Epigenetic Inhibitors Research Articles

Abstract LB-92: RVX-2135 is a novel, orally bioavailable epigenetic BET inhibitor that synergizes with cytarabine and idarubicin to inhibit proliferation of acute myeloid leukemia cells.

Abstract Current treatment of acute myeloid leukemia (AML) often includes a combination of intravenous cytarabine (Ara-C) and an anthracycline such as idarubicin. Bromodomain and extra-terminal domain (BET) proteins contain two bromodomains that bind to acetylated lysines in histones and regulate gene transcription by an epigenetic mechanism. Here we describe a novel small molecule BET inhibitor, RVX-2135, which selectively binds recombinant BET bromodomains of BRD2, BRD3, and BRD4 but not non-BET bromodomains. RVX-2135 inhibited BRD2, BRD3, and BRD4 dual bromodomains binding to acetylated histones in vitro with sub to low micromolar IC50 values. In the human AML cell line MV4-11, RVX-2135 displaced BET proteins from chromatin and significantly reduced expression of c-MYC and BCL-2 mRNAs, with IC50s of 4 and 5 μM, respectively. RVX-2135 inhibited proliferation of cultured MV4-11 cells with an IC50 value of 6 μM, and induced cell cycle arrest and apoptosis. In vivo, nude mice bearing MV4-11 xenografts were treated orally with RVX-2135 at doses of 75 and 120 mg/kg b.i.d, and showed tumor growth inhibition of 77 and 92% respectively. There was a corresponding inhibition of c-MYC and BCL-2 mRNA expression in the xenograft tumors. In combination therapy in vitro, RVX-2135 synergized with idarubicin and cytarabine, with Chou-Talalay's combination indices of 0.17 and 0.18, respectively. The synergistic effect also translated into increased apoptosis when cytarabine was combined with RVX-2135. Similar data were seen with BET inhibitors from different chemical scaffolds, suggesting that this may represent a promising new class of drugs for AML. Citation Format: Eric Campeau, Ravi Jahagirdar, Jin Wu, Emily M. Gesner, Olesya Kharenko, Raymond Yu, Sarah Attwell, Henrik C. Hansen, Gregory S. Wagner, Kevin G. McLure, Peter R. Young. RVX-2135 is a novel, orally bioavailable epigenetic BET inhibitor that synergizes with cytarabine and idarubicin to inhibit proliferation of acute myeloid leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-92. doi:10.1158/1538-7445.AM2013-LB-92

Abstract 1016: Combination low dose 5-azacytidine (AZA) and romidepsin (FK228) therapy re-sensitizes ovarian cancer cells to cisplatin.

Abstract Ovarian cancer is the deadliest gynecologic malignancy. Current options for treating platinum-resistant disease are limited by drug choices and toxicities. Epigenetic therapy is emerging as a strategy for reversing tumor resistance and a recent encouraging clinical trial in platinum-resistant ovarian cancer suggests a potential unique application in ovarian cancer. The epigenetic drugs histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) cause chromatin relaxation and multiple anti-tumor effects on cell growth, cell cycle, cell death and deregulation of DNA damage response and repair (DDR) pathways. Among HDACi we have tested in a panel of ovarian cancer cells, the depsipeptide FK228 exerts the most potent growth inhibitory effects in the majority of cells. Therefore, we aimed to determine whether the combination of FK228 and the DNMTi 5-azacytadine (AZA) can re-sensitize ovarian cancer cells to cisplatin more effectively than either agent alone. We found that FK228 synergized with low doses of the DNMTi 5-azacytadine (AZA) in SRB cell growth assays in vitro, and that low dose combinations of the drugs enhanced the growth inhibitory effects of cisplatin. Similar effects were observed when ovarian cancer cells were grown as xenografts in immunocompromised mice in vivo. Furthermore, a key DDR sensor, phosphorylated gamma-H2AX (pH2AX) activation was associated with the enhanced cytotoxic effects of the combination of the epigenetics and may be a marker of response. In conclusion, we have identified a novel combination of epigenetic drugs that can re-sensitize ovarian cancer cells to cisplatin treatment, at least in part through stimulation of pH2AX. Targeting DDR pathways in DDR-deficient ovarian cancer cells with low dose epigenetic drugs represents a promising strategy in ovarian cancer treatment. Citation Format: Andrew Wilson, Lanzi Sinaise, Jade M. Readus, Michelle Park, Anum S. Lalani, Jeanette Saskowski, Dineo Khabele. Combination low dose 5-azacytidine (AZA) and romidepsin (FK228) therapy re-sensitizes ovarian cancer cells to cisplatin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1016. doi:10.1158/1538-7445.AM2013-1016

Reversibility of Membrane N-Glycome of HeLa Cells upon Treatment with Epigenetic Inhibitors

Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.

Epigenetics and Novel Therapeutic Approaches

Epigenetic changes are targeted to the DNA, as well as chromatin core histones and unlike genetic mutations these changes are reversible, making them attractive targets for therapeutic purposes [1]. Multiple levels of epigenetic regulatory mechanisms provide orchestration of chromatin structure for gene expression in healthy cells; however, result inappropriate silencing or activation of gene expressions, finally leading to various disease states such as cancer [2]. Epigenetic modifications can be a determinant factor to characterize the disease states, and detailed profiling of these modifications may allow predictions of disease outcome or therapeutic strategies. Epigenetic therapy can offer promising tools to restore/reverse changes. Interestingly, several epigenetic inhibitors such as DNA methyltransferase inhibitors (DNMTis), and histone deacetylase inhibitors (HDACis) approved by the US Food and Drug Administration (FDA) are in clinics for the treatment of various hematological malignancies. Potential epigenetic modifications, such as DNA methylation, covalent histone modifications, such as histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation, non-covalent mechanisms, such as incorporation of histone variants, nucleosome remodeling and positioning, microRNAs (miRNAs) expression are important targets for therapeutic purposes [2,3]. How these effectors interact to each other to affect gene expression and cause disease is the active field of study in epigenetics.

Epigenetic therapy of hematological malignancies: where are we now?

A growing amount of evidence points towards alterations in epigenetic machinery as a leading cause in disease initiation and progression. Like genetic alterations, misregulation of the epigenetic regulators can lead to abnormal gene expression. However, unlike genetic events, the epigenetic machinery may be targeted pharmacologically, potentially resulting in the reversal of a particular epigenetic state. The success of DNA methyltransferase and histone deacetylase inhibitors represents a proof of concept for the use of therapies intended to target the epigenome in the treatment of hematological malignancies. Nevertheless, the molecular mechanisms underlying the efficacy of these agents have not been completely elucidated. Recently, a large number of studies sequencing cancer cell genomes identified recurring mutations of epigenetic regulators, providing new insights into the molecular underpinnings of cancer. Consequently, the efforts to identify specific epigenetic inhibitors have been expanded in order to target particular subsets of patients. This review will summarize the progress made using the currently available epigenetic therapies and discuss some of the more recently identified targets whose inhibition may present potential avenues for the treatment of hematologic malignancies.

An In Vitro Library Screen to Rapidly Determine Activity Against Normal and Mutant Bone Marrow Progenitor Cells

AbstractAbstract 4916 Background:Aberrant signal transduction plays a central role in the pathogenesis of MDS/MPN, as indicated by the high prevalence of mutations that activate Ras signaling. Yet despite the central role of Ras signaling in the pathogenesis of JMML, at this time there are no signal transduction inhibitors with established efficacy in JMML. A screen of inhibitors has the potential to reveal potential therapeutic strategies and inform efforts to treat other neoplasms driven by hyperactive Ras signaling, both in the hematopoietic system and elsewhere. Aim:To investigate novel therapeutic options for JMML by utilizing a novel, reproducible system for rapid screening in primary cells. Innovations include using flow cytometry to isolate a highly clonogenic, disease-relevant “PreGM” population of primary bone marrow cells that recapitulate the abnormal growth pattern characteristic of JMML and unsorted bone marrow, the use of a genetically engineered mouse model, and the development of automated microscopy protocols. Method:Unfractionated bone marrow cells harvested from Mx1-Cre, KrasD12 and wildtype mice were utilized in the screens. PreGM cells, identified as Lineage lo/- Sca1- c-kit+ CD34+ CD16/32- CD105- CD150-, were purified from harvested bone marrow using flow cytometry. The purified PreGM cells were sorted into 96 well plates containing various inhibitors at set concentrations ranging from 1X (5 μg/ml, approx. 10 μM for most compounds) to 10−7X (5×10−7 μg/ml). The freshly sorted PreGM cells were exposed to inhibitors for 3 days under standard culture conditions (at 37°C, 98% humidity and 5% CO2) in 80% IMDM, 20% FBS and saturating dose of 10ng/ml of GM-CSF. At the end of that period, cell growth was quantified using the IN Cell Analyzer 2000 (GE).A total of 94 different inhibitors were screened using this method. The screen included a negative control (DMSO) and cytotoxic positive controls (Cytarabine, Adriamycin and Gemcitabine). Compound families included cyotoxic agents, tyrosine kinase inhibitors, PI3K family inhibitors, mitotic kinase inhibitors, epigenetic modifiers, hedgehog signaling inhibitors, and others. The majority of compounds were either FDA approved drugs or agents used in recent clinical trials. Candidates were screened for preferential activity against Mx1-Cre, KrasD12 cells. Results:Primary bone marrow cells were harvested from a total of 28 mice, 18 wild type (WT) and 10 Mx1-Cre, KrasD12. PreGM growth was quantified and dose response curves constructed for WT and mutant cells. WT and mutant IC50s for each compound were calculated using the ‘drc' package from the R Project for Statistical Computing. Out of 94 candidates tested in this screen, none were found to demonstrate preferential inhibitory activity against Mx1-Cre, KrasD12 cells. Neither were any of the drugs found to be comparatively toxic to WT cells or to have significantly higher IC50s in mutant PreGM cells in comparison to WT cells. Some compounds of interest included Vorinostat, an epigenetic inhibitor, which was found to have robust inhibitory activity against both mutant and WT cells. It has comparable IC50s in mutant and WT cells with a calculated IC50 of 0. 0480X (std. error: 0. 135) in Mx1-Cre, KrasD12 cells and 0. 0244X (std. error: 0. 0293) in WT cells. Conclusion:None of the 94 compounds used in the screen were found to preferentially inhibit mutant or WT cell growth, indicating that Kras mutant cells have similar drug sensitivities to normal cells over a broad range of mechanistic approaches. These findings suggest that it may be difficult to find “synthetic lethal” opportunities for drugs that are selectively toxic to primary cells driven by hyperactive Ras signaling. Disclosures:No relevant conflicts of interest to declare.

Current Trends In Epigenetic Drug Discovery

Epigenetics is a major field of biomedical research, and epigenetic drug discovery shows great promise for new drugs. The first epigenetic inhibitors are already approved for human treatment. Here, we review a number of case studies that cover different aspects of epigenetic drug discovery spanning from sequencing of epigenetic modifications, assays development over screening to medicinal chemistry, in vivo testing and clinical application.

Differential Cellular and Molecular Effects of Butyrate and Trichostatin A on Vascular Smooth Muscle Cells

The histone deacetylase (HDAC) inhibitors, butyrate and trichostatin A (TSA), are epigenetic histone modifiers and proliferation inhibitors by downregulating cyclin D1, a positive cell cycle regulator, and upregulating p21Cip1 and INK family of proteins, negative cell cycle regulators. Our recent study indicated cyclin D1 upregulation in vascular smooth muscle cells (VSMC) that are proliferation-arrested by butyrate. Here we investigate whether cyclin D1 upregulation is a unique response of VSMC to butyrate or a general response to HDAC inhibitors (HDACi) by evaluating the effects of butyrate and TSA on VSMC. While butyrate and TSA inhibit VSMC proliferation via cytostatic and cytotoxic effects, respectively, they downregulate cdk4, cdk6, and cdk2, and upregulate cyclin D3, p21Cip1 and p15INK4B, and cause similar effects on key histone H3 posttranslational modifications. Conversely, cyclin D1 is upregulated by butyrate and inhibited by TSA. Assessment of glycogen synthase 3-dependent phosphorylation, subcellular localization and transcription of cyclin D1 indicates that differential effects of butyrate and TSA on cyclin D1 levels are linked to disparity in cyclin D1 gene expression. Disparity in butyrate- and TSA-induced cyclin D1 may influence transcriptional regulation of genes that are associated with changes in cellular morphology/cellular effects that these HDACi confer on VSMC, as a transcriptional modulator.

Identification of PR-SET7 and EZH2 selective inhibitors inducing cell death in human leukemia U937 cells

Chemical manipulations undertaken on some bis(bromo- and dibromo-phenol) compounds previously reported by us as wide-spectrum epigenetic inhibitors let us to identify bis (bromo- and dibromo-methoxyphenyl) derivatives highly selective for PR-SET7 and EZH2 (compounds 4, 5, 9, and 10). Western blot analyses were carried out in U937 cells to determine the effects of such compounds on the methyl marks related to the tested enzymes (H3K4me1, H3K9me2, H4H20me1, and H3K27me3). The 1,5-bis(3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one 4 (EC(50) vs EZH2 = 74.9 μM), tested in U937 cells at 50 μM, induced massive cell death and 28% of granulocytic differentiation, highlighting the potential use of EZH2 inhibitors in cancer.

Decitabine and Vorinostat Cooperate To Sensitize Colon Carcinoma Cells to Fas Ligand-Induced Apoptosis In Vitro and Tumor Suppression In Vivo

The death receptor Fas and its physiological ligand (FasL) regulate apoptosis of cancerous cells, thereby functioning as a critical component of the host cancer immunosurveillance system. To evade Fas-mediated apoptosis, cancer cells often downregulate Fas to acquire an apoptosis-resistant phenotype, which is a hallmark of metastatic human colorectal cancer. Therefore, targeting Fas resistance is of critical importance in Fas-based cancer therapy and immunotherapy. In this study, we demonstrated that epigenetic inhibitors decitabine and vorinostat cooperate to upregulate Fas expression in metastatic human colon carcinoma cells. Decitabine also upregulates BNIP3 and Bik expression, whereas vorinostat decreased Bcl-x(L) expression. Altered expression of Fas, BNIP3, Bik, and Bcl-x(L) resulted in effective sensitization of the metastatic human colon carcinoma cells to FasL-induced apoptosis. Using an experimental metastasis mouse model, we further demonstrated that decitabine and vorinostat cooperate to suppress colon carcinoma metastasis. Analysis of tumor-bearing lung tissues revealed that a large portion of tumor-infiltrating CD8(+) T cells are FasL(+), and decitabine and vorinostat-mediated tumor-suppression efficacy was significantly decreased in Fas(gld) mice compared with wild-type mice, suggesting a critical role for FasL in decitabine and vorinostat-mediated tumor suppression in vivo. Consistent with their function in apoptosis sensitization, decitabine and vorinostat significantly increased the efficacy of CTL adoptive transfer immunotherapy in an experimental metastasis mouse model. Thus, our data suggest that combined modalities of chemotherapy to sensitize the tumor cell to Fas-mediated apoptosis and CTL immunotherapy is an effective approach for the suppression of colon cancer metastasis.

Abstract 4888: Decitabine and vorinostat cooperate to sensitize metastatic human colon carcinoma cells to Fas-mediated apoptosis

Abstract The death receptor Fas and its physiological ligand, FasL, which is expressed on the surface of cytotoxic T-cells (CTLs), regulate apoptosis of cancerous cells. While normal colon epithelial cells express high levels of Fas, Fas expression is diminished in colon carcinomas. Metastatic colon cancers acquire an apopotosis-resistant phenotype often due to the loss of Fas expression and function. Previous studies have implicated Fas-mediated apoptosis as an important contributor of tumor regression. Therefore, targeting Fas resistance is of critical importance in treating colon cancer through Fas-based cancer therapy and immunotherapy. In this study, we explore whether epigenetic pharmacological agents can restore Fas expression and re-establish sensitivity to Fas-mediated apoptosis in metastatic human colon carcinoma cells. First we determined that the epigenetic drugs Decitabine, a DNA methyltransferase inhibitor, and Vorinostat, a HDAC inhibitor, each are capable of up-regulating Fas expression. When combined, these two inhibitors cooperate to increase Fas expression to an even greater extent than each agent alone. Pre-treatment with decitabine, vorinostat, or both drugs followed by incubation with FasL, was able to overcome resistance to FasL-induced apoptosis. Analysis of bisulfite-modified genomic DNA, isolated from human metastatic colon carcinoma cells revealed that the Fas promoter is sporadically methylated; therefore sensitization of Fas receptor expression on the cell surface by decitabine was unlikely through direct inhibition of Fas promoter meythlation. Although the Fas promoter was not heavily methylated, the promoters for BNIP3 and Bik, which are downstream of Fas signaling, were methylated. After treatment with these pharmacological epigenetic inhibitors, alone or in combination, protein levels of both BNIP3 and Bik were increased. Our data suggest that combined modalities of chemotherapy and Fas-based immunotherapy may be an effective approach for the suppression of colon cancer metastasis. 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 4888. doi:1538-7445.AM2012-4888

An inhibitor of histone demethylases specifically blocks cancer growth in vitro and in vivo

Histone modifying enzymes are highly deregulated in cancers, leading to aberrant transcriptional programs and thus constitute key therapeutic targets. Using an unbiased cell‐based screen, we have identified a novel compound that modulates the activity of Jumonji histone demethylases. Here, we report the anti‐cancer properties and the mode of action of this small molecule called Elizib. We show, through viability measurements across a large panel of human cancer and normal epithelial cells, that this drug has remarkable specificity for cancer with IC50 values as low as 2 nM. In vivo, it effectively reduces tumor burden in mouse xenograft models. Elizib causes cancer‐cell specific changes in cell cycle progression, upregulates pro‐apoptotic and differentiation genes and downregulates proliferative genes, partially re‐establishing a normal transcriptional program in cancer cells, leading to their demise. Enzyme activity assays and chromatin immunoprecipitation studies demonstrate that our small molecule exerts these activities in cell culture and in vivo at least in part through inhibition of demethylases of the Jumonji family. Thus, we have identified a novel epigenetic inhibitor with specific anti‐cancer activity in cells and in vivo. Elizib opens up a new therapeutic venue and is a novel chemical tool to unravel aspects of epigenetic transcriptional regulation in cancer.

Outcome of acute myeloid leukaemia following myelodysplastic syndrome after azacitidine treatment failure

Outcome of acute myeloid leukaemia following myelodysplastic syndrome after azacitidine treatment failure

Epigenetic modulation of the HeLa cell membrane N-glycome

BackgroundEpigenetic changes play a role in all major events during tumorigenesis and changes in glycan structures are hallmarks of virtually every cancer. Also, proper N-glycosylation of membrane receptors is important in cell to cell and cell–environment communication. To study how modulation of epigenetic information can affect N-glycan expression we analyzed effects of epigenetic inhibitors on HeLa cell membrane N-glycome. MethodsHeLa cells were treated with DNA methylation (zebularin and 5-aza-2-deoxycytidine) and histone deacetylation (trichostatin A and Na-butyrate) inhibitors. The effects on HeLa cell membrane N-glycome were analyzed by hydrophilic interaction high performance liquid chromatography (HILIC). ResultsEach of the four epigenetic inhibitors induced changes in the expression of HeLa cell membrane N-glycans that were seen either as an increase or a decrease of individual glycans in the total N-glycome. Compared to DNA methylation inhibitors, histone deacetylation inhibitors showed more moderate changes, probably due to their higher gene target selectivity. ConclusionsThe results clearly show that composition of HeLa cell membrane N-glycome can be specifically altered by epigenetic inhibitors. General significanceGlycans on the cell membrane are essential elements of tumor cell's metastatic potential and are also an entry point for nearly all pathogenic microorganisms. Since epigenetic inhibitors used in this work are registered drugs, our results provide a new line of research in the application of these drugs as anticancer and antimicrobial agents. This article is part of a Special Issue entitled Glycoproteomics.

Death Receptor Pathway Activation and Increase of ROS Production by the Triple Epigenetic Inhibitor UVI5008

Deregulation of the epigenome is recognized as cause of cancer and epigenetic factors are receiving major attention as therapeutic targets; yet, the molecular mode of action of existing epi-drugs is largely elusive. Here, we report on the decryption of the mechanism of action of UVI5008, a novel epigenetic modifier, that inhibits histone deacetylases, sirtuins, and DNA methyltransferases. UVI5008 highly efficiently induces cancer cell-selective death in a variety of models and exerts its activities in several human tumor xenografts and genetic mouse models of human breast cancer in vivo. Its anticancer activity involves independent activation of death receptors and reactive oxygen species production. Importantly, UVI5008 action is not critically dependent on p53, Bcl-2 modifying factor, and/or TNF-related apoptosis-inducing ligand as cell death is efficiently induced in cells mutated or deficient for these factors limiting the risk of drug resistance development and maximizing its application spectrum. The simultaneous modulation of multiple (epigenetic) targets promises to open new avenues with unanticipated potential against cancer.

Promises and challenges of anticancer drugs that target the epigenome

The occurrence of epigenetic aberrations in cancer and their role in promoting tumorigenesis has led to the development of various small molecule inhibitors that target epigenetic enzymes. In preclinical settings, many epigenetic inhibitors demonstrate promising activity against a variety of both hematological and solid tumors. The therapeutic efficacy of those inhibitors that have entered the clinic however, is restricted predominantly to hematological malignancies. Here we outline the observed epigenetic aberrations in various types of cancer and the clinical responses to epigenetic drugs. We furthermore discuss strategies to improve the responsiveness of both hematological and solid malignancies to epigenetic drugs.

Emerging therapeutic approaches in the management of metastatic castration-resistant prostate cancer.

Although treatment options for men with castration-resistant prostate cancer (CRPC) have improved with the recent and anticipated approvals of novel immunotherapeutic, hormonal, chemotherapeutic and bone-targeted agents, clinical benefit with these systemic therapies is transient and survival times remain unacceptably short. Thus, we devote the second section of this two-part review to discussing emerging therapeutic paradigms and research strategies that are entering phase II and III clinical testing for men with metastatic CRPC. We will discuss a range of emerging hormonal, immunomodulatory, antiangiogenic, epigenetic and cell survival pathway inhibitors in current clinical trials, with an emphasis on how these therapies may complement our existing treatment options.

Epigenetic Regulation of Glucose Transporters in Non-Small Cell Lung Cancer

Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy.

Computer- and structure-based lead design for epigenetic targets

The term epigenetics is defined as inheritable changes that influence the outcome of a phenotype without changes in the genome. Epigenetics is based upon DNA methylation and posttranslational histone modifications. While there is much known about reversible acetylation as a posttranslational modification, research on reversible histone methylation is still emerging, especially with regard to drug discovery. As aberrant epigenetic modifications have been linked to many diseases, inhibitors of histone modifying enzymes are very much in demand. This article will summarize the progress on small molecule epigenetic inhibitors identified by structure- and computer-based approaches.

HDAC Class I Inhibition Acetylates a Non-Histone Protein STAT3 by Modulating p300-STAT3-HDAC1 Interaction In Activated B- Cell Like (ABC) Diffuse Large B Cell Lymphoma

AbstractAbstract 115Patients with diffuse large B- cell lymphoma (DLBCL) tumors that have an activated B-cell like (ABC) gene expression profile have a poorer prognosis. Understanding the mechanism(s) used by ABC tumor cells to resist the effects of common chemotherapy agents may lead to alternative approaches for the treatment of these tumors. ABC cell lines have been shown to have high levels of phosphorylated STAT3 (pSTAT3); however, the mechanisms that regulate STAT3 signaling in ABC DLBCL remain unclear. Histone deacetylases (HDACs) are enzymes that can deacetylate both non-histone and histone substrates. In this study we tested the hypothesis that HDACs in the tumor cells target a non-histone protein STAT3 in ABC DLBCL.In studies of HDAC expression in DLBCL tumors, we found over-expression of the type 1 HDACs, specifically HDAC1and HDAC3, in the pSTAT3- positive ABC tumors as compared to germinal centre B like (GCB) tumors. We then performed a co-immunoprecipitation (Co-IP) assay to learn the functional interaction between STAT3 and HDAC1. We found that STAT3 formed complexes with HDAC1 or HDAC3. Further Co-IP studies demonstrated that p300, a histone acetyltransferase (HAT), STAT3, and HDAC1 are all in the same complex. To determine whether p300 acetylates STAT3 in ABC cells, we immuno-precipitated endogenous p300 and blotted with acetylated STAT3 and showed that p300 acetylates STAT3 at lysine 685. We next tested whether HDAC inhibition could affect p300 mediated STAT3 acetylation in ABC cells. Inhibition of HDAC activity through the HDAC inhibitor LBH589 (LBH, Novartis Pharmaceuticals) increased STAT3 acetylation in a dose- dependent manner. Similar results were obtained when we used antiacetyl- lysine antibody. Furthermore HDAC1 over-expression inhibits STAT3 acetylation at lysine 685. This data implies a tight regulation of STAT3 acetylation and deacetylases in vivo in ABC lymphoma. In addition to acetylation, STAT3 can be modified by phosphorylation, thus the effect of HDAC inhibition on pSTAT3 both at serine and tyrosine residues was studied. We observed a dose-dependent decrease in pSTAT3 with some inhibitory effect on total STAT3. LBH was found to mediate STAT3 dephosphorylation by inhibiting the tyrosine phosphorylation of JAK2 and TYK2, the STAT3 upstream activators, in a dose- dependent manner. Since ABC lymphoma has higher levels of HDAC1 or HDAC3 and pSTAT3/STAT3 than GCB, we hypothesized that ABC cells will be more sensitive to HDAC inhibition than GCB. In fact, when ABC and GCB DLBCL cells were treated with LBH we observed that LBH was more cytotoxic to ABC than GCB as evidenced by annexin/PI staining and PARP cleavage. LD90 was 25 nM for ABC cells, however GCB cells required 5 times more LBH to kill 90% cells. STAT3 activation regulates genes involved in cell survival, including Bcl-2, Mcl-1, Bcl-XL, and c-Myc. LBH treatment resulted in down-regulation of Mcl-1 and c-Myc in ABC cells but has no effect in GCB cells; however, Bcl-2 and Bcl-XL levels were not decreased in both the subtype. Having established that HDAC1 physically associated with STAT3 and that LBH treatment elevated STAT3 acetylation in ABC cells, we proceeded to deplete endogenous HDAC1 with siRNA in Ly3 cells and found that HDAC1 knockdown up-regulated STAT3 acetylation indicating that HDAC1 negatively regulates the acetylation in vivo. HDAC1 inhibition also prevented phopshorylation of STAT3 and induces aopotosis in ABC cells.In summary, we have demonstrated that a key consequence of HATs and HDACs expression and activity is modulation of the STAT3 pathway in ABC lymphoma. Inhibition of this pathway with the HDAC inhibitor LBH inhibits constitutive STAT3 signaling and induces Mcl-1 mediated apoptosis. These studies provide the rationale for targeting the poorly responsive ABC-type DLBCL by inhibiting HDAC activity with epigenetic inhibitors such as LBH. We are currently testing LBH589 in relapsed DLBCL in a phase I clinical trial. Disclosures:No relevant conflicts of interest to declare.