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BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

The loss of the retinoblastoma tumor suppressor gene (RB) is common in many human cancers, including prostate. We previously reported that engineered deletion of RB in prostate epithelial cells results in sustained cell growth in serum-free media, a predisposition to develop hyperplasia and dysplasia in prostate tissue recombinant grafts, and sensitization to hormonal carcinogenesis. Examining the molecular consequence of RB loss in this system, we show that cyclooxygenase-2 (COX-2) is significantly up-regulated following RB deletion in prostate tissue recombinants. To study the effect of RB deletion on COX-2 regulation, we generated wild-type (PrE) and Rb-/- (Rb-/-PrE) prostate epithelial cell lines rescued by tissue recombination. We show elevated COX-2 mRNA and protein expression in Rb-/-PrE cell lines with increased prostaglandin synthesis. We also find that loss of Rb leads to deregulated E2F activity, with increased expression of E2F target genes, and that exogenous expression of E2F1 results in elevated COX-2 mRNA and protein levels. COX-2 promoter studies reveal that E2F1 transcriptionally activates COX-2, which is dependent on the transactivation and DNA-binding domains of E2F1. Further analysis revealed that the E2F1 target gene, c-myb, is elevated in Rb-/-PrE cells and E2F1-overexpressing cells, whereas ectopic overexpression of c-myb activates the COX-2 promoter in prostate epithelial cells. Additionally, cotransfection with E2F1 and a dominant-negative c-myb inhibited E2F1 activation of the COX-2 promoter. Taken together, these results suggest activation of a transcriptional cascade by which E2F1 regulates COX-2 expression through the c-myb oncogene. This study reports a novel finding describing that deregulation of the Rb/E2F complex results in increased COX-2 expression and activity.

Tumor suppressor gene silencing by DNA hypermethylation contributes to tumorigenesis in many tumor types. This aberrant methylation may be due to increased expression and activity of DNA methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine to cytosines in CpG dinucleotides. Elevated expression of the maintenance DNA methyltransferase, DNA methyltransferase 1 (DNMT-1), has been shown in carcinomas of the colon, lung, liver, and prostate. Based on the nearly ubiquitous alterations of both DNA methylation and the retinoblastoma protein (pRb) pathway found in human cancer, we investigated a potential regulatory pathway linking the two alterations in murine and human prostate epithelial cells. Analysis of DNA methyltransferase levels in Rb-/- murine prostate epithelial cell lines revealed elevated Dnmt-1 levels. Genomic DNA sequence analysis identified conserved E2F consensus binding sites in proximity to the transcription initiation points of murine and human Dnmt-1. Furthermore, the Dnmt-1 promoter was shown to be regulated by the pRb/E2F pathway in murine and human cell lines of epithelial and fibroblast origin. In the absence of pRb, Dnmt-1 transcripts exhibited aberrant cell cycle regulation and Rb-/- cells showed aberrant methylation of the paternally expressed gene 3 (Peg3) tumor suppressor gene. These findings show a link between inactivation of the pRb pathway and induction of DNA hypermethylation of CpG island-containing genes in tumorigenesis.

The retinoblastoma tumor suppressor (pRb) has traditionally been studied as a negative regulator of cell cycle progression through its interactions with the E2F family of transcription factors. Utilizing prostate epithelial cell lines established from Rb+/+ and Rb-/- prostate tissues, we previously demonstrated that Rb-/- epithelial cells were not transformed and retained the ability to differentiate in vivo despite the lack of pRb. To further study the effects of pRb loss in an epithelial cell population, we utilized oligonucleotide microarrays to identify any pRb-dependent transcriptional regulation during serum depletion-induced growth arrest. These studies identified 120 unique transcripts regulated by growth arrest in Rb+/+ cells. In these wild-type cells, the majority (80%) of altered transcripts were downregulated, including 40 previously identified E2F target genes. Although the transcriptional repression of E2F target genes is characteristic of pRb pocket protein family activity, further analysis revealed that, compared to Rb+/+ cells, Rb-/- cells exhibited a nearly identical response for all transcripts including those of E2F target genes. These findings demonstrate that pRb is not strictly required for the vast majority of transcriptional alterations associated with growth arrest.