BaoChau Le

BET Inhibition Silences Expression of MYCN and BCL2 and Induces Cytotoxicity in Neuroblastoma Tumor Models

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.

Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762.

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

Development and Validation of Reagents and Assays for EZH2 Peptide and Nucleosome High-Throughput Screens

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [3H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

Activation of the p53-MDM4 regulatory axis defines the anti-tumour response to PRMT5 inhibition through its role in regulating cellular splicing

Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.

Abstract 5379: A potent EZH2 inhibitor exhibits long residence time and anti-tumor activity

The EZH2 histone methyltransferase is frequently mutated in diffuse large B-cell lymphoma leading to increased trimethylation of histone H3 lysine 27 (H3K27me3). Drug discovery efforts have previously identified a pyridone-based chemical series of EZH2 inhibitors that potently and selectively inhibit EZH2 catalytic activity. These compounds are capable of globally decreasing H3K27me3 levels, de-repressing EZH2 target genes, and inducing growth inhibition of many lymphoma cell lines both in cell culture and in vivo. Through medicinal chemistry optimization, we have developed EZH2 inhibitors with significantly improved potency in both biochemical and cellular assays. These compounds exhibit a prolonged enzyme residence time that can be further extended in vitro through the addition of an H3K27me3 peptide. Herein, we report the biochemical and cellular activity of these new EZH2 inhibitors. Citation Format: Heidi Ott, Glenn van Aller, Jessica Ward, BaoChau Le, Cynthia Rominger, James Foley, Susan Korenchuk, Charles McHugh, Michael Butticello, Charles Blackledge, James Brackley, Joelle Burgess, Celine Duquenne, Neil Johnson, Jiri Kasparec, Louis LaFrance, Mei Li, Kenneth McNulty, Kenneth Newlander, Stuart Romeril, Stanley Schmidt, Mark Schulz, Dai-Shi Su, Dominic Suarez, Xinrong Tian, Christopher Carpenter, Juan Luengo, Ryan Kruger, Steven Knight, Michael T. McCabe. A potent EZH2 inhibitor exhibits long residence time and anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5379. doi:10.1158/1538-7445.AM2015-5379

Abstract 2867: Inhibition of BET bromodomain proteins as a therapeutic approach in small cell lung cancer

BET (bromodomain and extra-terminal) family proteins are epigenetic readers that regulate expression of genes involved in cell growth and oncogenesis. Selective small molecule BET inhibitors, such as the GSK I-BETs (I-BET762, I-BET151), abrogate binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate proliferation and survival in a number of hematologic cancer models, partially through down-regulation of the critical oncogene, MYC. We and others have previously shown activity for BET inhibitors in solid tumor models such as neuroblastoma and prostate cancer, with concomitant down-regulation of MYC family gene expression. However, MYC or MYCN silencing only partially accounts for the activity of BET inhibitors in these models, suggesting that transcriptional regulation of multiple pathways promotes I-BET induced phenotypes. Here we describe the activity of the selective BET inhibitor, I-BET762, in small cell lung cancer (SCLC), a highly aggressive malignancy with few treatment options. We observe potent growth inhibition and apoptosis in a subset of cell lines and patient-derived SCLC models in vitro, as well as significant tumor growth inhibition in cell line and primary mouse xenografts. These anti-proliferative effects are likely mediated by inhibition of BRD2, BRD3, and BRD4, as siRNAs individually targeting all three proteins result in partial inhibition of SCLC cell line growth. Similar to our observations in neuroblastoma and prostate cancer, I-BET762 treatment in SCLC cell lines results in transcriptional changes affecting MYC and other pathways. We explore the contribution of these changes to the anti-proliferative effects observed in SCLC models, and identify potential combination strategies to enhance the activity of I-BET762. Taken together, our data highlight the potential of BET inhibitors as novel therapeutic agents to treat small cell lung cancer driven by various oncogenic pathways. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Citation Format: Anastasia Wyce, BaoChau Le, Yuchen Bai, David Soong, Xi-Ping Zhang, Jeanne J. Matteo, Susan Korenchuk, Michael F. Butticello, Ramona Plant, Maureen R. Bleam, Yan Degenhardt, Charles F. McHugh, Christopher Carpenter, Peter J. Tummino, Olena Barbash. Inhibition of BET bromodomain proteins as a therapeutic approach in small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2867. doi:10.1158/1538-7445.AM2015-2867

Abstract 382: Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer

BET (bromodomain and extra-terminal) family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in a number of hematologic cancer models, partially through down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will similarly regulate expression of MYC family genes (MYC, MYCN, MYCL1) in solid tumor models characterized by MYC family amplification or over-expression. We and others have recently shown activity for BET inhibitors in MYCN-amplified neuroblastoma models, with concomitant down-regulation of MYCN expression. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression and cell growth in prostate cancer models. I-BET762 treatment inhibited MYC expression accompanied by growth inhibition and decreased survival in prostate cancer cell lines that over-express MYC. In addition to MYC signatures, gene expression profiling in cell lines identified numerous cell cycle-associated genes as being significantly down-regulated by I-BET762. Importantly, our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes. Consistent with our in vitro observations, BET inhibition reduces MYC expression and tumor burden in a primary model of castration resistant prostate cancer that expresses high levels of MYC. Taken together, our data highlight the potential of BET inhibitors as a novel therapeutic approach to treat prostate tumors driven by MYC over-expression. Citation Format: Anastasia Wyce, Yan Degenhardt, Yuchen Bai, BaoChau Le, Susan Korenchuk, Ming-Chih Crouthamel, Charles F. McHugh, Robert Vessella, Caretha L. Creasy, Peter J. Tummino, Olena Barbash. Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 382. doi:10.1158/1538-7445.AM2014-382

Abstract 2939: Discovery and synthesis of highly potent and selective small molecule inhibitors of the histone methyltransferase EZH2

The histone methyltransferases are a group of enzymes which catalyze the transfer of a methyl group from the co-factor S-Adenosylmethionine (SAM) to the lysine and arginine residues of histone tails. This post-translational modification is a key event in maintaining gene expression patterns. In recent years, the relationships between aberrant histone methylation patterns and cancer progression have been recognized. These developments, along with an improved understanding of the underlying structural biology, have made histone methyltransferases highly attractive targets for therapeutic intervention. The histone lysine methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is frequently over-expressed in a wide variety of cancerous tissues. There is a strong correlation between overexpression of EZH2 and aberrant transcriptional signaling in cells, ultimately resulting in poor clinical prognosis. Inhibition of EZH2 is expected to alter transcriptional expression and ultimately lead to an improved clinical outcome. This presentation will describe medicinal chemistry efforts in the development of highly potent and selective small molecule inhibitors of EZH2. The synthesis, SAR, and identification of a clinical candidate will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2939. doi:1538-7445.AM2012-2939