Targeting MYCN in neuroblastoma by BET bromodomain inhibition

Abstract

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.

Figure 1

MYCN-amplified neuroblastoma is sensitive to the effects of BET bromodomain inhibition. (A) Anti-proliferative activity of JQ1 was profiled in over 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 wildtype for MYCN and poor MYCN expression. Drug response is presented as the natural log of the half-maximal effective concentration [Ln(IC50)], plotted against the maximum effect corresponding to the minimum measured viability (Emax). (B) Distribution of Emax and Ln(IC50) for MYCN wildtype versus MYCN amplified cancer cell lines based on SNP 6.0 copy number analysis. P value calculated using non parametric 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 normalized by Actin level (ratio MYCN/Actin). (D) Structures of BET bromodomain inhibitors. (+)-JQ1, I-BET, and I-BET151 are all active, structurally distinct, BET bromodomain inhibitors. JQ1R is the inactive (−)-JQ1 enantiomer. (E) Dose response of neuroblastoma cell line viability with BET bromodomain inhibitor treatment measured by a luminescent ATP detection assay. Data represent mean ± SEM of four biological replicates. (F) Four MYCN-amplified neuroblastoma cell lines were used to determine the effects of JQ1 on growth. Values over time are shown relative to the day zero values, with error bars representing the mean +/− SD of eight 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 μM 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 up-regulated genes following 24 hours of 1 μM JQ1 treatment of neuroblastoma cell lines based on an SNR Score and P < 0.05. Data are presented row normalized. (B) GSEA demonstrating 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 molecular signature data base (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, and FDR = false discovery rate). (D) GSEA showing downregulation of a custom MYCN gene set derived from the comparison of MYCN-amplified versus MYCN-non-amplified 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 μM 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 four technical replicates. *P < 0.001 calculated using a one-way ANOVA with Bonferroni correction comparing JQ1 treatment to DMSO within a cell line. (G) Western-blots for MYCN on whole cell extracts from four neuroblastoma cell lines treated with 1 μM 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 up-regulated genes after JQ1 treatment with a consistent direction of regulation in neuroblastoma, multiple myeloma, and AML and fold-change greater than two and P ≤ 0.05. Data are presented row normalized. (B) Heatmap of the JQ1 consensus signature developed in Fig 4A evaluated in a data set 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 row normalized. The neuroblastoma samples cluster into two groups which are associated with the MYCN amplification status of the tumors (P < 0.002 by Fisher exact test) and with high stage (stage 3 and 4) versus low stage (all other) (P < 0.002 by Fisher exact test). (C) A relaxed consensus JQ1 downregulation signature was identified based on the absolute FC ≥ 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 Fisher test. The hubs correspond to collections of genes sets with a unifying class label according to GO biological processes. The size of the nodes is correlated with the number of genes in the gene set. (D) Table describing the results of Fisher 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 row normalized.

Figure 5

(A) ChIP with a BRD4 antibody at two sites within the MYCN promoter region in BE(2)-C cells treated with 1 μM 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 ~1Mb away from MYCN. Error bars represent ± SEM of triplicate data. *P < 0.05, **P < 0.01 (paired t-test). (B) Western blot demonstrating effects of 1 μM 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 μM 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 +/minus; SD of four technical replicates. * P < 0.001 calculated using a one-way ANOVA with Bonferroni correction comparing JQ1 treatment to DMSO within a cell line.

Figure 6

Effect of BRD4 downregulation on neuroblastoma cell lines. (A–E) The three most JQ1-sensitive neuroblastoma cell lines were transduced with shRNAs targeting BRD4 or a control shRNA. (A) RNA was extracted at day three 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 four technical replicates. *P < 0.05 and **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 zero (time of seeding) values, with errors bars representing the mean +/− SD of eight replicates per condition. (C) Apoptosis analysis was performed on day 6 post-transduction. (D) Western blots showing BRD4 and MYCN protein levels on day four post-transduction with three BRD4-directed shRNAs. (E) MYCN transcript levels were quantified four days after transduction. The expression values are relative to cells infected with a control shRNA. Error bars represents mean +/− SD of four technical replicates. *P < 0.001 calculated using a one-way ANOVA, with Bonferroni correction, comparing each BRD4-directed shRNA to 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 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 five mice. *P = 0.01 calculated using non parametric 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 DAPI (blue) in GEMM tumors treated with either vehicle or JQ1 as indicated. (F) Western blot indicating the expression of MYCN and MCM7 in GEMM tumors treated with either vehicle or JQ1 as indicated.