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Elevated expression of the c-MYC oncogene is one of the most common abnormalities in human cancers. Unfortunately, efforts to identify pharmacological inhibitors that directly target MYC have not yet yielded a drug-like molecule due to the lack of any known small molecule binding pocket in the protein, which could be exploited to disrupt MYC function. We have recently described a strategy to target MYC indirectly, where a screening effort designed to identify compounds that can rapidly decrease endogenous c-MYC protein levels in a MYC-amplified cell line led to the discovery of a compound series that phenocopies c-MYC knockdown by siRNA. Herein, we describe our medicinal chemistry program that led to the discovery of potent, orally bioavailable c-MYC-reducing compounds. The development of a minimum pharmacophore model based on empirical structure activity relationship as well as the property-based approach used to modulate pharmacokinetics properties will be highlighted.

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

[Image: see text] We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure–activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.

BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. 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 several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. 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.

EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). Overexpression of EZH2 is implicated in tumorigenesis, and mutations within its catalytic domain occur in lymphoma. Here, Caretha Creasy and colleagues describe a potent small-molecule inhibitor of EZH2 methyltransferase activity that decreases levels of methylated H3K27 and reactivates silenced PRC2 target genes. It also inhibits the proliferation of EZH2 mutant cell lines and the growth of EZH2 mutant xenografts in mice. Pharmacological inhibition of EZH2 activity may therefore be a viable strategy for treating EZH2 mutant lymphoma.

Aberrant DNA hypermethylation within promoter regions and subsequent gene silencing are near universal hallmarks of human cancer. Reversal of DNA methylation by a hypomethylating agent, such as decitabine (Dacogen) or azacytidine (Vidaza), has shown clinical benefit for the treatment of heme malignancies. However, these agents have several limitations that preclude their full potential from being realized such as the requirement for IV administration, poor PK properties and a mechanism that requires incorporation into replicating DNA. This indirect, irreversible inhibition of the DNA methyltransferase (DNMT) family (DNMT1, 3a and 3b) and subsequent DNA damage induces significant dose-limiting toxicity thus preventing sufficient target engagement required for maximal demethylation. A drug discovery effort focused on DNMT1, the key family member responsible for maintaining the DNA methylation pattern, was initiated based on the compelling nature of the target coupled with the need for improved agents. A high-throughput screen identified a single dicyanopyridine (DCP) series of reversible, non-DNA incorporating, highly selective inhibitors for DNMT1 over DNMT3a or DNMT3b. Ensuing structure-activity relationship (SAR) optimization of the series led to the discovery of potent tool compounds that in cancer cells induced robust decreases in global DNA methylation, transcriptional activation of many silenced genes, and inhibition of cancer cell growth. In contrast to decitabine where most cells showed a cytotoxic response, our DNMT1 tool inhibitors primarily elicited a cytostatic response. Furthermore, studies in a mouse tumor model revealed decreased DNA methylation and a dose-dependent (1-45 mg/kg, BID) decrease in tumor growth with regression at the highest doses. In summary, a series of potent, selective DNMT1 inhibitors were discovered and refined delivering tool compounds capable of eliciting changes in DNA methylation, transcriptional activation, and tumor regression at well-tolerated doses. Thus demonstrating that selective, non-covalent inhibitors of DNMT1 may provide benefit over traditional DNA incorporating hypomethylating agents. Citation Format: Melissa B. Pappalardi, Mark Cockerill, Jessica L. Handler, Alexandra Stowell, Kathryn Keenan, Christian S. Sherk, Elisabeth A. Minthorn, Charles F. McHugh, Charlotte Burt, Kristen Wong, David T. Fosbenner, Mehul Patel, Jacques Briand, Helai Mohammad, Lourdes Rueda, Andrew Benowitz, Rab Prinjha, Dirk Heerding, Ryan G. Kruger, Ali Raoof, Allan Jordan, Bryan W. King, Michael T. McCabe. Discovery of selective, noncovalent small molecule inhibitors of DNMT1 as an alternative to traditional DNA hypomethylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2994.

SummaryEpigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.

SummaryThe EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

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.

NUT midline carcinoma (NMC) is a highly aggressive squamous cell cancer that responds poorly to standard chemotherapuetic approaches. NMC is characterized by translocations involving the NUT (nuclear protein in testes) protein, which in a majority of cases is fused to the BET (bromodomain and extra-terminal) protein family members BRD3 or BRD4. BET proteins (BRD2, BRD3, BRD4, BRDT) are epigenetic readers that modulate expression of genes involved in cell growth and oncogenesis. Selective small molecule inhibitors of BET proteins, such as the GSK I-BETs (I-BET762, I-BET151), abrogate binding of BET proteins to acetylated chromatin and inhibit the expression of BET target genes. Here we describe the activity in I-BET762 and other BET inhibitors in pre-clinical models of NMC. Consistent with previous reports, we observe profound growth inhibition and cytotoxicity in NMC cell lines in vitro, as well as significant tumor growth inhibition or tumor regression in cell line xenografts of NMC. I-BET762 treatment in NMC cell lines results in transcriptional changes affecting MYC and other pathways critical for cancer cell growth. We explore the contribution of these changes to the anti-proliferative effects observed in NMC models, and identify rational combinations to improve upon the efficacy of I-BET762 as a monotherapy. Taken together, our data highlight novel mechanisms through which BET inhibitors impact NMC cell growth and survival, and suggest potential treatment strategies to improve response in this highly aggressive disease. 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, Peter Soden, Daniel J. Felitsky, Jeanne J. Matteo, Susan Korenchuk, Gary Thripp, Kathryn Keenan, Charles F. McHugh, Rab Prinjha, Christopher Carpenter, Nicholas Smithers, Olena Barbash. Mechanism-based combination strategies for BET inhibitors in NUT midline carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4693.

BET (bromodomain and extra-terminal) family proteins are epigenetic readers that modulate expression 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 chromatin and inhibit transcription of BET target genes. We and others have previously demonstrated single agent activity for BET inhibitors in a number of pre-clinical solid and hematologic tumor models. Transcriptomics and mechanistic studies from several of these tumor types indicate that BET inhibitors influence numerous signaling pathways at the transcriptional level, including RAS/RAF/MEK signaling. Here we describe the synergistic effects of combining BET and MEK inhibitors in various solid and hematologic cancer models. We observe synergistic growth inhibition and apoptosis in a subset of cell lines representing multiple tumor types, as well as patient-derived xenograft models treated with the combination ex vivo. Additionally, combination of BET and MEK inhibitors results in improved tumor growth inhibition in cell line xenograft models compared to either single agent therapy. Further exploration of the combination in sensitive tumor types highlights multiple mechanisms potentially driving synergy, and suggests possible markers associated with sensitivity to the combination. Taken together, our data highlight the potential of BET/MEK inhibitor combinations to improve upon the efficacy observed for these agents as monotherapies in a wide variety of preclinical cancer models. 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. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents. Citation Format: Anastasia Wyce, Daniel J. Felitsky, Xi-Ping Zhang, Jeanne J. Matteo, Susan Korenchuk, Lijoy K. Mathew, Melissa Musso, Sakina Khaku, Victoria Ortiz, Kathryn Keenan, Melissa Stern, Yan Degenhardt, Ramona Plant, Charles F. McHugh, Peter J. Tummino, Christopher Carpenter, Olena Barbash. Broad activity for the combination of BET and MEK inhibitors across solid and hematologic cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4709.

The isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are mutated in acute myelogenous leukemia, low-grade glioma, intrahepatic cholangiocarcinoma, and chondrosarcomas. IDH1 and IDH2 normally function to convert isocitrate into alpha-ketoglutarate. However, when these enzymes are mutated at select residues the mutant enzymes now convert α-KG into 2-hydroxyglutarate (2-HG). In normal cells, 2-HG levels are typically extremely low, but IDH1/2 mutant cells can accumulate up to 10 mM 2-HG. In an effort to counteract the neomorphic activity of mutant IDH enzymes, we identified and developed potent inhibitors of IDH1. The compounds inhibit IDH1 catalytic activity in biochemical assays and reduce 2-HG production in IDH1-mutant cell lines. Consistent with the fact that 2-HG inhibits α-KG dependent enzymes including histone demethylases and Tet family hydroxylases, these IDH1 inhibitors induce a decrease in several histone methylation marks and also DNA methylation. These data demonstrate that small molecule inhibitors can reverse many of the epigenetic effects of mutant IDH1. Note: This abstract was not presented at the meeting. Citation Format: Cynthia Rominger, Chad Quinn, Enoch Gao, Beth Pietrak, Alan Rendina, Angela Smallwood, Arthur Groy, Susan Korenchuk, Charles McHugh, Ken Wiggall, Alexander Reif, Stanley Schmidt, Hongwei Qi, Huizhen Zhao, Nestor Concha, Christopher Carpenter, Juan Luengo, Ryan Kruger, Benjamin Schwartz, Nicholas Adams, Michael T. McCabe. A novel inhibitor of IDH1 abrogates 2-HG production and reverses aberrant epigenetic alterations in IDH1 mutant cells. [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 3514. doi:10.1158/1538-7445.AM2015-3514

PRMT5 is responsible for symmetric dimethylation of arginine residues in glycine and arginine rich (GAR) motifs on a variety of cytosolic and nuclear proteins including histones, spliceosome components, regulators of translation, transcription factors, kinases and others. PRMT5 driven methylation of some of these proteins has been implicated in tumorigenesis. For example, PRMT5 deposits repressive marks on histones and silences a subset of tumor suppressor genes, such as RB and ST7. PRMT5 methylation of non-histone substrates (such as E2F1 and p53) also contributes to cancer cell growth and death. PRMT5 driven methylation of spliceosome subunits and components of translational machinery has been well described but its connection to PRMT5's role in cancer has not been established. We have identified first-in-class small molecules that are highly potent, selective, reversible inhibitors of PRMT5. Cellular mechanistic studies revealed that PRMT5 inhibition decreases symmetric arginine dimethylation on a variety of cellular proteins including spliceosome components, histones and transcription factors. PRMT5 inhibition leads to gene expression and splicing changes ultimately resulting in the induction of p53 in lymphoma cell lines. In addition to impacting the p53 pathway, PRMT5 inhibition leads to attenuation of the expression of cell cycle related genes, genes involved in ribosome and spliceosome homeostasis, as well as genes important for cellular metabolism. PRMT5 inhibitor attenuates proliferation and induces cell death in a subset of mantle cell and diffuse large B-cell lymphoma cell lines and inhibits tumor growth in xenograft models of mantle cell lymphoma. These data underline the potential of PRMT5 inhibitors as a therapeutic strategy in mantle cell and diffuse large B-cell lymphoma. Citation Format: Olena Barbash, Sarah Gerhart, David Soong, Christine Thompson, Rocio Montes de Oca, Ping Zhang, Charles McHugh, Kristy Kuplast, Christina Majer, Richard Chesworth, Jesse Smith, Robert Copeland, Elayne Penebre, Kenneth Duncan, Neil Johnson, Chris Carpenter, Ryan Kruger. Protein arginine methyltransferase 5 (PRMT5) inhibition as a therapeutic strategy in B-cell lymphoma. [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 LB-248. doi:10.1158/1538-7445.AM2015-LB-248

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression and has been shown to play a role in hematopoietic differentiation. LSD1 is also overexpressed in multiple tumor types. These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel, irreversible, GSK LSD1 inhibitor (GSK2879552) in acute myeloid leukemia (AML) and small cell lung cancer (SCLC). GSK2879552 is a potent, selective, mechanism-based inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML and SCLC cells have a unique requirement for LSD1. While GSK2879552 treatment did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of genes whose expression increased with LSD1 inhibition. Treatment of AML cell lines with GSK2879552 increased cell surface expression of CD11b and CD86, markers associated with a differentiated immunophenotype. Six days of GSK2879552 treatment resulted in potent anti-proliferative growth effects in 19 of 25 AML cell lines representing a range of AML subtypes. Treating for longer time periods revealed sensitivity in all AML cell lines. AML blast colony forming ability was also inhibited in 4 of 5 bone marrow samples derived from primary AML patient samples. The effects of LSD1 inhibition were further characterized in vivo using a mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and were treated upon engraftment. After 17 days of treatment, control mice had 80% GFP+ cells in the bone marrow whereas treated mice had only 2.8% GFP positive cells (p Growth inhibition was also observed in a subset of SCLC cell lines. GSK2879552 treatment of mice engrafted with SCLC lines resulted in greater than 80% tumor growth inhibition. Studies using patient derived primary SCLC showed similar efficacy demonstrating the growth inhibition of SCLC with an LSD1 inhibitor extended beyond cell lines. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML and SCLC. A Phase I clinical trial using GSK2879552 was initiated in March, 2014. 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: Kimberly Smitheman, Monica Cusan, Yan Liu, Michael Butticello, Melissa Pappalardi, James Foley, Kelly Federowicz, Glenn Van Aller, Jiri Kasparec, Xinrong Tian, Dominic Suarez, Jess Schneck, Jeff Carson, Patrick McDevitt, Thau Ho, Charles McHugh, William Miller, Scott Armstrong, Christine Hann, Neil Johnson, Ryan G. Kruger, Helai P. Mohammad, Shekhar Kamat. Inhibition of LSD1 for the treatment of 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 3513. doi:10.1158/1538-7445.AM2015-3513

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

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

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

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression. LSD1 has been shown to play a role in development in various contexts. LSD1 can interact with pluripotency factors in human embryonic stem cells and is important for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 is overexpressed in multiple cancer types and recent studies suggest inhibition of LSD1 reactivates the all-trans retinoic acid receptor pathway in acute myeloid leukemia (AML). These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and through its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552) in AML. GSK2879552 is a potent, selective, mechanism-based, irreversible inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlated with a dose dependent increase in gene expression. Treatment with GSK2879552 promoted the expression of cell surface markers, including CD11b and CD86, associated with a differentiated immunophenotype in 12 of 13 AML cell lines. For example, in SKM-1 cells, increases in cell surface expression of CD86 and CD11b occurred after as early as one day of treatment with EC50 values of 13 and 7 nM respectively. In a separate study using an MV-4-11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing. GSK2879552 treatment resulted in a potent anti-proliferative growth effect in 19 of 25 AML cell lines (average EC50 = 38 nM), representing a range of AML subtypes. Potent growth inhibition was also observed on AML blast colony forming ability in 4 out of 5 bone marrow samples derived from primary AML patient samples (average EC50 = 205 nM). The effects of LSD1 inhibition were further characterized in an in vivo mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and upon engraftment, the mice were treated for 17 days. After 17 days of treatment, control treated mice had 80% GFP+ cells in the bone marrow whereas treated mice possessed 2.8% GFP positive cells (p Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. GSK2879552 is currently in late preclinical development and clinical trials are anticipated to start in 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Disclosures: Kruger: GlaxoSmithKline Pharmaceuticals: Employment. Mohammad:GlaxoSmithKline Pharmaceuticals: Employment. Smitheman:GlaxoSmithKline Pharmaceuticals: Employment. Liu:GlaxoSmithKline Pharmaceuticals: Employment. Pappalardi:GlaxoSmithKline Pharmaceuticals: Employment. Federowicz:GlaxoSmithKline Pharmaceuticals: Employment. Van Aller:GlaxoSmithKline Pharmaceuticals: Employment. Kasparec:GlaxoSmithKline Pharmaceuticals: Employment. Tian:GlaxoSmithKline Pharmaceuticals: Employment. Suarez:GlaxoSmithKline Pharmaceuticals: Employment. Rouse:GlaxoSmithKline Pharmaceuticals: Employment. Schneck:GlaxoSmithKline Pharmaceuticals: Employment. Carson:GlaxoSmithKline Pharmaceuticals: Employment. McDevitt:GlaxoSmithKline Pharmaceuticals: Employment. Ho:GlaxoSmithKline Pharmaceuticals: Employment. McHugh:GlaxoSmithKline Pharmaceuticals: Employment. Miller:GlaxoSmithKline Pharmaceuticals: Employment. Johnson:GlaxoSmithKline Pharmaceuticals: Employment. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other. Tummino:GlaxoSmithKline Pharmaceuticals: Employment.

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

EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) responsible for methylating histone H3 on lysine 27 (H3K27) and repressing transcription of target genes. Dysregulation of H3K27 methylation is implicated in tumorigenesis in both solid and hematopoietic tumors and occurs through multiple mechanisms including elevation of EZH2 due to loss of regulating miRNAs; inactivating mutations in UTX, an H3K27 demethylase which acts in opposition to EZH2; and somatic mutations in EZH2. Point mutations identified in EZH2 in both follicular lymphoma (FL) and GCB diffuse large B cell lymphoma (DLBCL) exhibit increased H3K27 tri-methylation due to an altered histone substrate preference. This suggests that inhibitors of EZH2 activity may be effective in treating lymphomas carrying activating mutations in EZH2. We have identified first-in-class inhibitors of EZH2 which are highly-potent, reversible, SAM competitive, selective and equipotent against both WT and mutant EZH2. Cellular mechanistic studies demonstrate that inhibition of EZH2 decreases global H3K27me3 in both WT and EZH2 mutant cell lines; however, the cell proliferation response is quite different. Overall, DLBCL cell lines harboring activating mutations in EZH2 are highly sensitive to EZH2 inhibition, while DLBCL cell lines with WT EZH2 are moderately sensitive or resistant. Transcriptional and ChIP-seq studies reveal differences in the transcriptional response of EZH2 target genes and methylation patterns among cell lines providing insight into the mechanism of sensitivity. In a mouse model bearing EZH2 mutant DLBCL tumor xenografts, inhibition of EZH2 methyltransferase activity reduces global H3K27me3, increases expression of EZH2 target genes and inhibits the growth of tumors in a dose dependent manner. Together, these data demonstrate for the first time that direct inhibition of EZH2 methyltransferase activity is effective at inhibiting tumor growth and suggests a promising path forward in the clinic for the treatment of EZH2 mutant lymphoma. 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 4700. doi:1538-7445.AM2012-4700