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Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition

Nature Chemical Biology volume 12, pages 672–679 (2016)Cite this article

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Abstract

Here we show that acute myeloid leukemia (AML) cells require the BRD9 subunit of the SWI−SNF chromatin-remodeling complex to sustain MYC transcription, rapid cell proliferation and a block in differentiation. Based on these observations, we derived small-molecule inhibitors of the BRD9 bromodomain that selectively suppress the proliferation of mouse and human AML cell lines. To establish these effects as on-target, we engineered a bromodomain-swap allele of BRD9 that retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells confers resistance to the antiproliferative effects of our compound series, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. To our knowledge, our study provides the first evidence for a role of BRD9 in cancer and reveals a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells.

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Figure 1: BRD9 is a subunit of SWI–SNF complexes in acute myeloid leukemia cells.
Figure 2: Brd9 supports acute myeloid leukemia growth by sustaining Myc expression and an undifferentiated cell state.
Figure 3: A chemical series that inhibits the BRD9 bromodomain.
Figure 4: A bromodomain-swap allele validates on-target activity of BRD9 inhibitors.
Figure 5: Chemical Brd9 inhibition mimics the transcriptional effects of Brd9 knockdown.
Figure 6: A SET domain-swap allele validates on-target activity of the EZH2 inhibitor GSK126.

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Gene Expression Omnibus

NCBI Reference Sequence

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Acknowledgements

We thank A. Bhagwat for critical reading of the manuscript and members of the Vakoc laboratory for discussion of the presented findings. The phylogenetic tree of human bromodomains was provided by S. Knapp. This work was supported by Cold Spring Harbor Laboratory US National Cancer Institute (NCI) Cancer Center Support grant CA455087 for developmental funds and shared resource support. Additional funding was provided by the Alex's Lemonade Stand Foundation, the V Foundation, Pershing Square Sohn Cancer Research Alliance and by Boehringer Ingelheim. A.F.H. is supported by a Boehringer Ingelheim Fonds PhD Fellowship. J.-S.R. is supported by the Martin Sass Foundation and the Lauri Strauss Leukemia Foundation. C.R.V. is supported by a Burroughs-Wellcome Fund Career Award and National Institutes of Health grant NCI RO1 CA174793.

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Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Anja F Hohmann, Jessica L Minder, Jae-Seok Roe, Junwei Shi & Christopher R Vakoc

  2. Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Anja F Hohmann & Christopher R Vakoc

  3. Boehringer Ingelheim Regional Center Vienna GmbH and Company KG, Vienna, Austria

    Laetitia J Martin, Steffen Steurer, Gerd Bader, Darryl McConnell, Mark Pearson, Thomas Gerstberger, Teresa Gottschamel, Diane Thompson & Manfred Koegl

  4. Molecular and Cellular Biology Program, Stony Brook University, Stony Brook, New York, USA

    Junwei Shi

  5. Department of Medical Genome Sciences, University of Tokyo, Kashiwa, Japan

    Yutaka Suzuki

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Contributions

A.F.H., L.J.M., M.K., C.R.V. designed experiments and analyzed results; A.F.H. and J.L.M. performed genetic characterization of BRD9, cellular evaluation of BRD9 inhibitors and domain-swap studies; J.-S.R. performed ChIP-seq; J.S. performed IP-MS; L.J.M., S.S., G.B., T.Ge., T. Go., D.T. and M.K. derived BRD9 inhibitors, carried out in vitro characterization and human cell line sensitivity profiling; Y.S. prepared and sequenced shRen and shBrd9 RNA-seq libraries; L.J.M., D.M., M.P., M.K. and C.R.V. supervised the research; A.F.H., L.J.M. and C.R.V. wrote the manuscript.

Corresponding author

Correspondence to Christopher R Vakoc.

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Competing interests

This study was funded in part via a sponsored research agreement with Boehringer Ingelheim.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–15. (PDF 9599 kb)

Supplementary Data 1

Custom gene sets used in GSEA. (XLSX 38 kb)

Supplementary Note 1

Diagram summarizing the small-molecule screening efforts. (PDF 73 kb)

Supplementary Note 2

Synthetic procedures. (PDF 531 kb)

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Hohmann, A., Martin, L., Minder, J. et al. Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition. Nat Chem Biol 12, 672–679 (2016). https://doi.org/10.1038/nchembio.2115

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