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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Accessions

Primary accessions

Gene Expression Omnibus

Protein Data Bank

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.).

Author information

Author notes

    • Henry E. Pelish
    •  & Brian B. Liau

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Henry E. Pelish
    • , Brian B. Liau
    • , Ioana I. Nitulescu
    • , Anupong Tangpeerachaikul
    • , Diogo H. Da Silva
    • , Brittany T. Caruso
    • , Alexander Arefolov
    • , Olugbeminiyi Fadeyi
    • , Karrie Du
    • , Ge Zou
    • , Chong Si
    • , Andrew G. Myers
    •  & Matthew D. Shair
  2. Department of Chemistry and Biochemistry, University of Colorado, Campus Box 596, Boulder, Colorado 80303, USA

    • Zachary C. Poss
    • , Christopher C. Ebmeier
    •  & Dylan J. Taatjes
  3. Lurie Family Imaging Center, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Amanda L. Christie
    •  & Nancy E. Kohl
  4. Division of Hematology/Oncology, Children's Hospital, Boston, Massachusetts 02215, USA

    • Deepti Banka
  5. Proteros Biostructures GmbH, Bunsenstrasse 7a, D-82152 Martinsried, Germany

    • Elisabeth V. Schneider
    •  & Anja Jestel
  6. Max-Planck-Institut für Biochemie, Am Kloperspitz 18, D-82152 Martinsried, Germany

    • Elisabeth V. Schneider
  7. Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Roderick T. Bronson
  8. Cancer Biology and Genetics Program and Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    • Andrei V. Krivtsov
    •  & Scott A. Armstrong
  9. Department of Pediatrics, Columbia University Medical Center, New York, New York 10032, USA

    • Andrew L. Kung
  10. Bioinfo, Plantagenet, Ontario K0B 1L0, Canada

    • Madeleine E. Lemieux

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthew D. Shair.

Extended data

Supplementary information

PDF files

  1. 1.

    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.

Excel files

  1. 1.

    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.

  2. 2.

    Supplementary Table 2

    This table shows Kinome profiling of cortistatin A in MOLM-14 cell lysate and in vitro with recombinant kinases.

  3. 3.

    Supplementary Table 3

    This table shows Genes differentially expressed in MOLM-14 cells upon 3h or 24h treatment with cortistatin A.

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https://doi.org/10.1038/nature14904

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