ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia

Journal name:
Nature
Volume:
543,
Pages:
265–269
Date published:
DOI:
doi:10.1038/nature21687
Received
Accepted
Published online

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.

At a glance

Figures

  1. AML growth is sensitive to ENL depletion in vitro and in vivo.
    Figure 1: AML growth is sensitive to ENL depletion in vitro and in vivo.

    a, Negative-selection competition assay that plots the relative percentage of RFP+sgRNA+ cells over time after transduction of different MLL-rearranged leukaemia cell lines with indicated sgRNAs targeting GFP control (GFP-sg), ENL (ENL-sg1, ENL-sg5) or AF9 (AF9-sg1, AF9-sg4). n = 3. b, Representative images (left) and quantification (right) of colonies formed by MOLM-13 cells transduced with indicated sgRNAs. n = 4. c, FACS analysis of CD11b surface expression after 4 days of Dox treatment (left) and quantification of CD11b median intensity (right). n = 4. d, Top, comparison of mouse and human ENL sequences at the indicated sgRNA recognition sites. Red nucleotides indicate mismatches. PAM, protospacer-adjacent motif. Bottom, negative-selection competition assay that plots the relative percentage of RFP+sgRNA+ cells after transduction of leukaemia cells with indicated constructs. n = 3. e, Percentage of human CD45+ cells in the peripheral blood of mice receiving MOLM-13 cells transduced with indicated sgRNAs at day 27 (n = 4) or 32 (n = 10) after injection. f, Kaplan–Meier survival curves of recipient mice (n = 10 per group) transplanted with MOLM-13 cells expressing indicated sgRNAs. P < 0.0001 using a log-rank test. All error bars represent mean ± s.d. and statistical significance was calculated using two-tailed unpaired Student’s t-test unless noted otherwise. **P < 0.01, ***P < 0.001, ****P < 0.0001.

  2. ENL modulates the recruitment of Pol II to activate oncogenic gene expression.
    Figure 2: ENL modulates the recruitment of Pol II to activate oncogenic gene expression.

    a, Heat map representation of genes differentially expressed in iCas9-MOLM-13 cells expressing sgRNAs targeting GFP control, ENL or AF9 (fold change > 1.5 and adjusted P < 0.05) 5 days after Dox treatment. Red and green indicate relative high and low expression, respectively. See Supplementary Table 1. b, GSEA plots evaluating the changes in monocyte differentiation (top) and leukaemia stem-cell (LSC) gene signatures (bottom) upon ENL depletion. FDR, false discovery rate; NES, normalized enrichment score. c, Genomic distribution of Flag–ENL ChIP–seq peaks in MOLM-13 cells. The peaks are enriched in the promoter regions (TSS ± 3 kb). P < 1 × 10−300 (binomial test). See Supplementary Table 3. d, Average genome-wide occupancies of Flag–ENL (blue) and Pol II (black) on Flag–ENL-bound genes along the transcription unit. The gene body length is aligned by percentage from the TSS to transcription termination site (TTS). 5 kb upstream of TSS and 5 kb downstream of TTS are also included. eg, Average occupancy of Pol II (e), Pol II S2P (f) or CDK9 (g) on Flag–ENL-bound or non-ENL bound genes (others) in iCas9-MOLM-13 cells expressing sgRNAs targeting GFP control or ENL. The gene body length is aligned as in d. RPM, reads per million.

  3. ENL binds and colocalizes with acetylated histone H3 genome-wide via its YEATS domain.
    Figure 3: ENL binds and colocalizes with acetylated histone H3 genome-wide via its YEATS domain.

    a, Peptide pull-down assay of indicated histone peptides and ENL YEATS domain. b, ITC titration and fitting curves of human ENL YEATS domain titrated with H3(17–28)K27ac, H3(1–10)K9ac, H3(1–25)K18ac or unmodified H3(1–34) peptides. c, Overall structure of the ENL YEATS domain bound to H3K27ac peptide. ENL YEATS is depicted as blue ribbons with key residues highlighted by green sticks. Histone H3K27ac peptide is shown as a yellow ribbon, with side chains highlighted by sticks. Red dashes, hydrogen bonds; red sphere, water molecule. d, Electrostatic potential surface of the ENL YEATS domain ranging from −10 to 10 kT e−1. Histone H3 peptide is depicted as space-filling spheres. e, Top, hydrogen bonding network between H3K27ac peptide and ENL. Hydrogen bonds are shown as red dashes. Key residues of ENL are depicted as green sticks and labelled black; the H3 peptide is shown as yellow sticks and labelled red. Grey meshes, Fo − Fc omit map countered at 2.0σ level. Bottom, sequence alignment of histone H3 sequences flanking residues K9, K18 and K27. Conserved residues are highlighted in pink. f, ITC titration fitting curves of ENL YEATS mutants with H3(17–28)K27ac peptide. g, Peptide pull-downs of ENL YEATS mutants and indicated histone H3 peptides. See Supplementary Fig. 1 for gel source data. h, Venn diagram showing the overlap of Flag–ENL-, H3K9ac- and H3K27ac ChIP–seq peaks in MOLM-13 cells. P < 1 × 10−300 (three-way Fisher’s exact test). See Supplementary Tables 4 and 5. i, Average occupancies of Flag–ENL (blue), H3K9ac (red), H3K27ac (green) on ENL-bound genes along the transcription unit. j, Average genome-wide occupancies of wild-type (blue) and mutant ENL (F59A in cyan or Y78A in purple) on ENL-bound genes along the transcription unit.

  4. Disrupting the YEATS-histone acetylation interaction inhibits the functionality of ENL and sensitizes leukaemia cells to BET inhibitors.
    Figure 4: Disrupting the YEATS-histone acetylation interaction inhibits the functionality of ENL and sensitizes leukaemia cells to BET inhibitors.

    a, Heat map representation of genes differentially expressed in iCas9-MOLM-13 cells expressing sgRNAs targeting luciferase control or ENL (fold change > 1.5 and adjusted P < 0.05) in the indicated rescue conditions. b, GSEA plots evaluating the changes in monocyte differentiation (top) and leukaemia stem-cell gene signatures (bottom) in the indicated comparisons. 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, FACS analysis of CD11b surface expression in iCas9-MOLM-13 cells expressing ENL sgRNA and indicated mouse Enl rescue constructs after 4 days of Dox treatment. See Extended Data Fig. 7b for quantification. n = 3. e, Kaplan–Meier survival curves of mice (n = 10 per group) transplanted with MOLM-13 cells transduced with indicated sgRNAs and rescue constructs. P < 0.0001 using a log-rank test. f, Effect of JQ1 on the proliferation (normalized to DMSO control) of MOLM-13 cells transduced with indicated sgRNAs and rescue constructs. n = 5. g, h, Leukaemia burden (g) and survival curves (h) of mice transplanted with iCas9-MOLM-13 cells expressing indicated sgRNAs. Treatment with JQ1 (or vehicle control) and doxycycline was initiated at day 10 after transplantation. g, Percentage of human CD45+ cells in the peripheral blood of mice (n = 5) at 40 days after transplantation. P < 0.01 (ENL sgRNA plus JQ1 versus all other groups) using Mann–Whitney test. h, Kaplan–Meier survival curves of JQ1-treated mice and vehicle controls (n = 10 per group). P < 0.0001 (ENL sgRNA plus JQ1 versus all other groups) using a log-rank test. i, j, RNA for RNA-seq experiments was obtained from sorted RFP+sgRNA+ iCas9-MOLM-13 cells treated with DMSO or 50 nM JQ1 for 24 h. Genes found to be more than twofold downregulated (i) or upregulated (j) after JQ1 treatment in ENL sgRNA-expressing cells were examined. Left, box plots comparing the JQ1-induced fold changes of these genes in either control (red) or ENL sgRNA-expressing (blue) cells. Error bars indicate minimum and maximum, lines denote median, and top and bottom of boxes denote first and third quartile, respectively. P = 2.29 × 10−7 (i) and P = 2.44 × 10−7 (j) by two-tailed paired Student’s t-test. Right, pie charts showing the categorization of these genes based on the relationship to ENL depletion. Genes in which the absolute JQ1-induced fold change was more than 1.2-fold higher or lower in ENL sgRNA-expressing cells compared to control were classified as enhanced (blue) or decreased (green) by ENL sgRNA, respectively. There are significantly more genes in the enhanced than the decreased by ENL sgRNA group (P < 0.0001 by Fisher’s exact test). See Supplementary Table 10. All error bars represent mean ± s.d. (n = 3) unless noted otherwise.

  5. Depletion of ENL impairs the growth of AML.
    Extended Data Fig. 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. lo, 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. pr, 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.

  6. ENL is required for AML growth in vivo.
    Extended Data Fig. 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.

  7. Depletion of ENL deregulates core cellular processes and oncogenic pathways that are required for AML maintenance.
    Extended Data Fig. 3: Depletion of ENL deregulates core cellular processes and oncogenic pathways that are required for AML maintenance.

    af, 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.

  8. ENL depletion decreases the occupancies of total Pol II and Pol II S2P on ENL-bound genes.
    Extended Data Fig. 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.

  9. Binding specificity and detail of H3K27ac-bound ENL YEATS complex.
    Extended Data Fig. 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.

  10. The YEATS domain is required for the chromatin localization of ENL.
    Extended Data Fig. 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.

  11. The YEATS domain-histone acetylation interaction is required for the role of ENL in leukaemias.
    Extended Data Fig. 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.

  12. Depletion of ENL increases sensitivity to JQ1 by potentiating JQ1-induced transcriptional changes.
    Extended Data Fig. 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. fi, 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.

Tables

  1. Data collection and refinement statistics
    Extended Data Table 1: Data collection and refinement statistics

Accession codes

Primary accessions

Gene Expression Omnibus

Protein Data Bank

References

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Author information

  1. Present addresses: Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA (A.L.S., J.E.B.).

    • Amanda L. Souza &
    • James E. Bradner
  2. These authors contributed equally to this work.

    • Liling Wan,
    • Hong Wen &
    • Yuanyuan Li
  3. These authors jointly supervised this work.

    • Haitao Li,
    • C. David Allis,
    • Scott A. Armstrong &
    • Xiaobing Shi

Affiliations

  1. Laboratory of Chromatin Biology & Epigenetics, The Rockefeller University, New York, New York 10065, USA

    • Liling Wan,
    • Julia K. Joseph &
    • C. David Allis
  2. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Liling Wan,
    • Takayuki Hoshii &
    • Scott A. Armstrong
  3. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02215, USA

    • Liling Wan,
    • Takayuki Hoshii &
    • Scott A. Armstrong
  4. Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    • Hong Wen,
    • Xiaolu Wang &
    • Xiaobing Shi
  5. Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    • Hong Wen &
    • Xiaobing Shi
  6. Beijing Advanced Innovation Center for Structural Biology, MOE Key Laboratory of Protein Sciences, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China

    • Yuanyuan Li &
    • Haitao Li
  7. Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing 100084, China

    • Yuanyuan Li &
    • Haitao Li
  8. Dan L. Duncan Cancer Center, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA

    • Jie Lyu,
    • Yuanxin Xi &
    • Wei Li
  9. Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA

    • Yong-Hwee E. Loh &
    • Li Shen
  10. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 0221, USA

    • Michael A. Erb,
    • Amanda L. Souza &
    • James E. Bradner
  11. Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA

    • James E. Bradner
  12. Genes and Development and Epigenetics & Molecular Carcinogenesis Graduate Programs, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas 77030, USA

    • Xiaobing Shi

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.

Competing financial 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.

Corresponding authors

Correspondence to:

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.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Depletion of ENL impairs the growth of AML. (267 KB)

    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. lo, 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. pr, 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.

  2. Extended Data Figure 2: ENL is required for AML growth in vivo. (158 KB)

    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.

  3. Extended Data Figure 3: Depletion of ENL deregulates core cellular processes and oncogenic pathways that are required for AML maintenance. (281 KB)

    af, 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.

  4. Extended Data Figure 4: ENL depletion decreases the occupancies of total Pol II and Pol II S2P on ENL-bound genes. (250 KB)

    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.

  5. Extended Data Figure 5: Binding specificity and detail of H3K27ac-bound ENL YEATS complex. (142 KB)

    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.

  6. Extended Data Figure 6: The YEATS domain is required for the chromatin localization of ENL. (144 KB)

    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.

  7. Extended Data Figure 7: The YEATS domain-histone acetylation interaction is required for the role of ENL in leukaemias. (245 KB)

    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.

  8. Extended Data Figure 8: Depletion of ENL increases sensitivity to JQ1 by potentiating JQ1-induced transcriptional changes. (222 KB)

    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. fi, 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.

Extended Data Tables

  1. Extended Data Table 1: Data collection and refinement statistics (177 KB)

Supplementary information

PDF files

  1. Supplementary Information (1.2 MB)

    This file contains Supplementary Figure 1, uncropped scans with size marker indications.

Excel files

  1. Supplementary Tables (28.9 MB)

    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.

Additional data