vulnerability of NPC cells to THZ1 treatment. Further characterization of SE-mediated networks identified many novel SE-associated oncogenic transcripts, such as BCAR1, F3, LDLR, TBC1D2, and the long noncoding RNA TP53TG1. These transcripts were highly and specifically expressed in NPC and functionally promoted NPC malignant phenotypes. Moreover, DNA-binding motif analysis within the SE segments suggest that several transcription factors (including ETS2, MAFK, and TEAD1) may help establish and maintain SE activity across the genome. Taken together, our data establish the landscape of SE-associated oncogenic transcriptional network in NPC, which can be exploited for the development of more effective therapeutic regimens for this disease. Cancer Res; 77(23); 6614-26. ©2017 AACR.

Figure 1.

High-throughput chemical screening identifies THZ1 as a potent inhibitor of NPC cells. Heatmap showing the sensitivity of seven NPC lines to 126 small-molecule chemicals in high-throughput screening (A; only partial list of inhibitors shown) and a panel of 14 different CDK inhibitors ranked by IC₅₀ values (B). The entire results of A and B are presented in Supplementary Fig. S1 and Supplementary Tables S1 and S2. C, Cell viability assay measuring the dose response of NPC cell lines to THZ1. Data are presented as mean ± SD of three replicates. D, Immunoblotting analysis quantifying the expression of CDK7 and cleaved caspase-3 (CC3) upon CDK7 knockdown. GAPDH was used as a loading control. E, Cell viability assays showing the effects of THZ1 treatment on HK1 NPC cells at indicated time points and doses. Data are presented as mean ± SD of three replicates. ***, P < 0.001. F, Apoptosis analysis in C666-1 and HK1 cells treated with THZ1. Data are presented as mean ± SD of three replicates. **, P < 0.01; ****, P < 0.0001.

Figure 2.

THZ1: selective Inhibition of RNAPII-mediated transcription. A, Tumor growth curve of mice injected with either THZ1 or vehicle. Data represent mean ± SD of each group. *, P < 0.05; ****, P < 0.0001. B, Hematoxylin and eosin (H&E) and IHC staining of Ki67 and CC3 in tumor tissue sections. Original magnification, ×400. C, Metastasis model. Representative images of mouse lungs after Bouin’s fixation (left) and hematoxylin and eosin staining (right). Arrows, tumor metastases nodule. D, Immunoblot analysis of the levels of RNAPII CTD phosphorylation in C666-1 and HK1 cells treated with either THZ1 or DMSO at indicated concentrations for 12 hours. E, Heatmaps of gene expression in C666-1, HK1, and HNE1 NPC cells treated with THZ1 (200 nmol/L for 6 hours) relative to DMSO group. F, GO enrichment analysis of THZ1-sensitive transcripts in three NPC cell lines.

Figure 3.

Profiling of the SE landscapes in NPC cell lines. A, Hockey stick plots in NPC cells showing input-normalized, rank-ordered H3K27ac signals, highlighting a number of SE-associated genes. B, Heatmaps of ChIP-Seq signal intensity of H3K27ac [±2-kb windows around the center of transcription start site (TSS)] for three NPC cell lines, ordered by their mean signal. C, ChIP-seq profiles of H3K27ac, H3K4me1, H3K4me3, ATAC, and RNA Pol II binding at representative SE (EGFR and MALAT1)- or TE (SKP1)-associated gene loci in C666-1 NPC cells. D, Heatmaps of ChIP-seq profiles of H3K27ac, H3K4me1, H3K4me3, ATAC, and RNA Pol II, ordered by H3K27ac signal (C666-1). E, GO enrichment analysis of SE-associated genes.

Figure 4.

Characterizing the biological features of SE-associated transcripts in NPC. A and B, Box plots showing RNA expression levels (A) of transcripts associated with total pool of enhancers (ALL), TEs, and SEs, as well as their fold changes upon THZ1 treatment (B; 6 hours). C, GSEA of the fold changes of either SE- or TE-associated transcripts following THZ1 treatment. D, Representative ChIP-seq profiles showing that RNA Pol II occupancy across promoters and gene bodies was reduced upon THZ1 treatment. E, mRNA expression levels of JAG2 and F3 across various types of human cancers. Data were retrieved from the dataset E-MTAB-2706, which contains RNA-seq of 675 human cancer cell lines. Arrow, NPC cells.

Figure 5.

Identification of novel SE-associated oncogenes in NPC. A, Outline of the integrative analysis to nominate novel SE-associated oncogenes in NPC. Heatmaps on the right show the expression level of candidate genes following THZ1 treatment and their SE status in each cell line. B, Representative H3K27ac ChIP-seq profiles of two novel candidates, F3 and TBC1D2, in HK1 cells. C, Cell viability and colony formation assays demonstrating that four novel SE-associated candidate oncogenes were essential for cell proliferation and clonogenic growth of HK1 cells. D, Immunoblotting analysis verifying that the protein levels of the four novel SE-associated oncogenes were rapidly decreased by THZ1. E, Immunoblotting showing the protein levels of these four candidates in seven NPC cell lines (left) and representative colony formation results of NPC cells transfected with indicated siRNAs (right). F, Representative IHC photos displaying the expression of four novel oncogenes in primary NPC samples and the adjacent normal tissue. G, Growth-suppressive effect of silencing TP53TG1 in three NPC cell lines as assayed by colony formation (left) and cell viability (right). Data of C, E, and G represent the mean ± SD (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 6.

Characterization of SE-associated master TFs in NPC. A, Strategy of identifying SE-promoting master TFs in NPC cell lines. Following de novo motif analysis of all SE genomic regions, master TFs were (i) associated with SE and (ii) actively expressed in all three NPC cells. B, TF motifs enriched at NPC super-enhancers compared with other enhancers. C, Cell viability and colony formation assays showing ETS2 and TEAD promoted cell proliferation (left) and clonogenic growth (right) in HK1 cells. D, Prominent sensitivity of ETS2 expression to THZ1 (6, 12, and 24 hours) was quantified by qRT-PCR (top) and immunoblotting (bottom). E, H3K27ac ChIP-seq profiles of ETS2 in C666-1, HK1, and HNE1 cells.

Figure 7.

Establishing ETS2 as an SE-associated master TF in NPC. A, ETS2 expression was silenced by two independent shRNAs as validated by immunoblotting (top). Number of NPC colonies decreased after silencing ETS2 (bottom). B, ETS2 knockdown potently inhibited growth of NPC xenografts in NSG mice (top); weight of tumors was calculated at the end of the experiment (bottom). C, Immunoblot analysis measuring ETS2 protein expression in different NPC cell lines (top); silencing ETS2 decreased colony formation of SUNE2 cells (bottom, constitutively high ETS2 level). D, Representative ETS2 IHC expression in NPC and normal adjacent tissue. E, Heatmap of the RNA-seq results following ETS2 silencing. F, GO enrichment analysis of downregulated genes upon ETS2 knockdown. G, GSEA of downregulated genes upon ETS2 silencing. Data of A–C represent mean ± SD (n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).