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1.  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.
SUMMARY
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.
doi:10.1016/j.ccr.2012.09.003
PMCID: PMC3973134  PMID: 23079660
2.  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.
doi:10.1158/0008-5472.CAN-12-2359
PMCID: PMC3910284  PMID: 23100468
3.  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.
doi:10.1093/neuonc/nos285
PMCID: PMC3548579  PMID: 23190500
atypical teratoid/rhabdoid tumor; DZNep, EZH2; histone lysine methylation; Polycomb repressive complex 2
4.  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.
doi:10.1038/jid.2012.400
PMCID: PMC3898712  PMID: 23190898
5.  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.
doi:10.3390/ijms141224154
PMCID: PMC3876102  PMID: 24351808
NAFLD; EZH2; DZNep; microRNAs
6.  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.
doi:10.1021/bi301043k
PMCID: PMC3563936  PMID: 23050956
DNA polymerase; nucleotide selection; X-ray crystallography
7.  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.
doi:10.1016/j.lungcan.2012.08.003
PMCID: PMC3472089  PMID: 22925699
3-deazaneplanocin A (DZNep); polycomb-group protein; EZH2; non-small cell lung cancer; epigenetics; proliferation; apoptosis
8.  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.
doi:10.1002/ijc.27455
PMCID: PMC3375399  PMID: 22287205
EZH2; Medulloblastoma; Polycomb repressive complex 2; histone lysine methylation; DZNEP
9.  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.
doi:10.1111/j.1582-4934.2011.01482.x
PMCID: PMC3309068  PMID: 22050776
Chromatin remodeling; histone modifications; DNA methylation; bivalent chromatin; poised chromatin; CCR5; T Cells; Monocytes
10.  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.
doi:10.1158/1535-7163.MCT-12-0037
PMCID: PMC3416916  PMID: 22622284
Pancreatic ductal adenocarcinoma; EZH2; DZNeP; gemcitabine
11.  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.
doi:10.1002/cbic.201000343
PMCID: PMC3729217  PMID: 20715268
diacylglycerol (DAG)-lactones; PKC; plasma membrane; membrane anchoring; vesicles
12.  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.
doi:10.1016/j.bmc.2012.06.005
PMCID: PMC3534854  PMID: 22727781
Quadruplex; Thrombin-binding aptamer; NMR; Oligonucleotide synthesis; Uridine; 2′-Deoxy-2′-fluorouridine; North-methanocarbathymidine
13.  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.
doi:10.1021/ja304176q
PMCID: PMC3392044  PMID: 22632140
DNA replication; polymerase; translesion DNA synthesis; X-ray crystallography
14.  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.
doi:10.1016/j.tet.2009.08.035
PMCID: PMC2900805  PMID: 20625519
Conformationally locked carbocyclic nucleosides; North-methanocarbathymidine; antiviral; enantioselective synthesis
15.  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.
doi:10.1038/srep01911
PMCID: PMC3665955  PMID: 23714854
16.  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
17.  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°).
doi:10.1021/bi300215k
PMCID: PMC3330753  PMID: 22409313
18.  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.
doi:10.1093/hmg/ddr506
PMCID: PMC3263987  PMID: 22058289
19.  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.
doi:10.1021/jo201716c
PMCID: PMC3534852  PMID: 22026578
20.  EZH2 mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3 and NGFR 
Cancer research  2011;72(1):315-324.
Neuroblastoma (NB) is the most common extracranial pediatric solid tumor with an undifferentiated status and generally poor prognosis, but the basis for these characteristics remains unknown. In this study, we show that upregulation of the Polycomb complex histone methytransferase EZH2, which limits differentiation in many tissues, is critical to maintain the undifferentiated state and poor prognostic status of NB by epigenetic repression of multiple tumor suppressor genes. We identified this role for EZH2 by examining the regulation of CASZ1, a recently identified NB tumor suppressor gene whose ectopic restoration inhibits NB cell growth and induces differentiation. Reducing EZH2 expression by RNAi-mediated knockdown or pharmacological inhibiton with 3-deazaneplanocin A (DZNep) increased CASZ1 expression, inhibited NB cell growth and induced neurite extension. Similarly, EZH2−/− mouse embryonic fibroblasts (MEFs) displayed 3-fold higher levels of CASZ1 mRNA compared to EZH2+/+ MEFs. In cells with increased expression of CASZ1, treatment with HDAC inhibitors decreased expression of EZH2 and the Polycomb complex component SUZ12. Under steady-state conditions H3K27me3 and PRC2 components bound to the CASZ1 gene were enriched, but this enrichment was decreased after HDAC inhibitor treatment. We determined that the tumor suppressors CLU, NGFR and RUNX3 were also directly repressed by EZH2 like CASZ1 in NB cells. Together, our findings establish that aberrant upregulation of EZH2 in NB cells silences several tumor suppressors, which contribute to the genesis and maintenance of the undifferentiated phenotype of NB tumors.
doi:10.1158/0008-5472.CAN-11-0961
PMCID: PMC3487161  PMID: 22068036
CASZ1; neuroblastoma; EZH2; NGFR; CLU; RUNX3
21.  Methanocarba Analogues of Purine Nucleosides as Potent and Selective Adenosine Receptor Agonists 
Journal of medicinal chemistry  2000;43(11):2196-2203.
Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A1 and A3 receptor agonists having favorable pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S) conformation, as defined in the pseudorotational cycle. In binding assays at A1, A2A, and A3 receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly at the human A3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various N6-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the parent compounds are potent agonists at either A1 or A3 receptors, respectively, were synthesized. The N6-cyclopentyl derivatives were A1 receptor-selective and maintained high efficacy at recombinant human but not rat brain A1 receptors, as indicated by stimulation of binding of [35S]GTP-γ-S. The (N)-methanocarba-N6-(3-iodobenzyl)adenosine and its 2-chloro derivative had Ki values of 4.1 and 2.2 nM at A3 receptors, respectively, and were highly selective partial agonists. Partial agonism combined with high functional potency at A3 receptors (EC50 < 1 nM) may produce tissue selectivity. In conclusion, as for P2Y1 receptors, at least three adenosine receptors favor the ribose (N)-conformation.
PMCID: PMC3471159  PMID: 10841798
22.  (-)-Epigallocatechin-3-gallate and DZNep reduce polycomb protein level via a proteasome-dependent mechanism in skin cancer cells 
Carcinogenesis  2011;32(10):1525-1532.
Polycomb group (PcG) protein-dependent histone methylation and ubiquitination drives chromatin compaction leading to reduced tumor suppressor expression and increased cancer cell survival. Green tea polyphenols and S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors are important candidate chemopreventive agents. Previous studies indicate that (-)-epigallocatechin-3-gallate (EGCG), a potent green tea polyphenol, suppresses PcG protein level and skin cancer cell survival. Inhibition of AdoHcy hydrolase with 3-deazaneplanocin A (DZNep) inhibits methyltransferases by reducing methyl group availability. In the present study, we examine the impact of EGCG and DZNep cotreatment on skin cancer cell function. EGCG and DZNep, independently and in combination, reduce the level of PcG proteins including Ezh2, eed, Suz12, Mel18 and Bmi-1. This is associated with reduced H3K27me3 and H2AK119ub formation, histone modifications associated with closed chromatin. Histone deacetylase 1 level is also reduced and acetylated H3 formation is increased. These changes are associated with increased tumor suppressor expression and reduced cell survival and are partially reversed by vector-mediated maintenance of Bmi-1 level. The reduction in PcG protein level is associated with increased ubiquitination and is reversed by proteasome inhibitors, suggesting proteasome-associated degradation.
doi:10.1093/carcin/bgr171
PMCID: PMC3179425  PMID: 21798853
23.  2-Substitution of Adenine Nucleotide Analogues Containing a Bicyclo[3.1.0]hexane Ring System Locked in a Northern Conformation: Enhanced Potency as P2Y1 Receptor Antagonists 
Journal of medicinal chemistry  2003;46(23):4974-4987.
Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of P2Y1 receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829–842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y1 receptor and by using the radiolabeled antagonist [3H]2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y1 receptor was an (N)-methanocarba N6-methyl-2-iodo analogue 12, which displayed a Ki value in competition for binding of [3H]5 of 0.79 nM and a KB value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y1 receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y1 receptors. An enzymatic method of synthesis of the 3′,5′-bisphosphate from the corresponding 3′-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.
doi:10.1021/jm030127+
PMCID: PMC3408611  PMID: 14584948
24.  Inhibition of Histone Lysine Methylation Enhances Expression of Cancer-Testis Genes in Lung Cancer Cells: Implications for Adoptive Immunotherapy of Cancer 
Cancer research  2011;71(12):4192-4204.
Cancer-testis antigens such as NY-ESO-1, MAGE-A1 and MAGE-A3 are immunogenic proteins encoded by genes, which are normally expressed only in male germ cells, but activated by ill-defined epigenetic mechanisms in human tumors including lung cancers. Previously we reported induction of these cancer-testis antigens in cancer cells, but not normal cells, by DNA demethylating agents and histone deacetylase inhibitors using clinically achievable exposure conditions. In the present study, we evaluated chromatin alterations associated with repression/activation of cancer-testis genes in lung cancer cells to further develop gene induction regimens for cancer immunotherapy. Repression of NY-ESO-1, MAGE-A1, and MAGE-A3 coincided with DNA hypermethylation, recruitment and binding of polycomb group proteins, and histone heterochromatin modifications within the promoters of these genes. De-repression coincided with DNA demethylation, dissociation of polycomb proteins, and presence of euchromatin marks within the respective promoters. ShRNAs were used to inhibit several histone methyl transferases (KMTs) and histone demethylases (KDMs) that mediate histone methylation and repress gene expression. Knockdown of KMT6, KDM1 or KDM5B markedly enhanced deoxyazacytidine (DAC)-mediated activation of these cancer-testis genes in lung cancer cells. DZNep, a pharmacologic inhibitor of KMT6 expression, recapitulated the effects of KMT6 knock-down. Following DAC-DZNep exposure, lung cancer cells were specifically recognized and lysed by allogeneic lymphocytes expressing recombinant T cell receptors recognizing NY-ESO-1 and MAGE-A3. Combining DNA demethylating agents with compounds such as DZNep that modulate histone lysine methylation may provide a novel epigenetic strategy to augment cancer-testis gene expression as an adjunct to adoptive cancer immunotherapy.
doi:10.1158/0008-5472.CAN-10-2442
PMCID: PMC3116976  PMID: 21546573
lung cancer; epigenetics; cancer-testis gene; KMT6; KDM1; KDM5B; SirT1; DZNep; immunotherapy
25.  Synthesis of Conformationally Locked L-Deoxythreosyl Phosphonate Nucleosides Built on a Bicyclo[3.1.0]hexane Template 
The Journal of organic chemistry  2010;75(22):7659-7669.
Two conformationally locked versions of L-deoxythreosyl phosphonate nucleosides (2 and 3) were synthesized to investigate the preference of HIV reverse transcriptase for a conformation displaying either a fully diaxial or fully diequatorial disposition of substituents. Synthesis of the enantiomeric 4-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexane-2-ol carbocyclic nucleoside precursors (diaxially disposed) proceeded straightforwardly from commercially available (1R,4S)-4-hydroxy-2-cyclopent-2-enyl-1-yl acetate employing a hydroxyl-directed Simmons-Smith cyclopropanation that culminated with a Mitsunobu coupling of the purine base. For the more complicated 1-(6-amino-9H-purin-9-yl)bicyclo[3.1.0]hexan-3-ol carbocyclic nucleoside precursors (diequatorially disposed), the obligatory linear approach required the syntheses of key 1-aminobicyclo[3.1.0.]hexan-3-yl benzoate precursors that were assembled via the amide variant of the Kulinkovich reaction involving the intramolecular cyclopropanation of a substitued δ-vinylamide. Completion of the purine ring was achieved by conventional approaches but with much improved yields through the use of a microwave reactor. The syntheses of the phosphonates and the corresponding diphosphates were achieved by conventional means. None of the diphosphates, which were supposed to act as nucleoside triphosphate mimics, could compete with dATP even when present in a ten-fold excess.
doi:10.1021/jo101475p
PMCID: PMC3041872  PMID: 20964394

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