Abstract
Whole-genome analysis approaches are identifying recurrent cancer-associated somatic alterations in noncoding DNA regions. We combined somatic copy number analysis of 12 tumor types with tissue-specific epigenetic profiling to identify significant regions of focal amplification harboring super-enhancers. Copy number gains of noncoding regions harboring super-enhancers near KLF5, USP12, PARD6B and MYC are associated with overexpression of these cancer-related genes. We show that two distinct focal amplifications of super-enhancers 3′ to MYC in lung adenocarcinoma (MYC-LASE) and endometrial carcinoma (MYC-ECSE) are physically associated with the MYC promoter and correlate with MYC overexpression. CRISPR/Cas9-mediated repression or deletion of a constituent enhancer within the MYC-LASE region led to significant reductions in the expression of MYC and its target genes and to the impairment of anchorage-independent and clonogenic growth, consistent with an oncogenic function. Our results suggest that genomic amplification of super-enhancers represents a common mechanism to activate cancer driver genes in multiple cancer types.
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Acknowledgements
We thank G. Ha and other members of the Meyerson laboratory for discussions. We acknowledge support from US Department of Defense grant W81XWH-12-1-0269 (M.M.), National Cancer Institute grant 1R35CA197568 (M.M.) and the American Cancer Society Research Professorship (M.M.). X.Z. is supported by the Lung Cancer Research Foundation and the American Association for Cancer Research–John and Elizabeth Leonard Family Foundation Basic Cancer Research Fellowship. P.S.C. is supported by a fellowship from the International Association for the Study of Lung Cancer and National Cancer Institute grant 1F32CA180662.
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X.Z., P.S.C., J.M.F. and M.M. designed the research and wrote the manuscript with input from the other authors. X.Z., P.S.C., J.M.F. and H.W. conducted the biological assays, and X.Z., P.S.C., J.M.F., M.I. and A.D.C. conducted the computational analysis.
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M.M. and A.D.C. have received a commercial research grant from Bayer.
Integrated supplementary information
Supplementary Figure 1 The expression level of genes in tumors with somatic focal amplification of each super-enhancer versus tumors without this focal amplification.
Genes that are expressed and closest to each amplification were analyzed. Box plot: middle bar, median; lower and upper box limits, 25th and 75th percentiles, respectively; whiskers, min and max values. The P value is derived from a t test: **P ≤ 0.01, ***P ≤ 0.001.
Supplementary Figure 2 GISTIC peak identified on chromosome 13q from esophageal carcinomas.
The H3K27ac ChIP-seq profile from esophageal cells is presented. Super-enhancers are also indicated. Expression level of KLF5 in tumors with and without amplified KLF5-ESSE (KLF5 esophageal carcinoma super-enhancer). The P value is derived from a t test.
Supplementary Figure 3 A focally amplified region (MYC-LASE) in lung adenocarcinomas is part of a super-enhancer.
(a) Copy number amplification of the noncoding region ~450 kb 3′ to MYC in lung adenocarcinoma primary tumors from TCGA (n = 11; sample IDs are listed in Supplementary Table 3) and (b) lung adenocarcinoma cell lines (NCI-H2009, HCC78, HOP92 and NCI-H358). Red lines represent relative focal amplification regions identified from each sample. The focal amplification peak called by GISTIC is highlighted. (c) Whole-genome sequencing rearrangement analysis of two lung adenocarcinoma tumors (sample IDs in Supplementary Table 3) identify tandem duplications, indicated by the red curves, as detected by the JaBbA structural rearrangement algorithm (Imielinski et al., in preparation). ChIP-seq profile of H3K27ac and super-enhancer (SE) regions in (d) A549, NCI-H358, NCI-H2009 and (e) HCC95 and NCI-H2171 cells. Thin bars above the ChIP-seq signal represent super-enhancers that are called by the ROSE pipeline. LUAD, lung adenocarcinomas; SqCC, squamous cell lung carcinomas; Small cell, small cell lung cancer.
Supplementary Figure 5 Cancer type–specific enhancer activity of the super-enhancer region.
(a) ChIP-seq profile of H3K27ac at the MYC locus. No enrichment of H3K27ac is detectable at the MYC-LASE region for HEK293 cells. (b) Luciferase reporter assay (n = 3) of the five constituent enhancers, e1–e5, in A549 lung adenocarcinoma cells and HEK293 cells. The P value is derived from a t test: **P ≤ 0.01, ***P ≤ 0.001.
Supplementary Figure 6 siRNA silencing of NFE2L2 and CEBPB in A549 cells.
The P value is derived from a t test (n = 3): **P ≤ 0.01, ***P ≤ 0.001.
Supplementary Figure 7 RNA-seq results in NCI-H2009 cells with and without KRAB-dCas9–mediated e3 enhancer repression.
Blue dots represent genes that are significantly differentially expressed (>25%) after e3 enhancer repression. Negative-control (NC) includes sg-Empty and sg-Control, while KRAB includes sg-e3KRAB 1 and sg-e3KRAB 2. RPKM mean indicates the mean expression levels (RPKM) normalized to the negative-control samples.
Supplementary Figure 8 KRAB-dCas9 fusion–mediated repression of the e3 enhancer in NCI-H2009 cells.
KRAB-dCas9 fusion–mediated repression of the e3 enhancer in NCI-H2009 cells leads to a significant reduction in cellular transformation efficiency as measured by anchorage-independent growth assay (a) and cellular proliferation rate as measured by clonogenic growth assay (b). Representative images are shown.
Supplementary Figure 9 CRISPR/Cas9-mediated deletion of the e3 enhancer in NCI-H2009 cells.
CRISPR/Cas9-mediated deletion of the e3 enhancer in NCI-H2009 cells leads to a significant reduction in cellular transformation efficiency as measured by anchorage-independent growth assay (a) and cellular proliferation rate as measured by clonogenic growth assay (b). Representative images are shown.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9. (PDF 1004 kb)
Supplementary Table 1: Pan-cancer copy number alteration analysis.
The number of noncoding focal amplification peaks identified and H3K27ac ChIP-seq data availability are highlighted in each tumor type. (XLSX 69 kb)
Supplementary Table 2: Accession numbers for public data sets used in the study.
A list of accession numbers for ENCODE data, Roadmap project data and other public data sets that were used in the study. (XLSX 44 kb)
Supplementary Table 3: Primers used in the study.
A list of PCR primers and CRISPR sgRNA sequences that were used in the study. (XLSX 13 kb)
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Zhang, X., Choi, P., Francis, J. et al. Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers. Nat Genet 48, 176–182 (2016). https://doi.org/10.1038/ng.3470
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DOI: https://doi.org/10.1038/ng.3470
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