Abstract
Cancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs1. Recognition of modified histones by ‘reader’ proteins constitutes a key mechanism underlying these processes; therefore, targeting such pathways holds clinical promise, as exemplified by the development of bromodomain and extra-terminal (BET) inhibitors2,3. We recently identified the YEATS domain as an acetyl-lysine-binding module4, but its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralogue AF9, is required for disease maintenance in acute myeloid leukaemia. CRISPR–Cas9-mediated depletion of ENL led to anti-leukaemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies and chromatin-immunoprecipitation followed by sequencing analyses revealed that ENL binds to acetylated histone H3, and co-localizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemia. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced the recruitment of RNA polymerase II to ENL-target genes, leading to the suppression of oncogenic gene expression programs. Notably, disrupting the functionality of ENL further sensitized leukaemia cells to BET inhibitors. Together, our data identify ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in acute myeloid leukaemia, and suggest that displacement of ENL from chromatin may be a promising epigenetic therapy, alone or in combination with BET inhibitors, for aggressive leukaemia.
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Acknowledgements
We thank H. Li, M. Armstrong, C.-W. Chen, M. Yu, H. Chu, K. Tanaka, D. Peng and T. Scott for discussions and technical supports. We are grateful to The Rockefeller University Genomic Resource Center, Flow Cytometry Resource Center, The MD Anderson Science Park Next-Generation Sequencing Facility (CPRIT RP120348), The Sequencing and Microarray Facility and The Flow Cytometry and Cellular Imaging Core Facility (NIH/NCI P30CA016672), The Shanghai Synchrotron Radiation Facility BL17U beamline, and The China National Center for Protein Sciences Beijing for facility supports. The research was supported by funds from NIH (1R01CA204639-01), the Leukaemia and Lymphoma Society (LLS-SCOR 7006-13), and The Rockefeller University to C.D.A.; NIH grants (CA66996 and CA140575) and the Leukaemia and Lymphoma Society to S.A.A.; grants from NIH/NCI (1R01CA204020-01), Cancer Prevention and Research Institute of Texas (RP160237 and RP170285) and Welch Foundation (G1719) to X.S.; grants from the Major State Basic Research Development Program in China (2016YFA0500700 and 2015CB910503), and the Tsinghua University Initiative Scientific Research Program to H.L.; and grants from NIH (R01HG007538 and R01CA193466) to W.L. X.S. is a recipient of a Leukaemia & Lymphoma Society Career Development Award and an R. Lee Clark Fellow and Faculty Scholar of MD Anderson Cancer Center. L.W. is a fellow of the Jane Coffin Childs Memorial Fund. Y.L. is a Tsinghua Advanced Innovation fellow.
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Authors and Affiliations
Contributions
L.W., H.W., Y.L., H.L., C.D.A., S.A.A. and X.S. designed the study, analysed the data and wrote the paper. L.W. and H.W. planned and performed all the molecular, cellular and genomic studies; Y.L. and H.L. performed structural and calorimetric studies; L.W., T.H., M.A.E., A.L.S. and J.E.B. performed mouse xenograft studies; L.W., J.L., Y.X., Y.-H.E.L., L.S. and W.L. performed bioinformatics analysis; J.K.J. and X.W. provided technical assistance; H.L., C.D.A., S.A.A. and X.S. supervised the research.
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Competing interests
C.D.A. is a co-founder of Chroma Therapeutics and Constellation Pharmaceuticals; C.D.A. and X.S. are Scientific Advisory Board members of EpiCypher; S.A.A. is a consultant for Epizyme, Inc.
Additional information
Reviewer Information Nature thanks R. Agami, J. L. Hess, R. Marmorstein and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 Depletion of ENL impairs the growth of AML.
a, Western blot demonstrating the knockdown efficiency of five independent sgRNAs (sg1–sg5) targeting ENL. b, Competition assay that plots the relative percentage of RFP+sgRNA+ cells after transduction of leukaemia cells with indicated sgRNAs. n = 3. c, Western blot demonstrating the knockdown efficiency of five independent sgRNAs targeting AF9. d, Competition assay that plots the relative percentage of RFP+sgRNA+ cells after transduction of leukaemia cells with indicated sgRNAs. n = 3. e, Western blot demonstrating the induction of Cas9 expression after Dox treatment in iCas9-MOLM-13 cells. f, Western blot demonstrating the decrease in ENL protein levels upon Dox treatment in iCas9-MOLM-13 cells. g, Competition assay that plots the relative percentage of RFP+sgRNA+ cells after Dox treatment in iCas9-MOLM-13 cells. n = 3. h, Relative ENL mRNA levels determined by quantitative PCR after reverse transcription (qRT–PCR) in MV4;11 cells transduced with non-targeting (NT) control or ENL shRNAs (shENL-1, shENL-2). i, Representative images (left) and quantification (right) of colonies formed by MV4;11 cells transduced with indicated shRNAs. j, Light microscopy of May-Grünwald/Giemsa-stained MV4;11 leukaemia cells transduced with control or ENL shRNAs. k, FACS analysis of CD11b surface expression after 4 days of Dox-induced Cas9 expression (left) and quantification of CD11b median intensity (right) in iCas9-MOLM-13 cells transduced with indicated sgRNAs and rescue constructs. n = 3. l–o, Negative-selection competition assay that plots the relative percentage of RFP+sgRNA+ cells after Dox treatment in iCas9-U-937 (l), iCas9-K562 (m), iCas9-HeLa (n) and iCas9-U2OS (o) cells. n = 3. p–r, LSK cells were sorted from bone marrow of C57BL/6 mice and transduced with luciferase (shLuc) or Enl shRNA (shEnl). p, Relative Enl mRNA levels determined by qRT–PCR quantification after 3 days of drug selection. q, Relative proliferation of control (shLuc) or Enl-knockout (shEnl) LSKs. n = 4. r, Quantification of colonies formed by LSK cells cultured for 7 days. n = 4. G, granulocyte; GM, colony-forming unit containing granulocyte and macrophage; M, macrophage. All error bars represent mean ± s.d. See Supplementary Fig. 1 for western blot gel source data.
Extended Data Figure 2 ENL is required for AML growth in vivo.
a, Representative flow cytometry plots of donor-derived (human CD45+) peripheral blood cells 27 or 32 days after transplantation. The gate shown includes RFP+sgRNA+ human leukaemia cells. b, Representative flow cytometry plots of bone marrow cells in terminally diseased mice receiving cells transduced with ENL sgRNA. Most outgrowing leukaemia cells were RFP+sgRNA+. c, Western blot of sorted RFP+sgRNA+ leukaemia cells from terminally diseased mice (n = 3) receiving cells transduced with control or ENL sgRNA. See Supplementary Fig. 1 for western blot gel source data.
Extended Data Figure 3 Depletion of ENL deregulates core cellular processes and oncogenic pathways that are required for AML maintenance.
a–f, RNA for RNA-seq experiments was obtained from sorted RFP+sgRNA+ iCas9-MOLM-13 cells after 3 or 5 days of Dox treatment. a, Venn diagram showing the number and overlap of genes for which expression is significantly changed (adjusted P < 0.05) upon expression of indicated sgRNAs as compared to GFP control. b, Dot plots showing a strong correlation of transcriptional changes (log2 fold change over GFP control) caused by two independent sgRNAs targeting ENL. r, correlation coefficient. c, Heat map representation of genes differentially expressed between iCas9-MOLM-13 cells expressing sgRNAs targeting GFP control, ENL or AF9 (fold change > 1.5 and adjusted P < 0.05) after 3 days of Dox induction. d, e, GSEA plots evaluating the changes in monocyte differentiation and leukaemia stem cell gene signatures (d) and the MYC pathways (e) upon ENL depletion. f, Gene Ontology (GO) term analyses of downregulated (ENL-KO-DN, top) or upregulated (ENL-KO-UP, bottom) genes in ENL sgRNA-expressing cells. The top five biological processes that each group of genes were enriched in were shown (details in Supplementary Table 2). Fold enrichment and P values are shown. g, h, RNA for RNA-seq experiments was obtained from MV4;11 transduced with non-targeting (NT) or ENL shRNAs. GSEA plots evaluating the changes in monocyte differentiation and leukaemia stem-cell gene signatures (g) and the oncogenic pathways (h) after ENL knockdown.
Extended Data Figure 4 ENL depletion decreases the occupancies of total Pol II and Pol II S2P on ENL-bound genes.
a, b, Venn diagram showing overlaps of Flag–ENL-occupied genes with those of MLL-AF9 in MOLM-13 (ref. 26) (a) or MLL-AF4 in MV4;11 cells (ref. 27) (b), respectively. c, Venn diagram showing overlaps of Flag–ENL-occupied genes in MOLM13, MV4;11 and HeLa cells. See Supplementary Table 7. d, IPA analysis of ENL-bound genes overlapped among leukaemia cells but not HeLa cells. e, Genomic distribution of Flag–ENL ChIP–seq peaks in MV4;11 cells. The peaks are enriched in the promoter regions (TSS ± 3 kb). P < 1 × 10−300 (binomial test). See Supplementary Table 6. f, Average occupancies of Flag–ENL (blue) and Pol II (black) on Flag–ENL-bound genes in MV4;11 cells along the transcription unit. g, Box plots showing the fold changes (normalized to GFP control) of Pol II occupancy at TSS (TSS −30 bp to TSS +300 bp) or the rest of the gene body on ENL-bound and activated genes upon the expression of ENL sgRNA. The fold changes at both TSS and gene body were significantly lower than 1 (P < 0.0001 by one sample, two-tailed Student’s t-test). h, The genome browser view of Pol II signals in a few of ENL-bound genes (MYC, HLX, SLC1A5) in cells expressing sgRNAs targeting GFP (red) or ENL (blue). TSS is indicated by an arrow. i, Western blot showing comparable cellular levels of Pol II S2P in MOLM-13 cells expressing sgRNAs targeting GFP or ENL. See Supplementary Fig. 1 for gel source data. j, k, Average H3K79me2 (j) and H3K79me3 (k) occupancy on Flag–ENL-bound or non-ENL-bound genes (others) in cells expressing sgRNAs targeting GFP control or ENL.
Extended Data Figure 5 Binding specificity and detail of H3K27ac-bound ENL YEATS complex.
a, Histone peptide microarray (detailed annotations on the left) probed with anti-GST antibody against GST–ENL YEATS domain. H3K9ac, H3K18ac and H3K27ac are highlighted in yellow boxes. b, LIGPLOT diagrams of H3K27ac-ENL YEATS complex, listing interactions between H3 peptide and ENL YEATS. H3 segments (orange) and key residues of ENL YEATS (blue) are depicted in ball-and-stick mode. Grey ball, carbon; blue ball, nitrogen; red ball, oxygen; large cyan sphere, water molecule. Hydrogen bonds are indicated as green dashed lines with bond length shown in ångströms. Hydrophobic contacts are represented by an arc with spokes radiating towards the ligand atoms they contact, and the contacted atoms are shown with spokes radiating back.
Extended Data Figure 6 The YEATS domain is required for the chromatin localization of ENL.
a, Box plots showing H3K9ac (red) and H3K27ac (green) occupancy in ENL-bound or unbound genes (others) in MOLM-13 cells. P < 8.1 × 10−152 (H3K9ac) and P < 2.2 × 10−136 (H3K27ac) by two-tailed unpaired Student’s t-test. b, Venn diagram showing the overlap of Flag–ENL (blue) and H3K27ac ChIP–seq peaks (green) at promoter or enhancer regions. Promoter H3K27ac is defined as H3K27ac peaks at TSS ± 3 kb regions co-occupied with H3K4me3; enhancer H3K27ac is defined as non-promoter H3K27ac peaks co-occupied with H3K4me1. There is a significant overlap between Flag–ENL and H3K27ac ChIP–seq peaks at TSS (P = 5.7 × 10−105, two-way Fisher exact test) but not at enhancer (P = 1.0, two-way Fisher exact test). c, Average genome-wide occupancies of Flag–ENL (blue), H3K9ac (red), H3K27ac (green) at Flag–ENL-bound genes along the transcription unit in MV4;11 cells. See Supplementary Tables 8 and 9. d, Western blot showing the protein levels of ectopically expressed wild-type or mutant Flag–ENL (marked by asterisk) and endogenous ENL (marked by double asterisk). e, The genome browser view of H3K27ac, H3K9ac, Flag–ENL signals in a few of ENL-bound genes (MYC, HLX). TSS is indicated by an arrow. f, Average occupancies of wild-type, F59A or Y78A mutant Flag–ENL on ENL-bound genes along the transcription unit in MV4;11 cells. g, Western blot analysis of co-immunoprecipitation using the M2 anti-Flag antibody in cells expressing Flag–ENL and Myc-tagged DOT1L, AFF4, CDK9 or ELL2 proteins. FL, full-length; ΔN, deletion of amino acids 1–113; ΔC, deletion of amino acids 430–559. h, Western blot analysis of immunoprecipitation using the M2 anti-Flag antibody in cells expressing wild-type or mutant Flag–ENL. Endogenous CDK9 and AFF4 were assessed. i, qPCR analysis of the Pol II ChIP signal in MYC gene in ENL sgRNA-expressing cells rescued by wild-type or mutant (F59A or Y78A) mouse ENL. Error bars represent mean ± s.e.m. *P < 0.5, ***P < 0.001 (two-tailed unpaired Student’s t-test). See Supplementary Fig. 1 for gel source data.
Extended Data Figure 7 The YEATS domain-histone acetylation interaction is required for the role of ENL in leukaemias.
a, GSEA plots evaluating the enrichment of signatures related to stem cells, cell cycle or the MYC pathway in the indicated comparisons. b, Quantification of CD11b median intensity 4 days after Dox induction in iCas9-MOLM-13 cells transduced with indicated sgRNAs and rescue constructs. n = 3. ***P < 0.001 by two-tailed unpaired Student’s t-test. c, Negative-selection competition assay that plots the relative percentage of RFP+sgRNA+ cells after transduction of leukaemia cells with indicated constructs. n = 3. d, Quantification of CD11b median intensity 6 days after Dox induction in iCas9-MOLM-13 cells transduced with indicated sgRNAs and rescue constructs. n = 3. ***P < 0.001 by two-tailed unpaired Student’s t-test. e, Percentage of human CD45+ cells in peripheral blood of mice transplanted with MOLM-13 cells expressing indicated sgRNAs and rescue constructs 30 days after injection (n ≥ 8). ****P < 0.0001 by two-tailed unpaired Student’s t-test. All error bars represent mean ± s.d.
Extended Data Figure 8 Depletion of ENL increases sensitivity to JQ1 by potentiating JQ1-induced transcriptional changes.
a, Effect of JQ1 on the proliferation (normalized to DMSO control) of MOLM-13 cells transduced with indicated sgRNAs targeting SEC components. n = 5. b, Effect of JQ1 on the proliferation of indicated MLL-rearranged leukaemia cells transduced with shNT (red) or shENL (blue) shRNAs. (n = 3). c, d, Effect of JQ1 on the proliferation of indicated non-leukaemia cells (U2OS and HeLa) transduced with GFP, AF9 or ENL sgRNAs. n = 5. e, Kaplan–Meier survival curves of mice (n = 10 per group) transplanted with iCas9-MOLM-13 cells expressing indicated sgRNAs and pretreated with doxycycline for 4 days and JQ1 (or DMSO control) for 2 days ex vivo. P values were calculated using a log-rank test. f–i, RNA for RNA-seq experiments was obtained from sorted RFP+sgRNA+ iCas9-MOLM-13 cells treated with DMSO (marked as ‘0’) or 50 nM JQ1 for 24 h. Row-normalized heat map (f and h) and box plots of relative expression levels (z-scores, g and i) of genes found to be twofold downregulated (f and g) or upregulated (h and i) after JQ1 treatment in ENL sgRNA-expressing cells. All error bars represent mean ± s.e.m.
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1, uncropped scans with size marker indications. (PDF 1248 kb)
Supplementary Tables
This file contains the following Supplementary Tables: Differentially expressed genes, Gene ontology analysis, ChIP-seq peaks, ChIP-seq occupied genes, and lists of shRNA/sgRNA sequences, oligos, antibodies and GSEA gene sets used in this study. (XLSX 29662 kb)
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Wan, L., Wen, H., Li, Y. et al. ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia. Nature 543, 265–269 (2017). https://doi.org/10.1038/nature21687
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DOI: https://doi.org/10.1038/nature21687
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