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1.  Molecular mechanisms involved in the synergistic interaction of the EZH2 inhibitor 3-deazaneplanocin A (DZNeP) with gemcitabine in pancreatic cancer cells 
Molecular cancer therapeutics  2012;11(8):1735-1746.
Pancreatic ductal adenocarcinoma (PDAC) is characterized by overexpression of Enhancer-of Zeste-Homolog-2 (EZH2), which plays a pivotal role in cancer-stem-cell (CSC) self-renewal through methylation of histone-H3-lysine-27 (H3K27m3). Against this background, EZH2 was identified as an attractive target and we investigated the interaction of the EZH2-inhibitor DZNeP with gemcitabine.
EZH2 expression was detected by quantitative-RT-PCR in 15 PDAC cells, including 7 primary cell cultures, showing expression values correlated with their originator tumors (Spearman-R2=0.89, P=0.01). EZH2 expression in cancer cells was significantly higher than in normal ductal pancreatic cells and fibroblasts.
DZNeP (5 μM, 72-hour-exposure) modulated EZH2 and H3K27m3 protein expression, and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.2 (PANC-1), 0.3 (MIA-PaCa-2) and 0.7 (LPC006). The drug combination reduced the percentages of cells in G2/M phase (e.g., from 27 to 19% in PANC-1, P<0.05), and significantly increased apoptosis compared to gemcitabine-alone. Moreover, DZNeP enhanced the mRNA and protein expression of the nucleoside transporters hENT1/hCNT1, possibly because of the significant reduction of deoxynucleotides content (e.g., 25% reduction of deoxycytidine-nucleotides in PANC-1), as detected by LC-MS/MS.
DZNeP decreased cell migration, which was additionally reduced by DZNeP/gemcitabine combination (-20% in LPc006, after 8-hour exposure, P<0.05), and associated with increased E-cadherin mRNA and protein expression. Furthermore, DZNeP and DZNeP/gemcitabine combination significantly reduced the volume of PDAC spheroids growing in CSC-selective-medium, and decreased the proportion of CD133+ cells.
All these molecular mechanisms underlying the synergism of DZNeP/gemcitabine combination support further studies on this novel therapeutic approach for treatment of PDAC.
PMCID: PMC3416916  PMID: 22622284
Pancreatic ductal adenocarcinoma; EZH2; DZNeP; gemcitabine
2.  A greener enantioselective synthesis of the antiviral agent North-methanocarbathymidine (N-MCT) from 2-deoxy-d-ribose 
Tetrahedron  2009;65(41):8461-8467.
An enantioselective synthesis of suitably protected (1R,2S,4S,5S)-4-amino-1-(hydroxymethyl)bicyclo[3.1.0]hexan-2-ol, a key starting material for the synthesis of conformationally locked carbocyclic nucleosides, including the antiviral active North-methanocarba thymidine, is reported. Starting from 2-deoxyribose the target Boc-protected amine was prepared in 33% overall yield under condition that are ecologically friendlier than previous methods.
PMCID: PMC2900805  PMID: 20625519
Conformationally locked carbocyclic nucleosides; North-methanocarbathymidine; antiviral; enantioselective synthesis
3.  Lysine Methylation Promotes VEGFR-2 Activation and Angiogenesis 
Science signaling  2013;6(304):ra104.
Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial cell receptor tyrosine kinase, promotes tumor angiogenesis and ocular neovascularization. We report the methylation of VEGFR-2 at multiple Lys and Arg residues, including Lys1041, a residue that is proximal to the activation loop of the kinase domain. Methylation of VEGFR-2 was independent of ligand binding and was not regulated by ligand stimulation. Methylation of Lys1041 enhanced tyrosine phosphorylation and kinase activity in response to ligands. Additionally, interfering with the methylation of VEGFR-2 by pharmacological inhibition or by site-directed mutagenesis revealed that methylation of Lys1041 was required for VEGFR-2–mediated angiogenesis in zebrafish and tumor growth in mice. We propose that methylation of Lys1041 promotes the activation of VEGFR-2 and that similar posttranslational modification could also regulate the activity of other receptor tyrosine kinases.
PMCID: PMC4108444  PMID: 24300896
4.  Activation of p16 gene silenced by DNA methylation in cancer cells by phosphoramidate derivatives of 2’-deoxyzebularine 
Journal of medicinal chemistry  2008;51(23):7593-7601.
We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2’-deoxyzebularine-5’-triphosphate (dZTP). Because 2’-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine-5’-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.
PMCID: PMC2659950  PMID: 19006382
5.  Superior efficacy of a combined epigenetic therapy against human Mantle Cell Lymphoma (MCL) cells 
A deregulated epigenome contributes to the transformed phenotype of Mantle Cell Lymphoma (MCL). This involves activity of the PRC (polycomb repressive complex) 2, containing three core proteins EZH2, SUZ12 and EED, in which the SET domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation (3Me) of lysine (K)-27 on histone H3 (3MeK27H3), regulates the expression of HOX genes and promotes cell proliferation and aggressiveness of the transformed cells. Here, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) depletes EZH2, SUZ12 and 3MeK27H3 in the cultured human MCL cells. Treatment with DZNep increased the expression of p21, p27 and FBXO32, while depleting Cyclin D1 and Cyclin E1 levels in MCL cells. Additionally, DZNep treatment induced cell cycle arrest and apoptosis in cultured and primary MCL cells. Further, as compared to treatment with each agent alone, co-treatment with DZNep and the pan-histone deacetylase inhibitor panobinostat (PS) caused greater depletion of EZH2, SUZ12, 3MeK27H3 and Cyclin D1 levels, while inducing greater expression of FBXO32, p16, p21 and p27. Combined treatment with DZNep and PS also synergistically induced apoptosis of cultured and primary MCL cells while relatively sparing normal CD34+ cells. Co-treatment with DZNep and PS also caused significantly greater inhibition of tumor growth of JeKo-1 xenografts in NOD/SCID mice. These preclinical in vitro and in vivo findings demonstrate that the co-treatment with DZNep and PS is an active combined epigenetic therapy worthy of further in vivo testing against MCL.
PMCID: PMC4113119  PMID: 22932665
EZH2; polycomb; mantle cell lymphoma; HDAC inhibitor
6.  Effects of Substitutions at the 4′ and 2 Positions on the Bioactivity of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine 
Antimicrobial Agents and Chemotherapy  2013;57(12):6254-6264.
Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of 4′-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2′-exo/C-3′-endo (north) conformation. Steady-state kinetic analysis of the incorporation of 4′-substituted NRTIs by RT reveals a correlation between the north conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4′-ethynyl and cyano substitutions decrease the susceptibility of adenosine-based compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pKa of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites.
PMCID: PMC3837839  PMID: 24100493
7.  Disruption Of MYC-MiRNA-EZH2 Loop To Suppress Aggressive B-Cell Lymphoma Survival And Clonogenicity 
Leukemia  2013;27(12):2341-2350.
c-MYC (hereafter MYC) overexpression has been recognized in aggressive B-cell lymphomas and linked to adverse prognosis. MYC activation results in widespread repression of miRNA expression and associated lymphoma aggressive progression. Our recent study identified a MYC-miRNAs-EZH2 feed-forward loop linking over-expression of MYC, EZH2, and miRNA repression. Here, using a novel small-molecule BET bromodomain inhibitor, JQ1 and the EZH2 inhibitor, DZNep, we demonstrated that combined treatment of JQ1 and DZNep cooperatively disrupted MYC activation, resulting in a greater restoration of miR-26a expression and synergistically suppressed lymphoma growth and clonogenicity in aggressive lymphoma cells. Furthermore, CHIP assay demonstrated that MYC recruited EZH2 to miR-26a promoter and cooperatively repressed miR-26a expression in aggressive lymphoma cell lines as well as primary lymphoma cells. Loss or gain-of-function approaches revealed that miR-26a functioned as a tumor suppressor miRNA and mediated the combinatorial effects of JQ1 and DZNep. These findings represent a novel promising approach for silencing MYC-miRNA-EZH2 amplification loop for combinatorial therapy of aggressive B-cell lymphomas.
PMCID: PMC4015113  PMID: 23538750
B-cell lymphoma; MYC; EZH2; miRNA-26a; survival and clonogenicity
8.  Coordinated Silencing of Myc-Mediated miR-29 by HDAC3 and EZH2 As a Therapeutic Target of Histone Modification in Aggressive B-Cell Lymphomas 
Cancer cell  2012;22(4):506-523.
We investigated the transcriptional and epigenetic repression of miR-29 by Myc, HDAC3, and EZH2 in mantle cell lymphoma and other Myc-associated lymphomas. We demonstrate that miR-29 is repressed by Myc through a co-repressor complex with HDAC3 and EZH2. Myc contributes to EZH2 upregulation via repression of the EZH2 targeting miR-26a, and EZH2 induces Myc via inhibition of the Myc targeting miR-494 to create positive feedback. Combined inhibition of HDAC3 and EZH2 cooperatively disrupted the Myc-EZH2-miR-29 axis, resulting in restoration of miR-29 expression, down-regulation of miR-29 targeted genes, and lymphoma growth suppression in vitro and in vivo. These findings define a Myc-mediated miRNA repression mechanism, shed light on Myc lymphomagenesis mechanisms and reveals promising therapeutic targets for aggressive B-cell malignancies.
PMCID: PMC3973134  PMID: 23079660
9.  Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS 
BMC Cancer  2014;14:139.
Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.
Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.
Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.
These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.
PMCID: PMC4016511  PMID: 24575771
EZH2; Histone methyltransferase; rhabdomyosarcoma; Polycomb proteins; Differentiation; DZnep; EZH2 catalytic inhibitors
10.  Host Immune Defense Peptide LL-37 Activates Caspase-Independent Apoptosis and Suppresses Colon Cancer 
Cancer research  2012;72(24):6512-6523.
Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin–sensitive G-protein–coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG–mediated apoptosis in colon cancer cells.
PMCID: PMC3910284  PMID: 23100468
11.  Inhibition of EZH2 suppresses self-renewal and induces radiation sensitivity in atypical rhabdoid teratoid tumor cells 
Neuro-Oncology  2012;15(2):149-160.
Introduction. Overexpression of the Polycomb repressive complex 2 (PRC2) subunit Enhancer of Zeste 2 (EZH2) occurs in several malignancies, including prostate cancer, breast cancer, medulloblastoma, and glioblastoma multiforme. Recent evidence suggests that EZH2 may also have a role in rhabdoid tumors. Atypical teratoid/rhabdoid tumor (ATRT) is a rare, high-grade embryonal brain tumor that occurs most commonly in young children and carries a very poor prognosis. ATRTs are characterized by absence of the chromatin remodeling protein SMARCB1. Given the role of EZH2 in regulating epigenetic changes, we investigated the role of EZH2 in ATRT.
Methods. Microarray analysis was used to evaluate expression of EZH2 in ATRT tumor samples. We used shRNA and a chemical inhibitor of EZH2 to examine the impact of EZH2 inhibition on cell growth, proliferation, and tumor cell self-renewal.
Results. Here, we show that targeted disruption of EZH2 by RNAi or pharmacologic inhibition strongly impairs ATRT cell growth, suppresses tumor cell self-renewal, induces apoptosis, and potently sensitizes these cells to radiation. Using functional analysis of transcription factor activity, we found the cyclin D1-E2F axis to be repressed after EZH2 depletion in ATRT cells.
Conclusions. Our observations provide evidence that EZH2 disruption alters cell cycle progression and may be an important new therapeutic target, particularly in combination with radiation, in ATRT.
PMCID: PMC3548579  PMID: 23190500
atypical teratoid/rhabdoid tumor; DZNep, EZH2; histone lysine methylation; Polycomb repressive complex 2
12.  MicroRNA-26a is Strongly Down-regulated in Melanoma and Induces Cell Death through Repression of Silencer of Death Domains (SODD) 
Melanoma is an aggressive cancer that metastasizes rapidly, and is refractory to conventional chemotherapies. Identifying miRNAs that are responsible for this pathogenesis is therefore a promising means of developing new therapies. We identified miR-26a through microarray and qRT-PCR experiments as an miRNA that is strongly down-regulated in melanoma cell lines as compared to primary melanocytes. Treatment of cell lines with miR-26a mimic caused significant and rapid cell death compared to a negative control in most melanoma cell lines tested. In surveying targets of miR-26a, we found that protein levels of SMAD1 and BAG-4/SODD were strongly decreased in sensitive cells treated with miR-26a mimic compared to the control. The luciferase reporter assays further demonstrated that miR-26a can repress gene expression through the binding site in the 3′UTR of SODD. Knockdown of these proteins with siRNA showed that SODD plays an important role in protecting melanoma cells from apoptosis in most cell lines sensitive to miR-26a, while SMAD1 may play a minor role. Furthermore, transfecting cells with a miR-26a inhibitor increased SODD expression. Our findings indicate that miR-26a replacement is a potential therapeutic strategy for metastatic melanoma, and that SODD in particular is a potentially useful therapeutic target.
PMCID: PMC3898712  PMID: 23190898
13.  EZH2 Down-Regulation Exacerbates Lipid Accumulation and Inflammation in in Vitro and in Vivo NAFLD 
Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent, chronic liver diseases, worldwide. It is a multifactorial disease caused by complex interactions between genetic, epigenetic and environmental factors. Recently, several microRNAs, some of which epigenetically regulated, have been found to be up- and/or down-regulated during NAFLD development. However, in NAFLD, the essential role of the Polycomb Group protein Enhancer of Zeste Homolog 2 (EZH2), which controls the epigenetic silencing of specific genes and/or microRNAs by trimethylating Lys27 on histone H3, still remains unknown. In this study, we demonstrate that the nuclear expression/activity of the EZH2 protein is down-regulated both in livers from NAFLD rats and in the free fatty acid-treated HepG2. The drop in EZH2 is inversely correlated with: (i) lipid accumulation; (ii) the expression of pro-inflammatory markers including TNF-α and TGF-β; and (iii) the expression of miR-200b and miR-155. Consistently, the pharmacological inhibition of EZH2 by 3-Deazaneplanocin A (DZNep) significantly reduces EZH2 expression/activity, while it increases lipid accumulation, inflammatory molecules and microRNAs. In conclusion, the results of this study suggest that the defective activity of EZH2 can enhance the NAFLD development by favouring steatosis and the de-repression of the inflammatory genes and that of specific microRNAs.
PMCID: PMC3876102  PMID: 24351808
NAFLD; EZH2; DZNep; microRNAs
14.  Differential furanose selection in the active sites of archaeal DNA polymerases probed by fixed-conformation nucleotide analogues 
Biochemistry  2012;51(45):9234-9244.
DNA polymerases select for the incorporation of deoxyribonucleotide triphosphates (dNTPs) using amino acid side-chains that act as a “steric-gate” to bar improper incorporation of rNTPs. An additional factor in the selection of nucleotide substrates resides in the preferred geometry for the furanose moiety of the incoming nucleotide triphosphate. We have probed the role of sugar geometry during nucleotide selection by model DNA polymerases from Sulfolobus solfataricus using fixed conformation nucleotide analogues. North-methanocarba-dATP (N-MC-dATP) locks the central ring into a RNA-type (C2′-exo, North) conformation near a C3′-endo pucker and South-methanocarba-dATP (S-MC-dATP) locks the central ring system into a (C3′-exo, South) conformation near a C2′-endo pucker. Dpo4 preferentially inserts N-MC-dATP and in the crystal structure of Dpo4 in complex with N-MC-dAMP, the nucleotide analogue superimposes almost perfectly with Dpo4 bound to unmodified dATP. Biochemical assays indicate that the S. solfataricus B-family DNA polymerase Dpo1 can insert and extend from both N-MC-dATP and S-MC-dATP. In this respect, Dpo1 is unexpectedly more tolerant of substrate conformation than Dpo4. The crystal structure of Dpo4 bound to S-MC-dADP shows that poor incorporation of the Southern pucker by the Y-family polymerase results from a hydrogen bond between the 3′-OH group of the nucleotide analogue and the OH group of the steric gate residue, Tyr12, shifting the S-MC-dADP molecule away from the dNTP binding pocket and distorting the base pair at the primer-template junction. These results provide insights into substrate specificity of DNA polymerases, as well as molecular mechanisms that act as a barrier against insertion of rNTPs.
PMCID: PMC3563936  PMID: 23050956
DNA polymerase; nucleotide selection; X-ray crystallography
15.  Epigenetic therapy with 3-deazaneplanocin A, an inhibitor of the histone methyltransferase EZH2, inhibits growth of non-small cell lung cancer cells 
EZH2 (enhancer of zeste homolog 2) is the catalytic subunit of PRC2 (polycomb repressive complex 2), which mediates histone methyltransferase activity and functions as transcriptional repressor involved in gene silencing. EZH2 is involved in malignant transformation and biological aggressiveness of several human malignancies. We previously demonstrated that non-small cell lung cancers (NSCLCs) also overexpress EZH2 and that high expression of EZH2 correlates with poor prognosis. Growing evidence indicates that EZH2 may be an appropriate therapeutic target in malignancies, including NSCLCs. Recently, an S-adenosyl-L homocysteine hydrolase inhibitor, 3-Deazaneplanocin A (DZNep), has been shown to deplete and inhibit EZH2. The aim of this study was to determine the effect of DZNep in NSCLC cells. Knockdown of EZH2 by small-interfering RNA (siRNA) resulted in decreased growth of four NSCLC cell lines. MTT assays demonstrated that DZNep treatment resulted in dose-dependent inhibition of proliferation in the NSCLC cell lines with a half maximal inhibitory concentration (IC50) ranging from 0.08 to 0.24 μM. Immortalized but non-cancerous bronchial epithelial and fibroblast cell lines were less sensitive to DZNep than the NSCLC cell lines. Soft agarose assays demonstrated that anchorage-independent growth was also reduced in all three NSCLC cell lines that were evaluated using this assay. Flow cytometry analysis demonstrated that DZNep induced apoptosis and G1 cell cycle arrest in NSCLC cells, which was partially associated with cyclin A decrease and p27Kip1 accumulation. DZNep depleted cellular levels of EZH2 and inhibited the associated histone H3 lysine 27 trimethylation. These results indicated that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treatment of NSCLCs.
PMCID: PMC3472089  PMID: 22925699
3-deazaneplanocin A (DZNep); polycomb-group protein; EZH2; non-small cell lung cancer; epigenetics; proliferation; apoptosis
16.  Targeting the Enhancer of Zeste Homologue 2 in Medulloblastoma 
Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors.
PMCID: PMC3375399  PMID: 22287205
EZH2; Medulloblastoma; Polycomb repressive complex 2; histone lysine methylation; DZNEP
17.  Epigenetic control of CCR5 transcript levels in immune cells and modulation by small molecules inhibitors 
Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4+ T lymphocytes as well as in CD14+ monocytes. In normal naïve T cells and CD14+ monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5 expressing CD14+ monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non–CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14+ monocytes, this indicates a dominant role for AcH3 in CCR5 transcription.
We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.
PMCID: PMC3309068  PMID: 22050776
Chromatin remodeling; histone modifications; DNA methylation; bivalent chromatin; poised chromatin; CCR5; T Cells; Monocytes
18.  Membrane-surface anchoring of charged diacylglycerol-lactones correlates with biological activities 
Synthetic diacylglycerol lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes and other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study examines the biological properties of charged DAG-lactones exhibiting different alkyl groups attached to the heterocyclic nitrogen of an α–pyridylalkylidene chain, and particularly the relationship between membrane interactions of the substituted DAG-lactones and their respective biological activities. Our results suggest that bilayer interface localization of the N-alkyl chain in the R2 position of the DAG-lactones inhibits translocation of PKC isoenzymes onto the cellular membrane. However, the orientation of a branched alkyl chain at the bilayer surface facilitates PKC binding and translocation. This investigation emphasizes that bilayer localization of the aromatic side residues of positively-charged DAG lactone derivatives play a central role in determining biological activity and that this factor contributes to the diversity of biological actions of these synthetic biomimetic ligands.
PMCID: PMC3729217  PMID: 20715268
diacylglycerol (DAG)-lactones; PKC; plasma membrane; membrane anchoring; vesicles
19.  The effect on quadruplex stability of North-Nucleoside derivatives in the loops of the thrombin-binding aptamer 
Bioorganic & medicinal chemistry  2012;20(14):4186-4193.
Modified thrombin-binding aptamers (TBAs) carrying uridine (U), 2′-deoxy-2′-fluorouridine (FU) and North-methanocarbathymidine (NT) residues in the loop regions were synthesized and analyzed by UV thermal denaturation experiments and CD spectroscopy. The replacement of thymidines in the TGT loop by U and FU results in an increased stability of the antiparallel quadruplex structure described for the TBA while the presence of NT residues in the same positions destabilizes the antiparallel structure. The substitution of the thymidines in the TT loops for U, FU and NT induce a destabilization of the antiparallel quadruplex, indicating the crucial role of these positions. NMR studies on TBAs modified with uridines at the TGT loop also confirm the presence of the antiparallel quadruplex structure. Nevertheless, replacement of two Ts in the TT loops by uridine gives a more complex scenario in which the antiparallel quadruplex structure is present along with other partially unfolded species or aggregates.
PMCID: PMC3534854  PMID: 22727781
Quadruplex; Thrombin-binding aptamer; NMR; Oligonucleotide synthesis; Uridine; 2′-Deoxy-2′-fluorouridine; North-methanocarbathymidine
20.  A nucleotide analogue induced gain of function corrects the error-prone nature of human DNA polymerase iota 
Journal of the American Chemical Society  2012;134(25):10698-10705.
Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol ι through use of the fixed-conformation nucleotide North-methanocarba-2′-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol ι in complex with DNA containing a template 2′-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle (χ), which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol ι by preventing the Hoogsteen base-pairing mode normally observed for hpol ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.
PMCID: PMC3392044  PMID: 22632140
DNA replication; polymerase; translesion DNA synthesis; X-ray crystallography
21.  PRC2 overexpression and PRC2-target gene repression relating to poorer prognosis in small cell lung cancer 
Scientific Reports  2013;3:1911.
Small cell lung cancer (SCLC) is a subtype of lung cancer with poor prognosis. Expression array analysis of 23 SCLC cases and 42 normal tissues revealed that EZH2 and other PRC2 members were highly expressed in SCLC. ChIP-seq for H3K27me3 suggested that genes with H3K27me3(+) in SCLC were extended not only to PRC2-target genes in ES cells but also to other target genes such as cellular adhesion-related genes. These H3K27me3(+) genes in SCLC were repressed significantly, and introduction of the most repressed gene JUB into SCLC cell line lead to growth inhibition. Shorter overall survival of clinical SCLC cases correlated to repression of JUB alone, or a set of four genes including H3K27me3(+) genes. Treatment with EZH2 inhibitors, DZNep and GSK126, resulted in growth repression of SCLC cell lines. High PRC2 expression was suggested to contribute to gene repression in SCLC, and may play a role in genesis of SCLC.
PMCID: PMC3665955  PMID: 23714854
22.  N-methyl-substituted fluorescent DAG-indololactone isomers exhibit dramatic differences in membrane interactions and biological activity 
N-methyl substituted diacylglycerol-indololactones (DAG-indololactones) are newly-synthesized effectors of protein kinase C (PKC) isoforms and exhibit substantial selectivity between RasGRP3 and PKC alpha. We present a comprehensive analysis of membrane interactions and biological activities of several DAG-indololactones. Translocation and binding activity assays underline significant variations between the PKC translocation characteristics affected by the ligands as compared to their binding activities. In parallel, the fluorescent properties of the ligands were employed for analysis of their membrane association profiles. Specifically, we find that a slight change in the linkage to the indole ring resulted in significant differences in membrane binding and association of the DAG-indololactones with lipid bilayers. Our analysis shows that seemingly small structural modifications of the hydrophobic regions of these biomimetic PKC effectors contribute to pronounced modulation of membrane interactions of the ligands
PMCID: PMC3665409  PMID: 23106081
protein kinase C; phorbol ester; diacylglycerol-lactone; vesicles; FRET; tryptophane fluorescence
23.  The Conformationally Constrained N-Methanocarba-dT Analog Adopts an Unexpected C4'-exo Sugar Pucker in the Structure of a DNA Hairpin 
Biochemistry  2012;51(13):2639-2641.
Incorporation of a bicyclo[3.1.0]hexane scaffold into the nucleoside sugar was devised to lock the embedded cyclopentane ring in conformations that mimic the furanose North and South sugar puckers. To analyze the effects of North-methanocarba-2'-deoxythymidine (N-MCdT) on the conformation of B-DNA, we crystallized d(CGCGAA[mcTmcT]CGCG) with two N-MCdTs. Instead of a duplex the 12mer forms a tetraloop-hairpin, whereby loop N-MCdTs adopt the C4'-exo pucker (NE; P=50°). This indicates that the bicyclic framework does not limit the sugar pucker to the anticipated C2'-exo range (NNW; P=−18°).
PMCID: PMC3330753  PMID: 22409313
24.  Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin 
Human Molecular Genetics  2011;21(4):751-764.
Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1–UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.
PMCID: PMC3263987  PMID: 22058289
25.  Synthesis of Conformationally North-Locked Pyrimidine Nucleosides Built on an Oxa-bicyclo[3.1.0]hexane Scaffold 
The Journal of organic chemistry  2011;77(2):815-824.
Beginning with a known 3-oxabicyclo[3.1.0]hexane scaffold (I), the relocation of the fused cyclopropane ring bond and the shifting of the oxygen atom to an alternative location engendered a new 2-oxabicyclo[3.1.0]hexane template (II) that mimics more closely the tetrahydrofuran ring of conventional nucleosides. The synthesis of this new class of locked nucleosides involved a novel approach that required the isocyanate II (B = NCO) with a hydroxyl-protected scaffold as a pivotal intermediate that was obtained in eleven steps from a known dihydrofuran precursor. The completion of the nucleobases was successfully achieved by quenching the isocyanate with the lithium salts of the corresponding acrylic amides that led to the uracil and thymidine precursors in a single step. Ring closure of these intermediates led to the target, locked nucleosides. The anti-HIV activity of 29 (uridine analogue), 31 (thymidine analogue), and 34 (cytidine analogue) was explored in human osteosarcoma (HOS) cells or modified HOS cells (HOS-313) expressing the herpes simplex virus 1 thymidine kinase (HSV-1 TK). Only the cytidine analogue showed moderate activity in HOS-313 cells, which means that the compounds are not good substrates for the cellular kinases.
PMCID: PMC3534852  PMID: 22026578

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