ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia - PubMed (original) (raw)

. 2017 Mar 9;543(7644):265-269.

doi: 10.1038/nature21687. Epub 2017 Mar 1.

Liling Wan 1 2 3, Yuanyuan Li 6 7, Jie Lyu 8, Yuanxin Xi 8, Takayuki Hoshii 2 3, Julia K Joseph 1, Xiaolu Wang 4, Yong-Hwee E Loh 9, Michael A Erb 10, Amanda L Souza 10, James E Bradner 10 11, Li Shen 9, Wei Li 8, Haitao Li 6 7, C David Allis 1, Scott A Armstrong 2 3, Xiaobing Shi 4 5 12

Affiliations

Liling Wan et al. Nature. 2017.

Abstract

Cancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs. 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) inhibitors. We recently identified the YEATS domain as an acetyl-lysine-binding module, 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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Conflict of interest statement

The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. 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.

Figures

Extended Data Figure 1

Extended Data Figure 1. Depletion of ENL impairs the growth of AML

a, Western blot demonstrating the knockdown efficiency of five independent sgRNAs targeting ENL. b, Competition assay that plots the relative % of RFP+sgRNA+ cells following 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 % of RFP+sgRNA+ cells following transduction of leukaemia cells with indicated sgRNAs. n = 3. e, Western blot demonstrating the induction of Cas9 expression upon Dox treatment in iCas9-MOLM-13 cells. f, Western blot demonstrating the decrease of ENL protein levels upon Dox treatment in iCas9-MOLM-13 cells. g, Competition assay that plots the relative % of RFP+sgRNA+ cells following Dox treatment in iCas9-MOLM-13 cells. n = 3. h, Relative ENL mRNA levels determined by qRT-PCR quantification in MV4;11 cells transduced with non-targeting (NT) control or ENL shRNAs. i, Representative images (left) and quantification (right) of colonies formed by MV4;11 cells transduced with indicated shRNAs. j, Light microscopy of May-Grunwald/Giemsa-stained MV4;11 leukaemia cells transduced 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 % of RFP+sgRNA+ cells following Dox treatment in iCas9-U-937 (l), iCas9-K562 (m), iCas9-HeLa (n) and iCas9-U2OS (o) cells. n = 3. p-r, lineage−Sca-1+c-Kit+ (LSK) cells were sorted from bone marrow of C57BL/6 mice and transduced with luciferase or Enl shRNA. p, Relative Enl mRNA levels determined by qRT-PCR quantification after 3 days of drug selection. q, Relative proliferation of control or _Enl_-KD LSKs. n = 4**. r**, Quantification of colonies formed by LSK cells cultured for 7 days. n = 4. GM, colony-forming unit containing granulocyte and macrophage. M, macrophage. G, granulocyte. All error bars represent mean ± s.d. See Supplementary Figure 1 for western blot gel source data.

Extended Data Figure 2

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 post 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. The majority of 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 Figure 1 for western blot gel source data.

Extended Data Figure 3

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 the overlap of genes whose expression is significantly changed (padj < 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**. Heatmap representation of genes differentially expressed between iCas9-MOLM-13 cells expressing sgRNAs targeting _GFP_ control, _ENL_ or _AF9_ (fold change > 1.5 and padj < 0.05) after 3 days of Dox induction. d and e, GSEA plots evaluating the changes in monocyte differentiation and LSC gene signatures (d) and the MYC pathways (e) upon ENL depletion. f, GO term analyses of downregulated (ENL-KO-DN, top) or upregulated (ENL-KO-UP, bottom) genes in ENL sgRNA-expressing cells. The top 5 biological processes that each group of genes were enriched in were shown (details in Supplementary Table S2). Fold enrichment and P values are shown at the right. g and 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 LSC gene signatures (g) and the oncogenic pathways (h) upon ENL knockdown.

Extended Data Figure 4

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

a and b, Venn diagram showing overlaps of Flag-ENL-occupied genes with those of MLL-AF9 in MOLM-13 (Bernt et al, Cancer Cell 2011) (a) or MLL-AF4 in MV4;11 cells (Guenther et al. Genes&Dev. 2008) (b), respectively**. c**, Venn diagram showing overlaps of Flag-ENL occupied genes in MOLM13, MV4;11 and HeLa cells. More details shown in Supplementary Table S7. 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] ±3kb). P < 1×10−300 (binomial test). More details shown in Supplementary Table S6. 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, Boxplots showing the fold changes (normalized to GFP control) of Pol II occupancy at TSS (TSS – 30bp to TSS + 300bp) or the rest of the gene body on ENL-bound and activated genes upon the expression of ENL sgRNA. The log2 fold changes at both TSS and genebody were significantly lower than 0 (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 with arrow. i, Western blot showing comparable cellular levels of Pol II S2P in MOLM-13 cells expressing sgRNAs targeting GFP or ENL. See Supplementary Figure 1 for gel source data. j and 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

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. Green ball, carbon; Blue ball, nitrogen; Red ball, oxygen; Big cyan sphere, water molecule. Hydrogen bonds are indicated as green dashed lines with bond length shown as values in the unit of angstrom. 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

Extended Data Figure 6. The YEATS domain is required for ENL’s chromatin localization

a, Boxplots 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] ±3kb 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, 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. More details shown in Supplementary Table S8, S9. d, Western blot showing the protein levels of ectopically expressed WT or mutant Flag-ENL (marked as *) and endogenous ENL (marked as **). e, The genome browser view of H3K27ac, H3K9ac, Flag-ENL signals in a few of ENL-bound genes (MYC, HLX). TSS is indicated with arrow. f, Average occupancies of WT, F59A or Y78A mutant Flag-ENL on ENL-bound genes along the transcription unit in MV4;11 cells. g, Western blot analysis of coIP 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 aa1-113; ΔC: deletion of aa430–559 of ENL. h, Western blot analysis of IP using the M2 anti-Flag antibody in cells expressing WT 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 WT or mutant (F59A or Y78A) murine ENL. Error bars represent mean ± s.e.m. * P < 0.5, *** P < 0.001 (two-tailed unpaired Student’s _t_-test). See Supplementary Figure 1 for gel source data.

Extended Data Figure 7

Extended Data Figure 7. The YEATS domain-histone acetylation interaction is required for ENL’s roles 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 % of RFP+sgRNA+ cells following 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 post 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

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 and 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 hours. Row-normalized heatmap (f and h) and boxplots of relative expression levels (z-scores, g and i) of genes found to be 2-fold downregulated (f and g) or upregulated (h and i) following JQ1 treatment in ENL sgRNA-expressing cells. All error bars represent mean ± s.e.m.

Figure 1

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 following transduction of different _MLL_-rearranged leukaemia cell lines with indicated sgRNAs. 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 % of RFP+sgRNA+ cells following 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 27 (n = 4) or 32 (n = 10) days post 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.

Figure 2

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

a, Heatmap representation of genes differentially expressed in iCas9-MOLM-13 cells expressing sgRNAs targeting GFP control, ENL or AF9 (fold change > 1.5 and padj < 0.05) 5 days post Dox treatment. Red and green indicate relative high and low expression, respectively. More details shown in Supplementary Table S1. b, GSEA plots evaluating the changes in monocyte differentiation and LSC gene signatures upon ENL depletion. NES, normalized enrichment score; FDR q-val, false discovery rate q value. c, Genomic distribution of Flag-ENL ChIP-seq peaks in MOLM-13 cells. The peaks are enriched in the promoter regions ([TSS] ± 3kb). P < 1×10−300 (binomial test). More details shown in Supplementary Table S3. 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 TTS. 5 kb upstream of TSS and 5 kb downstream of TTS are also included. e-g, Average occupancy of Pol II (e), Pol II Ser2P (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.

Figure 3

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 H317–28K27ac, H31–10K9ac, H3K1–2518ac or unmodified H31–34 peptides. c, Overall structure of the ENL YEATS domain bound to H3K27ac peptide. ENL YEATS is depicted as blue ribbons with key residues highlighted as green sticks. Histone H3K27ac peptide is shown as a yellow ribbon with side chains highlighted in 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. Histone H3 peptide is depicted as space-filling spheres. e, Hydrogen bonding network between H3K27ac peptide and ENL (top). Hydrogen bonds are shown as red dashes. Key residues of ENL are depicted as green sticks and labeled black; the H3 peptide is shown as yellow sticks and labeled red. Grey meshes, Fo-Fc omit map countered at 2.0 σ 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 H317–28K27ac peptide. g, Peptide pull-downs of ENL YEATS mutants and indicated histone H3 peptides. See Supplementary Figure 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 (3-way Fisher’s exact test). More details shown in Supplementary Table S4, S5. 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 WT (blue) and mutant ENL (F59A in cyan or Y78A in purple) on ENL-bound genes along the transcription unit.

Figure 4

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

a, Heatmap representation of genes differentially expressed in iCas9-MOLM-13 cells expressing sgRNAs targeting luciferase (Luc) control or ENL (fold change > 1.5 and padj < 0.05) in the indicated rescue conditions. **b**, GSEA plots evaluating the changes in monocyte differentiation and LSC gene signatures in the indicated comparisons. **c**, Negative-selection competition assay that plots the relative % of RFP+sgRNA+ cells following 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 murine _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** and **h**, Leukemia 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-sg/JQ1 vs. all other groups) using Mann-Whitney test. **h**, Kaplan–Meier survival curves of control and JQ1-treated mice (n = 10 per group). _P_ < 0.0001 (ENL-sg/JQ1 vs. all other groups) using a log rank test. **i** and **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 hours. Genes found to be > 2-fold downregulated (i) or upregulated (j) upon JQ1 treatment in ENL sgRNA-expressing cells were examined. (Left) Boxplots comparing the JQ1-induced fold changes of these genes in either control (red) or ENL sgRNA-expressing cells (blue). Error bars indicate min to max. 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 whose absolute JQ1-induced fold change was > 1.2 fold higher or lower in ENL sgRNA-expressing cells compared to control were classified as “enhanced by ENL-sg” (blue) and “decreased by ENL-sg” (green), respectively. There are significantly more genes falling into “enhanced by ENL-sg” group than “decreased by ENL-sg” group (P < 0.0001 by Fisher’s exact test). More details shown in Supplementary Table S10. All error bars represent mean ± s.d. (n = 3) unless noted otherwise.

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