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. 2015 Oct 8;526(7572):273-276.
doi: 10.1038/nature14904. Epub 2015 Sep 28.

Mediator Kinase Inhibition Further Activates Super-Enhancer-Associated Genes in AML

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Free PMC article

Mediator Kinase Inhibition Further Activates Super-Enhancer-Associated Genes in AML

Henry E Pelish et al. Nature. .
Free PMC article

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 oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. 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 6-8). 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.

Figures

Extended Data Figure 1
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 IP-western. IP was conducted with Bethyl A302-500A (2 μg) on MOLM-14 whole cell extract and Western blot (WB) was performed on split IP lysate or 5% input with either anti-CDK8 Bethyl A302-501A (left panel), anti-CDK8 Bethyl A302-500A (right panel), 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 (Corr = 0.86, R2 = 0.84) 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 centered around MED1-defined SE and regular enhancer regions. Flanking regions are 2.5kb.
Extended Data Figure 2
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 Figure 1). (b) CA inhibition in vitro of CDK8 module activity but not CDK12:Cyclin K or CDK13:Cyclin K 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. “ns” is no substrate (kinase only) and “GST-CTD-P” is phosphorylated GST-CTD. 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 did not inhibit capture of CDK9, CDK12, CDK13, ROCK1, ROCK2 or GSG2. One of two experiments shown, full scan in Supplementary Figure 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 Figure 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 (f) CDK8/CCNC complex (IC50 = 5 nM) and (g) GSG2 (IC50 = 130 nM) as measured for the (e) (n=1, experiment performed once). (h) Dendrogram representation of results shown in Fig. 2c for 1 μM CA.
Extended Data Fig. 3
Extended Data Fig. 3. CA/CDK8/CCNC ternary complex
(a) 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 1.0 s. 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 gray) with certain residues and CA in stick representation. Dotted red lines indicate H-bonds. Key residues and binding elements are labelled.
Extended Data Fig. 4
Extended Data Fig. 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 S727 phosphorylation equally well in cells sensitive or insensitive to the antiproliferative activity of CA (one of two experiments shown, full scan in Supplementary Figure 1). (c) Immunoblots showing CDK8 and CDK19 levels upon 24 h CA treatment in sensitive cell lines MV4;11 and MOLM-14 (one of two experiments shown, full scan in Supplementary Figure 1). (d) CD41 and CD61 (vehicle vs. 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). (e) DNA content and Annexin V staining of indicated cell lines upon 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 Figure 1).
Extended Data Figure 5
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 A155I, A155F, A155Q, H106K and D103E in MOLM-14 cells afforded only modest desensitization to CA. Differential sensitivity of MOLM-14 cells to CA upon 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 Figure 1). (c) Differential sensitivity of MV4;11 and SKNO-1 cells to CA upon 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 quantitation 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 Fig 6
Extended Data Fig 6. CA disproportionately affects expression of SE genes in MOLM-14 cells
(a) GSEA plots showing positive enrichment of SE-associated genes (SE genes), defined by ChIP-seq signal for indicated factors, with 3 h CA treatment in MOLM-14 cells (differential expression vs. DMSO controls). (b) Venn diagram showing the overlap between SE genes and genes upregulated ≥1.2-fold upon 3 h CA treatment in MOLM-14 cells (“CA upregulated genes”). Numbers in red indicate the percentage of CDK8-occupied genes (peak within ±5kb of the gene). (c,d) RNA pol II ChIP-seq metagene profile plots of unchanged genes (black), SE-associated genes (SE genes, yellow), CA upregulated genes with vehicle treatment (no CA; red), and CA upregulated genes with 6 h CA treatment (with CA; blue). (e) Cumulative distribution plot of RNA pol II traveling 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) and (f) across all genes (1.21-fold, p < 2.2 × 10−16, Kolmogorov-Smirnov test). (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 Figure 1).
Extended Data Figure 7
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 TFs. 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 TFs upon 3 h CA treatment (MOLM-14 differential expression). (c) Fold-change in mRNA copies per cell of selected SE-associated genes upon 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 (d) either 1 day (FLAG-IRF1, FLAG-IRF8) or 3 days (FLAG-CDKN1B, FLAG-FOSL2, FLAG-ETV6) after induction with doxycycline or (h) 2 days after siRNA electroporation (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 Figure 1. (f) ChIP-seq binding profiles at the FOSL2 and ETV6 loci. (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) Heatmaps showing BRD4 and CDK8 ChIP-seq on regions depleted of BRD4 >2-fold upon 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
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 following single IP 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 sacrificed 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) was determined (n=3 mice). Dotted purple lines mark the range within 1 s.d. of the mean for healthy 8-week old female NSG mice, p-values determined by one-way ANOVA, each treatment vs. vehicle. (d) Hematoxylin 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. In 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 healthy 8-week old female NSG mice. (h) Mean body weight ± s.e.m., n=10 mice, for study in Fig. 4c (experiment performed once). (i) Immunoblot of NK 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 Figure 1). (j) Body weight, (k) day 15 CBC, and (l) day 15 blood chemistry 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 IP once daily for 15 days. (k) RBC, Red Blood Cells (x106 cells/μL); HCT, Hematocrit (%); HGB, Hemoglobin (g/dL); WBC, White Blood Cells (x103 cells/μL); and PLT, Platelets (x105 platelets/μL). (l) CHOL, Total Cholesterol (mg/dL); TRIG, Triglycerides (mg/dL); ALT Alanine Aminotransferase (U/L); AST Aspartate Aminotransferase (U/L); ALK, Alkaline Phosphatase (U/L); GLU, Glucose (mg/dL); TP Total Protein (g/dL); ALB, Albumin (g/dL); GLOB, Globulin (calculated, g/dL); ALB, Albumin (g/dL); A/G, Albumin/Globulin; TBIL, Total Bilirubin (mg/dL); BUN, Urea Nitrogen (mg/dL); Ca, Total Calcium (mg/dL); PHOS, Phosphorus (mg/dL); Na, Sodium (mEq/L); K, Potassium (mEq/L); Cl, Chloride (mEq/L); and Na/K, Sodium/Potassium.
Figure 1
Figure 1. CDK8 is asymmetrically loaded at SEs in MOLM-14 cells
(a) Clustering of total ChIP-seq signal of CDK8, MED1, BRD4, H3K27ac, RNA pol II, and H3K4me1 on CDK8 positive regions. Each respective cluster is ordered by CDK8 signal. The red bar indicates the cluster most highly enriched for the factors listed above. (b) Overlap between SEs independently identified by ChIP-seq signal for CDK8, MED1 and BRD4 based on the collapsed superset of regions identified by any one factor. (c) ChIP-seq binding profiles at the CEBPA locus.
Figure 2
Figure 2. CA suppresses AML cell proliferation by inhibiting Mediator kinases
(a) CA structure with N,N-dimethylamine red, C5–C9 ethano bridge magenta, C13-methyl green and isoquinoline blue. (b) Phosphorylation of the RNA pol II C-terminal domain (mean ± s.e.m., n=3 biological replicates, one of two experiments shown, autorad in Supplementary Figure 1). (c) Kinome profiling in MOLM-14 lysate (mean, n=2 biological replicates, experiment performed once, values < 35% indicate no change). (d) CA binding pocket of CDK8 from CA-CDK8-CCNC crystal structure (semi-transparent surface; CA in gold, CDK8 in grey) with contact residues and CA in stick representation. Dotted red lines indicate H-bonds. (e) Effect of CA on growth of indicated cell lines (mean ± s.e.m., n=3 biological replicates, one of two experiments shown). (f) Sensitivity of MOLM-14 cells to CA upon expression of indicated kinases (mean ± s.e.m., n=3 biological replicates, one of two experiments shown). (g) Immunoblot showing IFN-γ-stimulated STAT1 S727 phosphorylation in MOLM-14 cells expressing indicated kinases and treated with CA (one of two experiments shown, full scan in Supplementary Figure 1).
Figure 3
Figure 3. CA disproportionately increases transcription of SE-associated genes
(a-b) GSEA plots show that genes upregulated upon 3h CA treatment of MOLM-14 cells are significantly enriched in (a) MOLM-14 SE-associated genes (SE genes) and (b) genes downregulated by IBET-151 ≥2-fold in MOLM-13 cells. Red bars in b indicate H3K27ac SE genes in MOLM-14 cells in GSEA leading edge (22 genes, Fisher's exact test, p = 1.2 × 10−3). (c) Scatterplot of false discovery rate (FDR-q) versus normalized enrichment score (NES) for indicated gene sets evaluated by GSEA (n = 3,867), including C2 of MSigDB. (d) Cumulative distribution plots of RNA pol II TR. (e) Change in mRNA copy number/cell of selected SE genes after 3 h treatment (red and blue bars) or after 6 h I-BET151 treatment with CA treatment for the final 3 h (green bar) (mean ± s.e.m., n=3 biological replicates, one of two experiments shown). (f) Effect of change in expression of selected SE genes on MOLM-14 cell growth (mean ± s.e.m., with n=3 biological replicates for siETV6 and siFOSL2 and 6 for other siRNA knockdowns, 24 for FLAG-CEBPA and 12 for other inducible expressions, one of 2-6 experiments shown). (g) GSEA of SE genes in CA-treated cells. Regions of CDK8 and H3K27Ac co-enrichment identify SE genes in each cell line.
Figure 4
Figure 4. CA inhibits AML progression and CDK8 in vivo
(a) Bioluminescent images of mice bearing MV4;11 leukaemia cells. Mouse with median bioluminescence shown, treatment as in (b). Color scale 1.00×106 to 1.00×108. (b) Mean ± s.e.m., n=11 mice; p < 0.0001 for both doses on day 33 vs. vehicle, two-way ANOVA. (c) Mice harbouring SET-2 AML xenograft tumours and treated as indicated. Mean ± s.e.m., n=10 mice; 71% tumour growth inhibition on day 33, p < 0.0001, two-tailed t-test. (d) Densitometric analysis of STAT1 pS727 in NK cells isolated from the spleen of C57BL/6 mice treated with CA or vehicle (n=3 mice), STAT1 pS727 normalized to actin, p = 0.011 for 0.625 mg kg−1, one-way ANOVA, experiment performed once.

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References

    1. Hnisz D, et al. Super-enhancers in the control of cell identity and disease. Cell. 2013;155:934–947. - PMC - PubMed
    1. Whyte WA, et al. Master Transcription Factors and Mediator Establish Super-Enhancers at Key Cell Identity Genes. Cell. 2013;153:307–319. - PMC - PubMed
    1. Lovén J, et al. Selective Inhibition of Tumor Oncogenes by Disruption of Super-Enhancers. Cell. 2013;153:320–334. - PMC - PubMed
    1. Dawson MA, et al. Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia. Leukemia. 2013;28:311–320. - PMC - PubMed
    1. Kwiatkowski N, et al. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature. 2014;511:616–620. - PMC - PubMed

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