Article Figures & Data
Figures
- Figure 1.
MYCN-amplified neuroblastoma is sensitive to the effects of BET bromodomain inhibition. A, antiproliferative activity of JQ1 was profiled in more than 650 cancer cell lines, revealing a broad range of sensitivity and resistance. Red dots are neuroblastoma cell lines with MYCN amplification based on SNP 6.0 arrays and/or high levels of protein expression. Black dots indicate neuroblastoma cell lines wild-type (WT) for MYCN and poor MYCN expression. Drug response is presented as ln(IC50), plotted against the maximum effect corresponding to the minimum measured viability (Emax). B, distribution of Emax and ln(IC50) for MYCN–wild type versus MYCN-amplified cancer cell lines based on SNP 6.0 copy number analysis. P value was calculated using nonparametric Mann–Whitney test. Red squares indicate MYCN-amplified neuroblastoma cell lines. C, Spearman correlation between minimum measured viability (Emax), and the expression level of MYCN was normalized by actin level (ratio of MYCN/actin). D, structures of BET bromodomain inhibitors. (+)-JQ1, I-BET, and I-BET151 are all active and structurally distinct, BET bromodomain inhibitors. JQ1R is the inactive (−)-JQ1 enantiomer. E, Dose response of neuroblastoma cell line viability with BET bromodomain inhibitor treatment was measured by a luminescent ATP detection assay. Data represent mean ± SEM of 4 biologic replicates. F, 4 MYCN-amplified neuroblastoma cell lines were used to determine the effects of JQ1 on growth. Values over time are shown relative to the day 0 values, with error bars representing the mean ± SD of 8 replicates per condition.
- Figure 2.
JQ1 treatment induces a G0–G1 arrest and apoptosis in neuroblastoma cell lines. Indicated neuroblastoma cell lines were treated with 1 μmol/L JQ1 for (A) 24, 48, and 72 hours before cell-cycle analysis or (B) 72 hours before measuring apoptosis by annexin V staining detected by flow cytometry.
- Figure 3.
Inhibition of MYCN- and c-MYC–dependent transcription by JQ1 treatment of neuroblastoma cells. A, heatmap of the top 50 down- and upregulated genes following 24 hours of 1 μmol/L JQ1 treatment of neuroblastoma cell lines based on a signal-to-noise ratio (SNR) score and P < 0.05. Data are presented as row normalized. B, GSEA showing downregulation of MYCN- and c-MYC–dependent gene sets and representative sets of genes with proximal promoter regions containing MYCN- or MYC-MAX–binding sites in the transcriptional profiles of neuroblastoma cell lines treated with JQ1. Depicted is the plot of the running sum for the MSigDB gene set within the JQ1 neuroblastoma dataset, including the maximum enrichment score and the leading edge subset of enriched genes. C, table of selective gene sets enriched among genes downregulated by JQ1 in neuroblastoma cell lines based on GSEA (size, number of genes in each set; NES, normalized enrichment score). D, GSEA showing downregulation of a custom MYCN gene set derived from the comparison of MYCN-amplified versus MYCN-nonamplified primary neuroblastoma tumors. E, quantitative comparison of all transcription factor target gene sets available from the MSigDB by GSEA for downregulation in JQ1-treated neuroblastoma cells. Data are presented as a scatterplot of FDR versus NES for each evaluated gene set. Red indicates sets for either MYCN or c-MYC and gray for other transcription factors. F, neuroblastoma cell lines were treated with 1 μmol/L JQ1 or DMSO for 8 hours. After RNA extraction, level of MYCN transcript was quantified. Expression values are shown relative to the DMSO condition for each cell line. Error bars represent mean ± SD of 4 technical replicates. *, P < 0.001 calculated using a one-way ANOVA with Bonferroni correction comparing JQ1 treatment with DMSO within a cell line. G, Western blot analyses for MYCN on whole-cell extracts from 4 neuroblastoma cell lines treated with 1 μmol/L JQ1 or DMSO for 24 hours.
- Figure 4.
Transcriptional changes associated with BET bromodomain inhibition by JQ1. A, heatmap of the 36 down- and 17 upregulated genes after JQ1 treatment, with a consistent direction of regulation in neuroblastoma, multiple myeloma, and AML and fold-change greater than 2 and P ≤ 0.05. Data are presented as row normalized. B, heatmap of the JQ1 consensus signature developed in A, evaluated in a dataset profiling genome-wide expression of primary neuroblastoma tumors. JQ1 consensus signature genes denoted in blue are downregulated and those in red are upregulated with JQ1 treatment. Data are presented as row normalized. The neuroblastoma samples cluster into 2 groups, which are associated with the MYCN amplification status of the tumors (P < 0.002 by a two-tailed Fisher exact test) and with high stage (stage III and IV) versus low stage (all others; P < 0.002 by a two-tailed Fisher exact test). C, a relaxed consensus JQ1 downregulation signature was identified on the basis of the absolute fold change ≥ 1.5 and P value and FDR ≤ 0.05 and was interrogated in a functional enrichment analysis across the MSigDB. The results were visualized with the Enrichment Map software, which organizes the significant gene sets into a network called an “enrichment map.” In the enrichment map, the nodes correspond to gene sets and the edges reflect significant overlap between the nodes according to a two-tailed Fisher exact test. The hubs correspond to collections of gene sets with a unifying class label according to gene ontology (GO) biologic processes. The size of the nodes is correlated with the number of genes in the gene set. D, table describing the results of a two-tailed Fisher exact tests for the MSigDB signatures enriched with genes selectively downregulated by JQ1 in neuroblastoma cells. Heatmap of the genes uniquely regulated by JQ1 in neuroblastoma in the neural development (E) and apoptosis-related (F) gene sets. Data are presented as row normalized.
- Figure 5.
A, ChIP with a BRD4 antibody at 2 sites within the MYCN promoter region in BE(2)-C cells treated with 1 μmol/L JQ1 for 24 hours. Enrichment is shown as the percentage of total input DNA. The negative control region primers amplify within a gene desert region approximately 1 Mbaway from MYCN. Error bars represent ± SEM of triplicate data. *, P < 0.05; **, P < 0.01 (paired t test). B, Western blot analysis showing effects of 1 μmol/L JQ1 treatment for 24 hours on the expression of MCM7 and MDM2 in MYCN-amplified neuroblastoma cells. C, neuroblastoma cell lines were treated with 1 μmol/L JQ1 or DMSO for 24 hours. After RNA extraction, level of PHOX2B transcript was quantified. Expression values are shown relative to the DMSO condition for each cell line. Error bars represent mean ± SD of 4 technical replicates. *, P < 0.001 calculated using a one-way ANOVA with Bonferroni correction comparing JQ1 treatment with DMSO within a cell line.
- Figure 6.
Effect of BRD4 downregulation on neuroblastoma cell lines. A–E, the 3 most JQ1-sensitive neuroblastoma cell lines were transduced with shRNAs targeting BRD4 or a control shRNA. A, RNA was extracted at day 3 post transduction and BRD4 transcript level was quantified. Shown are the expression values relative to the shRNA control–transduced cells. Error bars represent mean ± SD of 4 technical replicates. *, P < 0.05; **, P < 0.01, calculated using a one-way ANOVA, with Bonferroni correction. B, viability values over days post transduction are shown relative to the day 0 (time of seeding) values, with error bars representing the mean ± SD of 8 replicates per condition. C, apoptosis analysis was conducted on day 6 post transduction. D, Western blot analyses showing BRD4 and MYCN protein levels on day 4 post transduction with 3 BRD4-directed shRNAs. E, MYCN transcript levels were quantified 4 days after transduction. The expression values are relative to cells infected with a control shRNA. Error bars represent mean ± SD of 4 technical replicates. *, P < 0.001 calculated using a one-way ANOVA, with Bonferroni correction, comparing each BRD4-directed shRNA with the control shRNA within a cell line.
- Figure 7.
Testing of JQ1 in multiple in vivo models of MYCN-amplified neuroblastoma. A, mice were injected with BE(2)-C cells subcutaneously and treated with JQ1 or vehicle once tumors reached 100 mm3. After 15 days, tumor volume was measured. Error bars indicate mean ± SD of 5 mice. *, P = 0.01, calculated using nonparametric Mann–Whitney test. B, effects of JQ1 treatment on survival in the BE(2)-C xenograft model. Day 0 indicates the first day of treatment, and mice were treated until time of sacrifice. C, a human MYCN-amplified primary neuroblastoma tumor was implanted into kidney capsule of nude mice, and mice were treated once daily with JQ1 for 28 days, starting 7 days after orthotopic transplantation. D, TH-MYCN mice with palpable tumors were treated with JQ1 or vehicle once daily for 28 days. Statistical significance (A–C) was determined by log-rank (Mantel–Cox) test for the survival curves, as shown. Day 0 indicates the day of treatment initiation. E, staining for Ki-67 (red), cleaved caspase-3 (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue) in GEMM tumors treated with either vehicle or JQ1, as indicated. F, Western blot analysis indicating the expression of MYCN and MCM7 in GEMM tumors treated with either vehicle or JQ1, as indicated.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legend - PDF file - 35K
- Supplementary Figures 1 -11 - PDF file - 326K, This file contains 11 supplementary figures with biochemistry, computational analysis, in vitro and in vivo studies to support the role of targeting MYCN in neuroblastoma with BETi.
- Supplementary Tables 1 - 9 - PDF file - 93K, This file contains 9 supplementary tables including the full primary screening data and secondary testing of BETi in neuroblastoma, supporting tables for the expression profiling analyses, and the list of Affymetrix probes (MYCN) and shRNA sequences for BRD4 and MYCN.
Volume 3, Issue 3
