Figure 1.
I-BET726: a novel selective inhibitor of BET family proteins.
(a) Chemical structure of GSK1324726A (I-BET726). (b) Crystal structure of I-BET726 (magenta) bound to the acetyl-binding pocket of BRD4-BD1 (resolution: 1.6 Å). (c) Concentration response curves for determination of binding affinity of I-BET726 to BRD2, BRD3, and BRD4 bromodomains by ligand displacement detected using Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET). IC50 values for BRD2, BRD3, and BRD4 are indicated. (d) Selectivity profile of I-BET726 showing average temperature shifts (delta Tm) in degrees Celsius for a panel of bromodomain proteins using a fluorescent thermal shift assay. N= 2 for all proteins except CREBBP (n= 4).
Figure 2.
I-BET726 treatment results in potent growth inhibition and cytotoxicity in neuroblastoma cell lines.
(a) gIC50 values observed for I-BET726 in a panel of neuroblastoma cell lines obtained from a 6 day growth-death assay. (b) Concentration response curves for SK–N-AS and CHP-212 from 6 day growth-death assay. Black horizontal line indicates growth in DMSO-treated controls. Red line indicates T0 value (100%). gIC50 and Ymin-T0 values are indicated. Data presented as the average of two independent curves from a single experiment, and is representative of data from three independent biological replicates. (c) Ymin-T0 values observed for I-BET726 in the panel of neuroblastoma cell lines obtained from a 6 day growth-death assay. Gray bars indicate a cytotoxic response, defined by a Ymin-T0 value ≤ -50, with evidence of net cell death (Y < T0) along the growth curve at concentrations less than 6 µM. (d) Graph summarizing number of cells in G1 phase (as a percent of total cell population) in the indicated neuroblastoma cell lines following treatment with a titration of I-BET726 (5 nM-20 000 nM) for 2 days based on propidium iodide staining. V represents vehicle (DMSO) control sample. (e) Histograms generated from cell cycle analysis in the CHP-212 cell line following 4 days treatment with the indicated concentration of I-BET726. Percentage of cells in G1 phase and sub-G1 phase are indicated. (f) Caspase induction in the indicated neuroblastoma cell lines following treatment with a titration of I-BET726 for one, two, or three days. Data is presented as fold induction over DMSO controls, following normalization to total cell number as measured by CellTiter-Glo.
Figure 3.
Global transcript profiling in neuroblastoma cell lines treated with I-BET726 reveals gene expression changes in apoptotic and signaling pathways.
(a) Hierarchical clustering of statistically significant probes that were differentially expressed in 100 nM or 1 µM treatments of I-BET726 relative to vehicle in CHP-212 and SK–N–SH. (b) Venn analysis for up-regulated and down-regulated probes described in (a) in the CHP-212 cell line. (c) Venn analysis for overlap of up-regulated and down-regulated probes in the SK–N–SH and CHP-212 cell lines. (d) Functional analyses of expression changes were performed by GO and canonical pathway enrichment at the gene level. A subset of statistically significant categories for GO Biological Process (>100 genes) and canonical pathways (>20 genes) from KEGG and BioCarta that were common among the two cell lines are shown. (e) qRT-PCR confirmation of a subset of genes selected from the functional analyses described in (d). Data represent mean value ± standard deviation for three independent biological replicates. Asterisks indicate statistical significance as measured by t-test (p <0.05).
Figure 4.
Global transcript profiling reveals gene expression changes in MYC-family pathways.
(a) qRT-PCR confirmation of changes in MYCN and MYC expression following treatment with the indicated concentration of I-BET726. Data represent mean value ± standard deviation for three independent biological replicates. Asterisks indicate statistical significance as measured by t-test (p <0.05). (b) GSEA enrichment plots showing the down-regulation of gene sets associated with Myc/Max and N-Myc binding motifs in I-BET726-treated CHP-212 cells. Normalized enrichment scores (NES) and FDR q values are indicated. (c) A MYCN transcriptional regulation network was constructed (see Materials and Methods) to depict N-Myc pathway genes that were modulated by I-BET726 treatment. Red and blue circles represent increased and decreased expression changes, respectively. Green, red and grey edges are shown for activation, inhibition and unspecified interaction types, respectively. (d) qRT-PCR confirmation of a subset of genes selected from the N-Myc network analysis described in (c). Data presented as described in (a).
Figure 5.
MYCN expression is directly regulated by BRD4 and repressed by treatment with I-BET726.
(a) Left: Concentration response curve for MYCN RNA expression following 24 hour treatment with I-BET726 in the CHP-212 cell line. Data was normalized to GAPDH and is presented as expression relative to DMSO-treated controls. Data presented as the average of two independent curves from a single experiment, and is representative of data from three independent biological replicates. Right: Table of IC50 and percent inhibition values for MYCN suppression following 24 hour treatment with I-BET726 in the indicated cell lines. (b) BRD4 ChIP in the non-MYCN-amplified cell line SK–N–SH. Binding of BRD4 to the MYCN promoter or to an intergenic region on Chromosome 12 following treatment with vehicle or 1µM I-BET726 for six hours. Data is presented as fold enrichment over signal generated from IgG control immunoprecipitations. Data shown is from a single experiment representative of typical results. (c) BRD4 ChIP data at the MYCN promoter, presented as percent of vehicle control signal in the non-MYCN-amplified cell line SK–N–SH (left) and the MYCN-amplified cell line CHP-212 (right). SK–N–SH data represents the mean value ± standard deviation for three independent biological replicates. Asterisk indicates statistical significance as measured by t-test (p= 0.005). CHP-212 data represents the mean value ± standard deviation for two independent biological replicates. (d) Western blot analysis of N-Myc expression in the MYCN-amplified cell lines CHP-212 and IMR32 following 24 or 48 hour treatment with vehicle or 1µM I-BET726. Actin expression included as a loading control. (e) gIC50 values obtained from CHP-212 cells overexpressing GFP or MYCN following treatment with I-BET726 in a 6 day growth-death assay. Data represents the mean value ± standard deviation from four independent experiments. (f) Concentration response curves for GFP or MYCN–overexpressing CHP-212 cells from a 6 day growth-death assay. Horizontal line indicates T0 measurement (normalized to 100%). Data shown was from a single experiment representative of typical results. (g) Left: Histograms generated from cell cycle analysis in GFP- or MYCN-overexpressing CHP-212 cells following 4 days treatment with 5 µM I-BET726. Right: Percentage of cells in subs G1, G1, S, and G2 phases from the cell cycle experiment.
Figure 6.
Suppression of BCL2 expression by I-BET726.
(a) Left: Concentration response curve for BCL2 RNA expression following 24 hour treatment with I-BET726 in the CHP-212 cell line. Data was normalized to GAPDH and presented as expression relative to DMSO-treated controls. Data presented as the average of two independent curves from a single experiment, and is representative of data from two independent biological replicates. Right: Table of IC50 values and percent inhibition of BCL2 expression following 24 hour treatment with I-BET726. (b) BRD4 ChIP in the non-amplified neuroblastoma cell line SK–N–SH. Binding of BRD4 to the BCL2 promoter or to an intergenic region on Chromosome 12 following treatment with vehicle or 1 µM I-BET726 for six hours. Data is presented as fold enrichment over signal generated from IgG control immunoprecipitations. Data shown was from a single experiment representative of typical results. (c) BRD4 ChIP data at the BCL2 promoter, presented as percent of vehicle control signal. Data represent the mean value ± standard deviation for three independent biological replicates. Asterisk indicates statistical significance as measured by T-test (p= 0.002). (d) Western blot analysis of Bcl-2 expression in the MYCN-amplified cell lines CHP-212 and IMR32 following 48 hour treatment with vehicle or 1 µM I-BET726. Tubulin expression included as a loading control. (e) gIC50 values obtained from CHP-212 or LA-N-2 cells overexpressing GFP or BCL2 following treatment with I-BET726 in a 6 day growth-death assay. Data represents the mean value ± standard deviation from three independent experiments. (f) Ymin-T0 values for CHP-212 or LA-N-2 cells overexpressing GFP or BCL2. Data represents the mean value ± standard deviation from three independent experiments. Asterisk indicates statistical significance as measured by t-test (p= 0.02). (g) Western blot analysis of Bcl-2 expression in CHP-212 or LA-N-2 cells overexpressing GFP or BCL2 following 48 hour treatment with DMSO or 1 µM I-BET726. Actin expression included as a loading control.
Figure 7.
Analysis of I-BET726 activity in vivo.
(a) Mean absolute body weight ± SD for mice in the SK–N-AS (left) and CHP-212 (right) xenograft studies treated with vehicle, 5 mg/kg, or 15 mg/kg I-BET726. (b) Mean absolute tumor volumes ± SEM for SK–N-AS subcutaneous xenografts following treatment with 5 mg/kg or 15 mg/kg I-BET726. Asterisks indicate statistical significance as measured by t-test (p <0.05). Tumor growth inhibition (TGI) for the 15 mg/kg group was 58% on day 14 (n= 9; p= 0.0060). (c) Mean absolute tumor volumes ± SEM for CHP-212 subcutaneous xenografts following treatment with 5 mg/kg or 15 mg/kg I-BET726. Asterisks indicate statistical significance as measured by T-test (p <0.05). TGI for 5 mg/kg was 50% on Day 42 (n= 8; p= 0.1816). TGI for 15 mg/kg was 82% on Day 42 (n=5; p =0.0488). (d) Pharmacodynamic analysis in CHP-212 and SK–N-AS xenografts 8 hours after initial dose of I-BET726. qRT-PCR analysis of MYCN, MYC, and BCL2 expression following I-BET726 treatment in the indicated models. Data is presented as fold induction compared to vehicle treated controls, and represents the average ± SD of data from three animals. (e) qRT-PCR analysis of apoptotic pathway and N-Myc pathway genes in CHP-212 xenografts 8 hours following treatment with I-BET726 on day 8 of study. Data is presented as described in (d).