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

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Science Advances  12 Oct 2016:
Vol. 2, no. 10, e1600760
DOI: 10.1126/sciadv.1600760
  • Fig. 1 BSP is a pan-BRD inhibitor in vitro.

    (A) Triazolopyridazine scaffold of BSP. (B) 2FoFc map of BSP bound to BRD4(1) contoured at 2σ. (C) Complex of BSP with the BRD of BRD9. The compound adopts an acetyl-lysine mimetic pose within the BRD cavity, initiating interactions with the conserved asparagine (N100). The sulfonamide function initiates contacts with ZA-loop residues (G43), further stabilizing the interaction without displacing any of the structurally conserved water molecules (red spheres). (D) BSP binding with low micromolar to nanomolar affinity to most structural classes within the human BRD family. Dissociation constants (KD) measured in-solution using ITC are displayed on the human BRD tree as spheres (size and color as indicated in the inset). BRD structural classes are annotated with roman numerals. (E) Overlay of ITC measurements of typically strong BSP interactions with the BRDs of CECR2, BRD9, and TAF1L(2). Raw injection heats for the titrations of proteins into solutions of BSP are shown on the left panels. The right panel shows the normalized binding enthalpies corrected for the heat of protein dilution as a function of binding site saturation (symbols as indicated in the figure). Solid lines represent a nonlinear least-squares fit using a single-site binding model. All titrations were carried out in 50 mM Hepes buffer (pH 7.5; 25°C) and 150 mM NaCl at 15°C while stirring at 1000 rpm.

  • 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).

  • Fig. 3 BSP inhibits growth in leukemia cell lines.

    (A) BSP inhibits clonogenic growth in leukemia cell lines. K562, KASUMI-1, MV4;11, and OCI-AML3 in methylcellulose were treated with vehicle (DMSO) or BSP (0.1, 0.5, or 1 μM) (n = 4). (B) Colony formation assay in K562, KASUMI-1, MV4;11, and OCI-AML3 cells using 0.1, 0.5, or 1.0 μM BSP (top) and the number of cells counted after treatment of cells with BSP for 6 to 10 days (n = 4) (bottom). CFU, colony-forming units; ns, not significant. (C) Similarity comparison of significantly expressed genes (P < 0.001 and fold change > 1.5) in the four cell lines after drug treatment. The heat map represents the intersect matrix for all pairwise comparisons (cell lines and treatments) using euclidean distances and complete linkage after transformation of the intersect counts into similarity Jaccard measures. (D) Venn diagrams showing overlap of the top statistically significant (Benjamini-Hochberg adjusted P < 0.001) genes (up- or down-regulated with a fold change of >1.5) differentially expressed by BSP or the pan-BET inhibitor JQ1 in four leukemia cell lines (K562, KASUMI-1, OCI-AML3, and MV4;11) after 8 hours of treatment with the inhibitor (0.5 μM) (top) and breakdown of the expression in terms of up- and down-regulated genes for each cell line (bottom). (E) Heat map of log fold changes in the expression of the top 50 statistically significant genes in the four cell lines tested, identified using Benjamini-Hochberg adjusted P < 0.001. Data in (B) represent means ± SEM (n = 4) and are annotated with P values obtained from a two-tailed t test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

  • Fig. 4 Comparison of the effects of BET inhibition on cell cycle and transcription in leukemias.

    (A and B) Cell cycle analysis of BSP (blue scale) and JQ1 (red scale) inhibition in resistant cells (A; K562) or sensitive cells (B; MV4;11) after 48 hours of treatment with inhibitors (amounts as indicated in the inset). The quantification given below each graph depicts the percent S-phase content measured under each condition, indicating a clear arrest in the sensitive line with minor effects on the resistant line. Data are means ± SEM (n = 3) and are annotated with P values obtained from analysis of variance (ANOVA) followed by Dunnett’s test (*P < 0.05 and **P < 0.01). (C) Heat map of fold changes (expressed in log2 scale as indicated in the inset) in the top 1000 significantly differentially expressed genes (Benjamini-Hochberg adjusted P < 0.001) in K562 cells (top) and MV4;11 cells (bottom) after 6 hours of treatment with selective BRD inhibitors or DMSO. The effects of JQ1 and BSP are very similar and much stronger than the effects of any other compounds targeting non-BET BRDs. (D) A published set of genes constituting a “JQ1 signature” is only attenuated by BSP and JQ1 in K562 cells (left) and MV4;11 cells (right) after 6 hours of treatment with a series of inhibitors (500 nM). The heat map depicts row-normalized values for gene expression, as indicated in the inset.

  • Scheme 1 Synthesis of BSP.
  • Table 1 ITC of human BRDs with BSP.

    Titrations were carried out in 50 mM Hepes buffer (pH 7.5; 25°C) and 150 mM NaCl at 15°C while stirring at 1000 rpm. Proteins were titrated into the ligand solution (reverse titration). Values are means ± SD.

    Protein[P] (μM)[L] (μM)KD (nM)ΔHobs (kcal/mol)NTΔS (kcal/mol)ΔG (kcal/mol)
    BAZ2A433153,745 ± 291−3.04 ± 0.0951.05 ± 0.0254.12−7.16
    BAZ2B60715Weak binding
    BRD1440131,653 ± 66−7.32 ± 0.0781.01 ± 0.0080.30−7.62
    BRD2(1)2712597.1 ± 6.7−7.90 ± 0.0341.00 ± 0.0031.34−9.25
    BRD2(2)2352550.3 ± 5.0−5.46 ± 0.0281.10 ± 0.0034.18−9.64
    BRD3(1)2752091.7 ± 5.3−10.02 ± 0.0391.00 ± 0.002−0.95−9.08
    BRD3(2)3052550.0 ± 4.7−8.62 ± 0.0411.11 ± 0.0031.01−9.63
    BRD4(1)2582041.8 ± 2.8−11.09 ± 0.0380.94 ± 0.002−1.36−9.73
    BRD4(2)2702039.7 ± 2.2−6.60 ± 0.0180.94 ± 0.0013.17−9.77
    BRDT(1)2282040.2 ± 2.8−13.16 ± 0.0471.02 ± 0.002−3.40−9.76
    BRDT(2)27120172.1 ± 10.6−5.61 ± 0.0281.00 ± 0.0033.31−8.92
    BRD92512541.7 ± 3.8−8.75 ± 0.0391.00 ± 0.0020.98−9.73
    BRPF1B40620311.5 ± 11.2−6.12 ± 0.0211.00 ± 0.0032.45−8.57
    BRPF3400158,621 ± 381−4.20 ± 0.1231.08 ± 0.0252.48−6.68
    CECR2202168.0 ± 1.0−17.28 ± 0.0621.04 ± 0.002−6.60−10.68
    CREBBP617251,524 ± 116−2.91 ± 0.0411.03 ± 0.0114.72−7.64
    EP300460157,194 ± 501−5.65 ± 0.2650.97 ± 0.0361.13−6.78
    BPTF230151,887 ± 53−10.09 ± 0.0741.07 ± 0.005−3.28−6.80
    GCN5L233615Weak binding
    PB1(3)38915Weak binding
    PB1(5)6041514,225 ± 802−3.19 ± 0.1831.09 ± 0.0533.20−6.39
    PCAF610134,762 ± 459−7.85 ± 0.4540.95 ± 0.044−0.83−7.02
    SMARCA222225Weak binding
    SMARCA44001319,685 ± 838−9.05 ± 0.5800.99 ± 0.055−2.84−6.21
    TAF1(1)460235,525 ± 199−3.34 ± 0.0460.98 ± 0.0103.60−6.94
    TAF1(2)2302016.6 ± 2.7−14.05 ± 0.0950.98 ± 0.003−3.80−10.25
    TAF1L(1)6101325,000 ± 2,027−4.78 ± 0.7781.00 ± 0.1461.29−6.07
    TAF1L(2)2502042.7 ± 4.4−12.48 ± 0.0690.99 ± 0.003−2.76−9.72
    TIF1A400158,475 ± 431−2.61 ± 0.0881.09 ± 0.0294.06−6.67
  • Table 2 ΔTm shifts (°C) of biotinylated BSP (BSP-a and BSP-b) tested against a panel of BET and other diverse BRDs.

    Compounds (final concentration, 10 μM) were added to the proteins (final concentration, 2 μM); the temperature was increased from 25° to 96°C at a step of 3°C/min; excitation and emission filters for the SYPRO Orange dye were set to 465 and 590 nm; and experiments were performed in triplicate. Values are means ± SD.

    ProteinΔTmobs (°C)
    BSP-aBSP-b
    BRD2(1)−7.5 ± 0.71.9 ± 0.5
    BRD2(2)−0.4 ± 0.23.0 ± 0.2
    BRD3(1)−2.7 ± 0.22.3 ± 0.3
    BRD3(2)−3.0 ± 0.13.1 ± 0.0
    BRD4(1)−2.7 ± 0.23.8 ± 0.3
    BRD4(2)−5.1 ± 0.01.8 ± 0.3
    BRDT(1)−5.0 ± 0.11.3 ± 0.3
    BRDT(2)0.5 ± 0.10.9 ± 0.4
    CECR24.3 ± 0.07.4 ± 0.1
    CREBBP−3.1 ± 0.60.1 ± 0.4
    TAF1(2)−12.4 ± 0.11.9 ± 0.1
    TAF1L(2)0.2 ± 0.12.9 ± 0.4
  • Table 3 Relative abundance of BRD-containing proteins in HEK293 cells (data taken from Proteomics DB; https://www.proteomicsdb.org/).

    Pull-down of human BRD-containing proteins with biotinylated BSP (BSP-a and BSP-b), followed by competitive elution with BSP and MS, resulted in enrichment of BSP-targeted BRDs.

    ProteinRelative peptide abundanceProteinRelative peptide abundance
    293BSP-aBSP-b293BSP-aBSP-b
    CECR2BRD74.51105
    BPTF4.31BRD94.577.68
    GCN5L23.75SP140L
    PCAFSP140
    BRD25.5982SP1003.43
    BRD35.0422SP1102.71
    BRD45.1732TIF1a4.91
    BRDTTRIM335.34
    BAZ1A5.19TRIM66
    BAZ1B5.630.641.33BAZ2A4.253
    BRWD33.61BAZ2B3.21
    PHIP5.051.091.15MLL3.67
    BRWD13.244TRIM287.05
    CREBBP4.61ZMYND84.4
    EP3004.87TAF116
    BRD84.48TAF1L
    ATAD24.650.980.87ZMYND11
    ATAD2B3.680.901ASH1L
    BRD13.48PB15.238.288
    BRPF13.6SMARCA24.44
    BRPF33.42SMARCA45.538.85

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/10/e1600760/DC1

    fig. S1. Topology of BRD cavity and binding of chemical scaffolds containing different potential expansion vectors.

    fig. S2. Structure-activity relationship of the triazolopyridazine class leading to BSP.

    fig. S3. BSP inhibits growth of cancer cell lines.

    fig. S4. Effect of BSP and JQ1 on leukemia cell lines.

    fig. S5. Effect of BSP and JQ1 on leukemia cell lines.

    fig. S6. Gene expression GO enrichment (biological processes).

    fig. S7. Gene expression after inhibition of leukemia cell lines with BSP or JQ1.

    fig. S8. GSEA of K562 and KASUMI-1 cell lines after BSP treatment.

    fig. S9. GSEA of MV4;11 and OCI-AML3 cell lines after BSP treatment.

    fig. S10. Effect of BSP on BET-specific genes.

    fig. S11. Expression of BRD-containing proteins in leukemic cell lines.

    fig. S12. Effects of the selective inhibition of different BRD subfamilies on transcriptional programs in leukemias.

    fig. S13. Transcriptional response in leukemia cell lines and inhibitor combination.

    fig. S14. GSEA comparison of BSP and JQ1 effects on leukemias.

    fig. S15. BSP profile of cellular receptor activity (ExpresSProfile; CEREP).

    table S1. Differential scanning fluorimetry profiling of triazolopyridazines against a panel of BRD modules.

    table S2. MetaCore analysis of gene expression data.

    table S3. BSP profile of cellular receptor activity data (ExpresSProfile; CEREP).

    table S4. Data collection and refinement statistics for BRD-BSP complexes.

    table S5. Primers used for qRT-PCR.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Topology of BRD cavity and binding of chemical scaffolds containing different potential expansion vectors.
    • fig. S2. Structure-activity relationship of the triazolopyridazine class leading to BSP.
    • fig. S3. BSP inhibits growth of cancer cell lines.
    • fig. S4. Effect of BSP and JQ1 on leukemia cell lines.
    • fig. S5. Effect of BSP and JQ1 on leukemia cell lines.
    • fig. S6. Gene expression GO enrichment (biological processes).
    • fig. S7. Gene expression after inhibition of leukemia cell lines with BSP or JQ1.
    • fig. S8. GSEA of K562 and KASUMI-1 cell lines after BSP treatment.
    • fig. S9. GSEA of MV4;11 and OCI-AML3 cell lines after BSP treatment.
    • fig. S10. Effect of BSP on BET-specific genes.
    • fig. S11. Expression of BRD-containing proteins in leukemic cell lines.
    • fig. S12. Effects of the selective inhibition of different BRD subfamilies on transcriptional programs in leukemias.
    • fig. S13. Transcriptional response in leukemia cell lines and inhibitor combination.
    • fig. S14. GSEA comparison of BSP and JQ1 effects on leukemias.
    • fig. S15. BSP profile of cellular receptor activity (ExpresSProfile; CEREP).
    • Legends for tables S1 and S2
    • table S3. BSP profile of cellular receptor activity data (ExpresSProfile; CEREP).
    • table S4. Data collection and refinement statistics for BRD-BSP complexes.
    • table S5. Primers used for qRT-PCR.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). Differential scanning fluorimetry profiling of triazolopyridazines against a panel of BRD modules.
    • table S2 (Microsoft Excel format). MetaCore analysis of gene expression data.

    Download tables S1 and S2

    Files in this Data Supplement:

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