Abstract
Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes1,2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6, 7, 8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.
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Data deposits
The atomic coordinates of CDK8–CCNC in complex with cortistatin A have been deposited in the Protein Data Bank (PDB) with accession number 4CRL. MIAME-compliant microarray data as well as aligned and raw ChIP-seq data were deposited to the Gene Expression Omnibus (GEO) with accession GSE65161.
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Acknowledgements
We thank R. Levine, R. King, B. Ebert, B. Bernstein, S. Gillespie, M. Galbraith, M. Patricelli and T. Nomanbhoy for discussions. Lentiviral packaging was completed at the University of Massachusetts Medical School RNAi core facility. Microarray data collection was performed at DFCI MicroArray Core Facility and UMass Medical School Genomics Core Facility. Formulation was performed at VivoPath. In-vivo portions of pharmacokinetic, natural killer and SET-2 studies were performed at Charles River. We thank S. Trauger and G. Byrd of Harvard FAS Small Molecule Mass Spectrometry for PK data acquisition and Harvard FAS Center for Systems Biology for flow sorting and high-throughput sequencing. Recombinant expression of CDK8 module subunits was completed at the Tissue Culture Shared Resource at the University of Colorado Cancer Center, supported by the NCI (P30 CA046934). HCT116 RNA-seq was carried out at the Genomics Shared Resource at the University of Colorado Cancer Center and supported by grant P30-CA046934. We thank A. Odell and R. Dowell for HCT116 RNA-seq data analysis, the R. Levine laboratory (MSKCC) for carrying out the SET-2 RNA-seq acquisition, the M. Geyer laboratory for purified CDK12–CCNK and CDK13–CCNK complexes, and P. Kovarik for STAT1 plasmids. This work was supported by NIH grant CA66996 (S.A.A.), NCI grants R01 CA170741 (D.J.T.) and F31 CA180419 (Z.C.P.), NIH T32 GM08759 (Z.C.P.), a Leukemia and Lymphoma Society Translational Research Program Grant (M.D.S.), the Blavatnik Biomedical Accelerator Program at Harvard (M.D.S.) and the Starr Cancer Consortium (M.D.S.).
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Contributions
H.E.P., B.B.L. and M.D.S. designed the research and analysed data. H.E.P., B.B.L., I.I.N., A.T., D.H.D., B.T.C. and K.D. performed cell-based and biochemical experiments not otherwise specified, and analysed data under guidance from M.D.S. Z.C.P. and C.C.E. performed in vitro kinase assays and HCT116 gene expression under guidance from D.J.T. A.A. and O.F. synthesized CA under guidance from M.D.S. C.S. and G.Z. synthesized CA under guidance from A.G.M. A.L.C. performed MV4;11 in vivo efficacy and safety studies under guidance from N.E.K. D.B. performed early MOLM-14 cell growth assays under guidance of S.A.A. E.V.S. and A.J. performed X-ray crystallography. R.T.B. performed mouse histopathology. A.L.K. advised on in vivo studies. S.A.A. and A.V.K. advised on AML studies. M.E.L. performed computational biology studies. H.E.P., B.B.L., D.J.T. and M.D.S. wrote the manuscript. M.D.S. supervised the research.
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Extended data figures and tables
Extended Data Figure 1 CDK8 ChIP-seq defines SE-associated genes.
a, The antibody used for CDK8 ChIP-seq (Bethyl A302-500A) was validated by immunoprecipitation (IP) and western blot (WB). Immunoprecipitation was conducted with Bethyl A302-500A (2 μg) on MOLM-14 whole-cell extract, and western blot was performed on split immunoprecipitation lysate or 5% input with either anti-CDK8 Bethyl A302-501A (left), anti-CDK8 Bethyl A302-500A (right), or normal rabbit IgG (CST, 2729), experiment performed once. b, MED1 and CDK8 density is highly correlated on active enhancer regions marked by H3K4me1 and H3K27ac (correlation = 0.86, R2 = 0.74) in MOLM-14 cells. The pink box represents SEs. c, Hierarchical clustering dendrogram of CDK8, MED1, BRD4, H3K27ac, RNA pol II and H3K4me1 ChIP-seq signal. d, Distribution of CDK8 signal with input subtracted across CDK8 bound regions. Regions to the right of inflection point are considered SEs. e, Distribution of CDK8, MED1, BRD4 and H3K27ac signal across putative enhancer regions. Regions to the right of the distribution inflection point are considered SEs. f, ChIP-seq profile plots centred around MED1-defined SE and regular enhancer regions. Flanking regions are 2.5 kb.
Extended Data Figure 2 CA inhibition of and binding to CDK8.
a, CA inhibition of CDK8 module phosphorylation of CDK8 and STAT1-S727 substrate (mean ± s.e.m., n = 3 biological replicates, one of two experiments shown, autorad in Supplementary Fig. 1). b, CA inhibition in vitro of CDK8 module activity but not CDK12–CCNK or CDK13–CCNK activity up to 10 μM. Equal amounts (silver stain) of GST–CTD were used as the substrate in in vitro kinase assays. The amount of each kinase used was empirically determined to give approximately the same GST–CTD signal under the assay conditions. GST–CTD-P, phosphorylated GST-CTD; ns, no substrate (kinase only). One of four experiments shown. c, Immunoblot showing that CA selectively and dose-dependently inhibits capture of native CDK8 (IC50 ≈ 10 nM) and CDK19 (IC50 ≈ 100 nM) from MOLM-14 lysates but does not inhibit capture of CDK9, CDK12, CDK13, ROCK1, ROCK2 or GSG2. One of two experiments shown, full scan in Supplementary Fig. 1. d, Immunoblots showing CA inhibition of CDK8-dependent IFN-γ-stimulated STAT1-S727 phosphorylation in MOLM-14 cells and CA inhibition of TGF-β-stimulated Smad2-T220 and Smad3-T179 phosphorylation in HaCaT cells (IC50 < 100 nM). One of two experiments shown, full scan in Supplementary Fig. 1. e, In vitro kinase activity profiling (mean for kinase reaction, n = 2 biological replicates, experiment performed once). f, g, CA dose-dependent inhibition of CDK8–CCNC complex (IC50 = 5 nM) (f) and GSG2 (IC50 = 130 nM) (g) as measured in e (n = 1, experiment performed once). h, Dendrogram representation of results shown in Fig. 2c for 1 μM CA.
Extended Data Figure 3 CA–CDK8–CCNC ternary complex.
a, The 2.4 Å crystal structure of the human CA–CDK8–CCNC ternary complex shown as a Corey–Pauling–Koltun (CPK) model. b, CA and neighbouring protein side chains are shown as a stick model coloured according to the chemical atom type (CA in cyan, CDK8–CCNC in grey, N in blue, O in red and S in yellow). CA is shown superimposed with the refined 2Fo − Fc electron density map contoured at 1.0σ. Hydrogen bonds are indicated as green dotted lines. c, A portion of the CA–CDK8–CCNC crystal structure showing the CA binding pocket of CDK8 (with and without a semi-transparent surface; CA in gold, CDK8 in grey) with certain residues and CA in stick representation. Dotted red lines indicate H-bonds. Key residues and binding elements are labelled.
Extended Data Figure 4 Antiproliferative activity of CA and I-BET151.
a, Plots showing antiproliferative activity of CA over time for selected sensitive cell lines and concentrations (mean ± s.e.m., n = 3 biological replicates, one of two experiments shown). b, Immunoblots showing that CA inhibits CDK8-dependent IFN-γ-stimulated STAT1-pS727 phosphorylation equally well in cells sensitive or insensitive to the antiproliferative activity of CA (one of two experiments shown, full scan in Supplementary Fig. 1). c, Immunoblots showing CDK8 and CDK19 levels after 24 h CA treatment in sensitive cell lines MV4;11 and MOLM-14 (one of two experiments shown, full scan in Supplementary Fig. 1). d, CD41 and CD61 (vehicle versus CA, P = 0.04 and 0.005, respectively, two-tailed t-test) on SET-2 cells after 3 days of indicated treatment (mean ± s.e.m., n = 3 biological replicates, one of two experiments shown). Phorbol 12-myristate 13-acetate (PMA) was used as positive control. e, DNA content and annexin V staining of indicated cell lines after treatment with CA (mean ± s.e.m., n = 3 biological replicates, one of two experiments shown). f, Immunoblots of CA dose- and time-dependent induction of PARP and caspase-3 cleavage for indicated cell lines (one of two experiments shown, full scan in Supplementary Fig. 1).
Extended Data Figure 5 Mediator kinases mediate the antiproliferative activity of CA.
a, We evaluated point mutations to CDK8 residues lining the CA-binding pocket: Ala155, His106, Asp103 and Trp105. Expression of CDK8 mutants A155I, A155F, A155Q, H106K and D103E in MOLM-14 cells afforded only modest desensitization to CA. Differential sensitivity of MOLM-14 cells to CA after expression of indicated mutant Flag–CDK8 proteins (mean ± s.e.m., n = 3 biological replicates, experiment performed once). b, Immunoblots showing that Flag–CDK8 or Flag–CDK19 and Flag–CDK8(W105M) or Flag–CDK19(W105M) are expressed at similar levels in MOLM-14, MV4;11 and SKNO-1 cells (experiment performed once, full scan in Supplementary Fig. 1). c, Differential sensitivity of MV4;11 and SKNO-1 cells to CA after expression of Flag–CDK8, Flag–CDK19, Flag–CDK8(W105M) and Flag–CDK19(W105M), legend as in d (mean ± s.e.m., n = 3 biological replicates, one of two experiments shown). d, Control showing that expression of Flag–CDK8(W105M) or Flag–CDK19(W105M) in MOLM-14, MV4;11 and SKNO-1 cells does not confer resistance to antiproliferative agents paclitaxel and doxorubicin (mean ± s.e.m., n = 3 biological replicates, one of two experiments shown). e, Purified Flag–CDK8(W105M) and Flag–CDK19(W105M) remain catalytically active for phosphorylation of CTD in vitro but are resistant to inhibition by CA (mean ± s.e.m., n = 3 biological replicates, experiment performed once). f, Representative autorad and silver stain images supporting quantification shown in e. g, Sequence alignment of human CDKs. Sequence alignment was performed on segments of CDK1-20 using Clustal Omega. The unique Trp105 residue in CDK8 and CDK19 is highlighted in red, and is absent from other CDKs (orange box). UniProt Knowledgebase entries: CDK1, P06493; CDK2, P24941; CDK3, Q00526; CDK4, P11802; CDK5, Q00535; CDK6, Q00534; CDK7, P50613; CDK8, P49336; CDK9, P50750; CDK10, Q15131; CDK11A, Q9UQ88; CDK11B, P21127; CDK12, Q9NYV4; CDK13, Q14004; CDK14, O94921; CDK15, Q96Q40; CDK16, Q00536; CDK17, Q00537; CDK18, Q07002; CDK19, Q9BWU1; CDK20, Q8IZL9.
Extended Data Figure 6 CA disproportionately affects expression of SE genes in MOLM-14 cells.
a, GSEA plots showing positive enrichment of SE-associated genes, defined by ChIP-seq signal for indicated factors, with 3 h CA treatment in MOLM-14 cells (differential expression versus DMSO controls). b, Venn diagram showing the overlap between SE genes and genes upregulated ≥ 1.2-fold after 3 h CA treatment in MOLM-14 cells. Numbers in red indicate the percentage of CDK8-occupied genes (peak within ±5 kb of the gene). c, d, RNA pol II ChIP-seq metagene profile plots of unchanged genes (black), SE-associated genes (yellow), CA-upregulated genes with vehicle treatment (no CA; red), and CA-upregulated genes with 6 h CA treatment (with CA; blue). e, f, Cumulative distribution plot of RNA pol II travelling ratio (TR) after treatment with CA (25 nM, 6 h) or vehicle across genes ≥1.2-fold downregulated by CA after 3 h (1.16-fold, P = 0.31, Kolmogorov–Smirnov test) (e) and across all genes (1.21-fold, P < 2.2 × 10−16, Kolmogorov–Smirnov test) (f). g, CA does not significantly change the total amount of RNA or mRNA in MOLM-14 or MV4;11 cells (mean ± s.e.m., n = 3 biological replicates, experiment performed once) after treatment with CA (25 nM, 3 h). h, Global levels of RNA pol II pS2 or RNA pol II pS5 do not change after treatment with CA by immunoblot analysis. Flavopiridol (FP) was used at 300 nM as a positive control (experiment performed twice, full scan in Supplementary Fig. 1).
Extended Data Figure 7 Effects of SE-associated gene expression levels on MOLM-14 AML cell proliferation.
a, Venn diagram showing overlap between CA-upregulated genes and CD14+ master transcription factors. Overlapping genes are listed; SE-associated genes identified by one (purple) or more (red) marks in MOLM-14 are indicated. b, GSEA plot showing positive enrichment of CD14+ master transcription factors after 3 h CA treatment (MOLM-14 differential expression). c, Fold-change in mRNA copies per cell of selected SE-associated genes after 3 h treatment with 100 nM CA, 500 nM I-BET151 or 3 h I-BET151 followed by addition of CA for 3 h (mean ± s.e.m., n = 3 biological replicates, experiment performed twice). d, h, mRNA expression levels either 1 day (Flag–IRF1, Flag–IRF8) or 3 days (Flag–CDKN1B, Flag–FOSL2, Flag–ETV6) after induction with doxycycline (d) or 2 days after siRNA electroporation (h) (mean, Poisson error, n = 15,000–20,000 technical replicates, experiment performed twice) corresponding to Fig. 3f. e, Immunoblot showing protein levels of CEBPA 4 days after siRNA electroporation or 1 day after doxycycline-induced expression (experiment performed once) corresponding to Fig. 3f, full scan in Supplementary Fig. 1. f, ChIP-seq binding profiles at the FOSL2 and ETV6 loci. Red bars denote SEs while grey bars denote regular enhancers. g, mRNA levels of indicated genes in MOLM-14 cells expressing Flag–CDK8 (grey) or Flag–CDK8(W105M) (red) after 3 h 25 nM CA treatment (mean ± s.e.m., n = 3 biological replicates, one of two experiments shown). i, Heat maps showing BRD4 and CDK8 ChIP-seq on regions depleted of BRD4 > 2-fold after I-BET151 treatment for 6 h before and after drug treatment. j, Effect of 3-day treatment with CA, I-BET151 or the combination of CA and I-BET151 on proliferation of MOLM-14 (mean ± s.e.m., n = 6 biological replicates, one of two experiments shown).
Extended Data Figure 8 CA inhibits AML progression and CDK8 in vivo and is well-tolerated at its efficacious dose.
a, Plasma concentration of CA after single intraperitoneal administration of 1 mg kg−1 CA to male CD-1 mice (mean ± s.e.m., n = 3 mice, experiment performed once). b–g, MV4;11 disseminated leukaemia study (experiment performed once). b, Bioluminescence images with the median bioluminescence for each treatment group on treatment day 1, showing engraftment of MV4;11 leukaemia cells. c, 30 days after treatment initiation, the mouse with the highest, lowest, and median day 29 bioluminescence for each treatment group was euthanized and the spleen weight (P < 0.05) and percentage of MV4;11 cells (mCherry-positive) in the spleen (P < 0.03) and femur bone marrow (P < 0.02) were determined (n = 3 mice). Dotted purple lines mark the range within 1 s.d. of the mean for the related healthy 8-week-old female NOD–SCID mice, P values determined by one-way ANOVA, each treatment versus vehicle. d, Haematoxylin and eosin staining of day-30 lung, spleen and bone marrow samples of the median mice in c. Hypercellular alveoli, evidence of leukaemia infiltration, are only observable with vehicle treatment. Spleen sample from the vehicle-treated mouse reveals a large population of cells with a round nucleus and relatively abundant cytoplasm. Similarly, all cells in the vehicle-treated bone marrow have round to oval nuclei and abundant cytoplasm, while normal erythroid or myeloid cells are not observed, suggesting that the spleen and the bone marrow have been dominated by the leukaemia cells. By contrast, the red pulp from the CA-treated mouse spleen shows a heterogeneous population of mature red blood cells, nucleated red blood cells, immature myeloid cells and megakaryocytes. The bone marrow from a CA-treated mouse also exhibits a mixture of erythroid precursors, myeloid precursors, and megakaryocytes. Scale bars, 250 μm. e, Kaplan–Meier survival analysis (n = 8 mice, P < 0.0001, log-rank test). f, Mean body weight ± s.e.m., n = 11 mice, for study in Fig. 4b. g, Complete blood count (CBC) analysis 30 days after first treatment for the mice analysed in c (n = 3 mice). Dotted purple lines mark the range within 1 s.d. of mean for the related healthy 8-week-old female NOD–SCID mice. h, Mean body weight ± s.e.m., n = 10 mice, for study in Fig. 4c (experiment performed once). i, Immunoblot of natural killer cell lysate from C57BL/6 mice treated as indicated in Fig. 4d. Each lane represents a distinct mouse sample with 1 = STAT1-pS727, 2 = STAT1, and 3 = β-actin (experiment performed once, full scan in Supplementary Fig. 1). j–l, Body weight (j), day 15 CBC (k), and day 15 blood chemistry (l) for healthy CD-1 mice (n = 3 mice, experiment performed once) treated with vehicle (20% hydroxypropyl-β-cyclodextrin) or 0.16 mg kg−1 CA intraperitoneally once daily for 15 days. k, l, A/G, albumin/globulin; ALB, albumin (g dl−1); ALK, alkaline phosphatase (U l−1); ALT, alanine aminotransferase (U l−1); AST, aspartate aminotransferase (U l−1); BUN, urea nitrogen (mg dl−1); Ca, total calcium (mg dl−1); CHOL, total cholesterol (mg dl−1); Cl, chloride (mEq l−1); GLOB, globulin (calculated, g dl−1); GLU, glucose (mg dl−1); HCT, haematocrit (%); HGB, haemoglobin (g dl−1); K, potassium (mEq l−1); Na, sodium (mEq l−1); Na/K, sodium/potassium; PHOS, phosphorus (mg dl−1); PLT, platelets (×105 platelets μl−1); RBC, red blood cells (×106 cells per μl); TBIL, total bilirubin (mg dl−1); TP, total protein (g dl−1); TRIG, triglycerides (mg dl−1); WBC, white blood cells (×103 cells μl−1).
Supplementary information
Supplementary Information
This file contains Supplementary Text with an additional reference and Supplementary Figure 1, which shows the un-cropped scans for Figure 2 and Extended Data Figures 2, 4, 5, 6, 7, 8. (PDF 3157 kb)
Supplementary Table 1
This table shows Super-Enhancer mapping in AML cell lines and Gene Ontology analysis on Super-Enhancer associated genes in MOLM-14 cells. (XLSX 12030 kb)
Supplementary Table 2
This table shows Kinome profiling of cortistatin A in MOLM-14 cell lysate and in vitro with recombinant kinases. (XLSX 76 kb)
Supplementary Table 3
This table shows Genes differentially expressed in MOLM-14 cells upon 3h or 24h treatment with cortistatin A. (XLSX 99 kb)
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Pelish, H., Liau, B., Nitulescu, I. et al. Mediator kinase inhibition further activates super-enhancer-associated genes in AML. Nature 526, 273–276 (2015). https://doi.org/10.1038/nature14904
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DOI: https://doi.org/10.1038/nature14904
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