Sci Adv. 2016 Oct 12;2(10):e1600760. eCollection 2016 Oct.

Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia.

Picaud S¹, Leonards K², Lambert JP³, Dovey O⁴, Wells C¹, Fedorov O⁵, Monteiro O⁵, Fujisawa T⁶, Wang CY⁶, Lingard H¹, Tallant C⁵, Nikbin N⁷, Guetzoyan L⁷, Ingham R⁷, Ley SV⁷, Brennan P⁸, Muller S⁵, Samsonova A⁹, Gingras AC¹⁰, Schwaller J², Vassiliou G¹¹, Knapp S¹², Filippakopoulos P¹³.

Author information

1: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.
2: Laboratory of Childhood Leukemia, Department of Biomedicine, University of Basel and Basel University Children's Hospital, Hebelstrasse 20, CH-4031 Basel, Switzerland.
3: Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada.
4: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, U.K.
5: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.; Target Discovery Institute, University of Oxford, Oxford OX3 7FZ, U.K.
6: Ludwig Institute for Cancer Research, University of Oxford, Oxford OX3 7DQ, U.K.
7: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
8: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.; Target Discovery Institute, University of Oxford, Oxford OX3 7FZ, U.K.; Alzheimer's Research UK Oxford, Nuffield Department of Medicine Research Building, University of Oxford, Oxford OX3 7FZ, U.K.
9: Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford OX3 7DQ, U.K.
10: Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada.; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
11: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, U.K.; Department of Haematology, Cambridge University Hospitals NHS Trust, Cambridge CB2 0QQ, U.K.; Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, U.K.
12: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.; Target Discovery Institute, University of Oxford, Oxford OX3 7FZ, U.K.; Institute for Pharmaceutical Chemistry and Buchmann Institute for Life Sciences, Goethe University, Max-von Laue Str. 9, 60438 Frankfurt am Main, Germany.
13: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.; Ludwig Institute for Cancer Research, University of Oxford, Oxford OX3 7DQ, U.K.

Abstract

Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.

KEYWORDS:

BET; Bromodomains; epigenetics; inhibition; leukemias

PMID:: 27757418
PMCID:: PMC5061470
DOI:: 10.1126/sciadv.1600760

[Indexed for MEDLINE]

Free PMC Article

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Fig. 2

BSP engages its target BRDs in cells.

(A) BSP binds to the BRD acetyl-lysine cavity, allowing for further functionalization toward the front channel within the ZA loop (Ra vector annotated in orange) or the back of the pocket (Rb vector annotated in orange). The vectors are shown in the complex of BSP with BRD4(1). (B) Two variants of biotinylated BSP (BSP-a and BSP-b) were prepared to explore binding to human BRDs in cells by pull-down experiments. (C) Biotinylated BSP (BSP-a or BSP-b) immobilized on magnetic beads was used to pull down human CECR2 from Flp-In T-REx HEK293 cells stably expressing 3×FLAG CECR2. The protein captured from whole-cell lysate was identified using anti-FLAG. (D) Cell lysate from HEK293T cells was incubated with biotinylated BSP (BSP-a or BSP-b) immobilized on magnetic streptavidin beads in the presence or absence of 30 nmol of BSP for 2 hours at 4°C. After pull-down and tryptic digestion with trypsin, proteins were identified in a TripleTOF 5600 mass spectrometer. (Top) Normalized abundance of each BRD-containing protein in HEK293 cells (data from Proteomics DB; https://www.proteomicsdb.org/). (Bottom) Ratio of peptide to peptide abundance in the presence and absence of competing BSP, shown as a bar graph. BRD families are annotated with roman numerals. (E) FRAP evaluation of full-length GFP-tagged BRD4 dissociation from chromatin in U2OS cells. Nuclei of DMSO-treated (top) or BSP-treated (1 μM; bottom) cells. Target regions of photobleaching are indicated with a white circle. Scale bar, 10 μm. FL-BRD4, full-length BRD4; FL-BRD9, full-length BRD9. (F) Quantitative comparison of time to half-maximal fluorescence recovery for BRD4 FRAP studies using BSP (red bars) as a function of ligand concentration. (G) FRAP evaluation of full-length GFP-tagged BRD9 dissociation from chromatin in U2OS cells. Nuclei of DMSO-treated (top) or BSP-treated (1 μM; bottom) cells in the presence of 10 μM SAHA (added to increase the experimental window). Target regions of photobleaching are indicated with a white circle. Scale bars, 10 μm. (H) Quantitative comparison of time to half-maximal fluorescence recovery for BRD9 FRAP studies using BSP (red bars) as a function of ligand concentration. Data in (F) and (H) represent means ± SEM (n = 30) and are annotated with P values obtained from a two-tailed t test (*P < 0.05 and ***P < 0.001).

Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia

Sci Adv. 2016 Oct;2(10):e1600760.