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RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia

Abstract

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

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Figure 1: AML growth is sensitive to Brd4 inhibition.
Figure 2: Brd4 is required for AML progression in vivo.
Figure 3: Brd4 inhibition leads to myeloid differentiation and leukaemia stem-cell depletion.
Figure 4: JQ1 suppresses the Myc pathway in leukaemia cells.

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Acknowledgements

We thank B. Ma, S. Muller and M. Weissenboeck for technical assistance; J. Simon, E. Earl and L. Bianco for support with mouse work; C. dos Santos for assistance with LSK FACS analysis; S. Hearn for microscopy support; G. Hannon, K. Chang, and E. Hodges for shRNA technology support; A. Gordon and M. Hammell for bioinformatics support; L. Dow for assistance with mouse pathology sample preparation; and G. Blobel for comments on the manuscript. We thank the Don Monti Memorial Research Foundation and Laurie Strauss Leukemia Foundation for research support. J.Z. was supported by a research fellowship from the German Research Foundation (DFG) and by the Andrew Seligson Memorial Clinical Fellowship at CSHL; A.R.R. was supported by an NIH traineeship and the Barbara McClintock fellowship; J.E.B and J.Q. are supported by the Damon-Runyon Cancer Research Foundation and Smith Family Foundation. P.B. is supported by a Damon Runyon-Lilly Clinical Investigator Award and a Leukemia and Lymphoma Society (LLS) Translational Research Program Grant, and is an LLS Scholar in Clinical Research. S.W.L. is supported by a Specialized Center of Research (SCOR) grant from the Leukemia and Lymphoma Society of America, a Cancer Target Discovery and Development (CTD2) grant from the National Cancer Institute, and by the Howard Hughes Medical Institute; C.R.V., J.S. and E.W. are supported by the CSHL President’s Council and the SASS Foundation for Medical Research.

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Authors

Contributions

J.Z., J.S. and C.R.V. designed research, performed experiments and analysed data. E.W., A.R.R. and M.J.T. performed experiments and analysed data. C.J. performed microarray experiments. A.C. assisted with shRNA library design. H.H., E.A.S., D.M., K.B., P.B. and P.V. performed experiments with primary leukaemia specimens. J.C.M. and M.W. generated and provided engineered AML lines. S.C.K. performed histological analysis. J.Q. and J.E.B. designed research and synthesized and supplied JQ1. J.Z., S.W.L. and C.R.V. co-wrote the paper. S.W.L. and C.R.V. supervised the research.

Corresponding authors

Correspondence to Scott W. Lowe or Christopher R. Vakoc.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains sequences of primers used in this study and Supplementary Figures 1-26 with legends. (PDF 24057 kb)

Supplementary Table 1

This file contains the sequences and primary screening performance of the custom shRNA library, including 1094 negative control shRNAs (designated PC and NC, respectively). shRNAs were designated by the number of the first nucleotide of the 22-nt target site in the mRNA transcript at the time of design. Provided are sequences of the 22-nt guide strands as well as a 97-nt oligonucleotides serving as templates for shRNA cloning using PCR. The relative abundance of each shRNA in the pool at the start (T0) and the endpoint (T14) of the screen is provided as normalized deep sequencing reads in two independent replicates (A and B). The relative change in representation is calculated as the ratio of reads in T14 compared to reads in T0 for both replicates. Only shRNAs with sufficient read numbers for evaluating depletion (>500 reads in both replicates, 1072 shRNAs = 97%) were analyzed. Scorers were defined as genes for which at least 2 shRNAs showed more than 20fold (T14/T0 < 0.05) in both replicates. (XLS 444 kb)

Supplementary Table 2

This file contains the gene sets used for Gene Set Enrichment Analysis (GSEA). Mouse genesets were obtained were converted into human gene names using bioDBNet dbWalk (http://biodbnet.abcc.ncifcrf.gov/db/dbWalk.php) or manually using the NCBI database. (XLS 84 kb)

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Zuber, J., Shi, J., Wang, E. et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478, 524–528 (2011). https://doi.org/10.1038/nature10334

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