Telomere Maintenance as a Target for Drug DiscoveryVijay Sekaran, Joana Soares, and Michael B. Jarstfer*

Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill,Chapel Hill, North Carolina 27599, United States

ABSTRACT: The observation that the enzyme telomerase is up-regulated in 80−90% ofcancer cells isolated from primary human tumors but is absent in neighboring cells ofhealthy tissue has resulted in significant efforts to validate telomerase as an anticancer drugtarget and to develop effective approaches toward its inhibition. In addition to inhibitorsthat target the enzymatic function of telomerase, efforts toward immunotherapy usingpeptides derived from its catalytic subunit hTERT and hTERT-promoter driven genetherapy have made significant advances. The increased level of telomerase in cancer cellsalso provides a potential platform for cancer diagnostics. Telomerase inhibition leads todisruption of a cell’s ability to maintain the very ends of the chromosomes, which are calledtelomeres. Thus, the telomere itself has also attracted attention as an anticancer drug target.In this Perspective, interdisciplinary efforts to realize the therapeutic potential of targetingtelomere maintenance with a focus on telomerase are discussed.

■ INTRODUCTIONPathways exhibiting differential activity in cancer cells whencompared to healthy cells are attractive targets for therapeuticintervention. Accordingly, the observation that the enzymetelomerase is up-regulated in 80−90% of all cancer cells isolatedfrom primary human tumors but is absent in neighboring cellsof healthy tissue has resulted in significant efforts to validatetelomerase as an anticancer drug target and to develop effectiveapproaches toward its inhibition.1 In addition to inhibitors thattarget the enzymatic function of telomerase, efforts towardimmunotherapy using peptides derived from the catalytictelomerase subunit hTERT (human telomerase reverse tran-scriptase)2 and hTERT-promoter driven gene therapy3 havemade significant advances. The increased level of telomeraseactivity in cancer cells also provides a potential platform forcancer diagnostics.4 Telomerase inhibition leads to disruptionof a cell’s ability to maintain the very ends of the chromosomes,which are called telomeres. Thus, the telomere itself has alsoattracted attention as an anticancer drug target. Here, wediscuss the interdisciplinary efforts to realize the therapeuticpotential of targeting telomere maintenance with a specificfocus on telomerase.

■ TELOMERE MAINTENANCE AND TELOMERASE INHUMAN CANCER CELLS

Telomeres are the physical ends of linear chromosomes, andtheir importance to human health is underscored by the NobelPrize in Physiology or Medicine awarded to ElizabethBlackburn, Jack Szostak, and Carol Grieder in 2009 for theirwork on telomere biology and biochemistry. Telomeres are soimportant because proper telomere maintenance is an absoluterequirement for continued cellular proliferation. Functionaltelomeres require a specific and repetitive DNA sequence andseveral telomere-specific proteins.5,6 These telomere-associatedproteins function in two primary capacities: maintaining the

structural and functional integrity of the telomere and ensuringcomplete replication of telomeric DNA. In the absence offunctional telomeres, chromosome ends are recognized as DNAdamage, and the ensuing DNA damage response generallyresults in either senescence or apoptosis, depending on the celltype.7 Mammalian telomeric DNA contains several thousandbase pairs of repetitive DNA with the sequence 5′-TTAGGG/3′-AATCCC and a single stranded 3′ overhang ofthe G-rich sequence.8,9 The canonical DNA polymerasesresponsible for DNA replication cannot efficiently replicatethe very ends of linear DNA templates. As a result, proliferativecells must employ additional pathways to overcome this end-replication problem. In human cells, this additional activityresides primarily in telomerase. Telomerase is a ribonucleo-protein complex that contains several protein subunits and oneRNA subunit.10,11 Human telomerase RNA (hTER) is a highlystructured 451-nucleotide long RNA that contains severalfunctional domains in addition to a templating sequence usedto code for the G-rich telomeric DNA strand.12 The proteinsubunit hTERT functions as a reverse transcriptase by using thetemplating portion of hTER to synthesize telomeric DNA.13 Inthe majority of cancer cells, hTERT is up-regulated, increasingthe telomerase activity required for continuous proliferation.8,14

In contrast, telomerase activity is down-regulated in mostnormal human diploid cells, with the exception of germline andstem cells, primarily owing to inhibition of hTERT expressionduring cellular differentiation.Because telomerase activity is absent in most differentiated

human cells, telomeres erode during the cellular aging process.This erosion allows telomere length to function like a mitotichourglass that regulates cellular replication and arguablyfunctions as a tumor suppressor mechanism.5 Cells lacking

Received: April 11, 2013

Perspective

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telomerase activity experience erosion of their telomeric DNAwhen they divide. At a critical length, the shortened telomeresare recognized as DNA damage and form a telomeredysfunction induced focus (TIF). Typically, cells respond toTIFs through senescence or apoptosis. Importantly, thetelomere-length dependence of proliferation appears to be aresult of the structure and not the length of the telomeric DNAper se. This is evidenced in part by experiments in the de Langelab showing that the negative consequences of short telomerescan be overcome by the overexpression of the telomere-bindingprotein TRF2.15 It is likely that as telomeres erode in cellslacking an active telomere maintenance pathway, the telomerestructure, predicted to be a lasso-like fold back structure termeda t-loop,16 becomes increasingly difficult to sustain eventuallyleading to TIFs and senescence or apoptosis if telomerefunction is not reestablished.6

Telomere maintenance directly impacts cancer in twospecific ways. The most obvious is the absolute requirementfor telomeric DNA maintenance to offset telomere loss fromthe end-replication problem, processing, and degradation. Mostcancer cells up-regulate expression of hTERT, in order toincrease telomerase activity.17 In 10−15% of cancers, arecombination based telomere lengthening pathway calledalternative lengthening of telomeres (ALT) supplants the needfor telomerase.18 The ALT pathway is particularly prevalent inastrocytic brain tumors and osteosarcomas.19 A secondconnection between telomere biology and neoplasias is theeffect of overall length of telomeric DNA and the actions oftelomere-associated proteins on genetic stability. Telomereerosion has been argued to serve as a tumor suppressionmechanism because it can activate the senescence pathway,which suppresses neoplastic transformation.5 However, even inthe presence of telomere erosion, senescence is not activated inthe absence of the Rb/p16 or p53 pathways, leading to geneticinstability and increased potential for transformation. Thesepivotal roles of telomere maintenance in tumorigenicity areevidenced by the ability to inhibit tumor growth by inhibitingtelomerase in cancer cells and by the ability to convert primaryhuman cells to cancer cells by combining constitutivetelomerase expression with oncogene expression and/or lossof a tumor repressor gene. For example, the simian virus 40large-T oncoprotein together with an oncogenic allele of H-rasand ectopic expression of hTERT has been demonstrated totransform a variety of cell types.20 For more comprehensivediscussions on the roles of telomerase and telomeremaintenance on tumor biology, several recent reviews arerecommended.21−24

■ TARGETING TELOMERE MAINTENANCE FORCANCER THERAPY

When considering anticancer drug targets, several advantagesfor targeting telomerase and telomere maintenance exist whencompared to other targets and pathways. Most important is theapparently universal requirement for telomere maintenance incancer cells including putative cancer stem cells.25,26 Inaddition, normal human cells including stem cells expresslower telomerase activity and generally maintain telomeres atlonger lengths when compared to cancer cells.27,28 Thesefeatures provide a level of specificity with telomerase inhibition.Telomerase activity itself was validated as an anticancer drugtarget in 1999 when two independent studies reported thatoverexpressing dominant-negative-hTERT mutants resulted intelomerase inhibition, telomere shortening, morphological

changes, growth arrest, and apoptosis in a variety of tumorcell lines.29,30 Since that time, several strategies for targetingtelomere maintenance and telomerase-positive cancer cells havebeen explored (Figure 1). The initial approach explored was the

direct inhibition of telomerase’s enzymatic activity. Asvalidation of the approach, the telomerase inhibitor imetelstat(1, GRN163L) has been explored in anticancer therapy inseveral clinical trials.31 Direct targeting of the telomere itself hasalso been accomplished with specific DNA binding agents thatbind to and stabilize the G-quadruplex structures formed bytelomeric DNA.32,33 The high activity of the hTERT promoterin cancer cells has resulted in the exploration of hTERT-promoter driven expression of cancer killing genes andviruses,34 and one clinical trial for hTERT-driven replicationof an oncolytic virus is underway.35 Immunotherapy targetinghTERT-derived peptides has also been explored leading toseveral clinical trials.36 Each of these platforms will be discussedfurther below.

■ CONSEQUENCE OF TELOMERASE INHIBITIONAND TELOMERE DISRUPTION

If telomere maintenance is to be considered a viable anticancerdrug discovery platform, it is paramount to understand theconsequences of disrupting telomere maintenance for bothhealthy and cancerous cells. The simplest and best exploredtelomere-targeted anticancer drug platform is the inhibition oftelomerase enzymatic activity. When telomerase activity isinhibited, DNA replication attending cellular proliferationresults in telomeric DNA erosion.29,37 Erosion of telomericDNA eventually results in dysfunctional telomeres that initiateTIF formation.5,6 Generally, proliferating human cells lose 50−200 base pairs of telomeric DNA during each cell cycle in theabsence of telomerase activity.38 Therefore, a lag between thephenotypic consequences of telomerase inhibition and theinitiation of telomerase inhibition therapy is likely to occur. Theduration of this phenotypic lag depends on both the initiallength of telomeric DNA, which ranges from 5000 to 15 000base pairs in human cancer cells, and the rate of erosion.Importantly, it is suspected that the length of a few telomeres,

Figure 1. Many ways to target the overexpression of telomerase incancer cells have been developed.

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not the average length of all telomeres, dictates the cellularresponse to telomerase inhibition.39 This suggests that sometumor types may be especially sensitive to telomeraseinhibition. Once telomeres erode to a critically short length,they cannot function properly,40 perhaps because they cannotbe folded into the proper protected state, presumably a t-loop.16 In most cancer cells, the loss of telomere functionresults in apoptosis, whereas in normal cells, telomere lossresults in cell cycle arrest. Notably, telomerase inhibition hasalso been observed to promote the ALT phenotype in tumorsconcomitant with up-regulation of the PGC-1β, a masterregulator of mitochondrial biogenesis.41 This report offerscritical insight into the mechanisms of adaptation that may bein play when tumors are subjected to telomerase inhibition.Inhibition of telomerase by some methods, for example,

antisense oligonucleotides targeting the hTERT mRNA,42 canresult in immediate effects that are not preceded by thephenotypic lag characteristic of telomerase activity inhibitorsand are not coupled to erosion of telomeric DNA. While aprecise explanation remains missing, one intriguing possibility isthat hTERT has several noncanonical properties that areseparate from its enzymatic activity, and inhibition of these mayhave immediate effects.43 Proposed additional activities oftelomerase include telomere capping, inhibition of apoptosis,activation of the Wnt pathway, and activation of the NFκBpathway.44,45 Thus, down-regulation of hTERT can potentiallylead to immediate antiproliferative effects. Understanding whysome cells are immediately impacted by the loss of hTERT andothers are not remains to be determined. Perhaps some cancercells become oncogenically addicted to hTERT and requirehTERT to maintain elevated growth signaling.Currently, one of the most studied small-molecule

telomerase inhibitors are G-quadruplex stabilizers.46 This classof molecules can impact telomere functions in at least twoprimary mechanisms. One is by inhibiting the enzymaticactivity of telomerase by preventing telomerase from binding toits primer or by interacting with the nascent DNA telomeraseproduct. The second is to dissociate or block telomere-bindingproteins from the telomere, disrupting the formation of theprotective cap. As expected, the former mechanism results in adelayed but highly telomerase-expression-specific effect whilethe second mechanism is more immediate but may suffer fromreduced specificity because formation of proper telomerestructure is required in all cells including normal somatic andstem cells. Early evidence suggests that the therapeutic indexfor telomere disrupters is surprisingly high at least in a mousemodel.47,48 One potential reason that telomere disruption maybe well tolerated in normal cells is that the DNA damageresponse at the telomere may be transient and reversible.Evidence for this hypothesis comes from experiments using aligand-stabilized version of the telomere binding proteinProtection of Telomeres 1 (POT1). POT1 is essential fortelomere protection, and the ligand-stabilized POT1 allowed

POT1 to be inhibited transiently and reversibly. In cancer cells,POT1 inhibition resulted in cell death. But in normal cells,POT1 inhibition resulted in arrest that was reversed byreactivating POT1.49

■ SMALL-MOLECULE TELOMERASE INHIBITORS

Once the importance of telomerase activity for cancer cellgrowth was recognized, several academic and pharmaceuticalgroups initiated programs to discover small moleculetelomerase inhibitors. Approaches toward the identification ofeffective telomerase inhibitors have included repurposing ofviral reverse transcriptase inhibitors, screening of naturalproducts, high throughput screens of diverse compoundlibraries, and more recently, the beginnings of structure-baseddiscovery and optimization. Despite these efforts, smallmolecule telomerase inhibitors have yet to reach the clinic. Inthis section, the varieties of approaches toward directtelomerase activity inhibition with small molecules will bediscussed.

■ NUCLEOSIDE ANALOGUES

Nucleoside analogues developed as viral reverse transcriptaseinhibitors have been investigated as telomerase inhibitors(Figure 2). Nucleoside analogues can hypothetically affect thetelomere through several mechanisms, including telomeraseinhibition and chain termination that can lead to telomereshortening and perturbation of telomere sequence that can leadto disruption of telomere function. The chain terminator 3′-azido-2′,3′-dideoxythymidine (2, AZT), the first nucleosidereverse transcriptase inhibitor approved for the treatment ofHIV-1, inhibits telomerase in vitro and in vivo, giving rise totelomere shortening.50,51 In cultured cells, treatment with 2resulted in telomere shortening, cell-cycle perturbation, andincreased p14ARF expression,52 and 2 exhibited synergisticeffects when combined with other chemotherapeuticdrugs.53−55 Several nucleoside HIV reverse transcriptaseinhibitors including stavudine, tenofovir, didanosine, andabacavir also inhibit telomerase activity and lead to telomereerosion in cultured cells, but non-nucleoside HIV RT inhibitorsdid not inhibit telomerase in these studies.56 HIV patients havebeen taking these and other nucleoside reverse transcriptaseinhibitors for decades, leading to possible effects from long-term telomerase inhibition. This potential side effect has beeninvestigated in a small patient population, and it was found thattelomere length was inversely correlated with total timepatients were exposed to nucleoside reverse transcriptaseinhibitors.57 Although other nucleoside triphosphate analogues,including arabinofuranylguanosine and 6-thio-7-deaza-2′-deox-yguanosine 5′-triphosphate, ddGTP, ddATP, and ddTTP,produced substantial inhibition of telomerase and telomereshortening, they have not been further tested in cell cultures orin animal models and are likely to be nonspecific, thus limiting

Figure 2. Representative nucleoside analogues identified as telomerase inhibitors.

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their clinical utility.51,58 As telomerase structural biologyincreases, it may be possible to rationally design nucleosideanalogues with the requisite telomerase specificity for thisplatform to be viable.

■ NON-NUCLEOSIDE ANALOGUESNon-nucleoside, small-molecule telomerase inhibitors havebeen identified by a variety of approaches. High throughputscreening of chemical libraries has been used to identifytelomerase inhibitors (Figure 3). The isothiazolone derivative

2-[3-(trifluoromethyl)phenyl]isothiazolin-3-one (3, IC50 = 1μM) was identified from a screen of a 16 000-member, chemicallibrary using rat telomerase (Figure 3).59 The reducing agentdithiothreitol was found to protect telomerase from inhibitionby 3, suggesting that inhibition resulted from interactionbetween the isothiazolone moiety of 3 with the sulfhydrylgroup of key cysteine residue(s).59 The nitrostyrene derivative3-(3,5-dichlorophenoxy)nitrostyrene (4, IC50 = 0.4 μM)60 andthe quinoxaline derivative 2,3,7-trichloro-5-nitroquinoxaline(5),61 two noncompetitive telomerase inhibitors, were alsoidentified by screening chemical libraries. At low concentrations(1 μM), 4 was not acutely cytotoxic to HeLa cells, but chronicexposure led to progressive telomere erosion followed by theinduction of senescence consistent with telomerase inhibition.60

Long-term treatment of the breast cancer cell line MCF7 with 5(1 μM) also resulted in telomere erosion, chromosomeabnormalities, and senescence.61

The crystal structure of TERT from the beetle Triboliumcastaneum62 has been used in limited structure based design oftelomerase inhibitors. Because 2H-pyrazole and dihydropyr-azole derivatives have been shown to have strong anticancer,antmalarial, antiviral, and anti-inflammatory activity, they wereselected as a starting scaffold for telomerase inhibition.63−65

Aryl-2H-pyrazoles with 1,3-benzodioxoles and electron with-drawing aryls groups and sulfonyl-4,5-dihydropyrazoles dis-played telomerase inhibition with IC50 values ranging from 0.8to 10 μM in a variety of cancer cells (Figure 4).63−65 Sinceprevious studies have shown that tetracyclic coumarins havenotable anti-HIV activities and that the HIV and telomerasereverse transcriptase proteins display structural homology,dihydropyrazolepiperidine derivatives containing coumarinmoieties were also synthesized.63,66 These molecules showed

anticancer activity against gastric cancer and the most potentinhibited telomerase in crude cell extracts with IC50 values inthe 1−2 μM range.63,66 Results from docking studies were usedto suggest that these molecules bind the hTERT active sitethrough key intermolecular hydrogen bonding and π−cationinteractions with invariant residues Lys189, Asp254, andGln308 and by projecting aryl and dihyrdopyrazole rings intothe hydrophobic dNTP-binding pocket.63,65,66 The π−cationinteraction is consistent with the structure−activity relationship(SAR) trend in which electron withdrawing groups on thearomatic ring attenuate inhibition, whereas electron donatinggroups increased inhibition. 3D quantitative SAR (QSAR) wasused to rationalize the proposed binding mechanism, and acorrelation coefficient of r2 = 0.966 was found for predicted andobserved IC50 for a small, chemically similar test set.65 Othertelomerase inhibitors proposed to bind telomerase usingdocking studies include 2-chloropyridines and 2-aminomethyl-5-(quinolin-2-yl)-1,3,4-oxadiazole-2(3H)-thionequinoline de-rivatives, although these studies did not offer strong validationof the binding models.67,68

Several natural products have been identified as telomeraseinhibitors as well (Figure 5). Helenalin (6), a natural

sesquiterpene lactone, was found to inhibit telomeraseapparently by reaction with a cysteine residue similar to 3.69

o-Quinone containing compounds, for example, tanshinone 2A(7), a plant diterpene known to exhibit diverse pharmacologicalactivities, also inhibit telomerase (IC50 = 3 μM). The quinonewas fully responsible for inhibition that was time- and DTT-dependent. It was also found that 7 undergoes redox cycling toproduce hydrogen peroxide. The data suggest that quinoneredox cycling inhibits telomerase, perhaps by oxidation ofconserved cysteine residues.70 Another apparently reversibleinhibitor is 8 (U-73122), commonly used as an inhibitor of

Figure 3. Examples of small-molecule telomerase inhibitors identifiedby screening small molecule libraries.

Figure 4. Telomerase inhibitors reported to bind the telomerase active site based on docking studies.

Figure 5. Natural product and natural product derivatives identified astelomerase inhibitors.

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phospholipase C, which was reported to be a potent (IC50 = 0.2μM) telomerase inhibitor.71 Inhibition was shown to bedependent on the pyrrole-2,5-dione functional group, suggest-ing potential alkylation of telomerase. β-Rubromycin (9), aquinone antibiotic, inhibited telomerase with an IC50 of 3 μM.72

Mechanistically, 9 appears to be a competitive inhibitor versusthe telomerase primer, it also inhibits retroviral reversetranscriptases, mammalian DNA polymerases, and terminaldeoxynucleotidyl transferase,73 indicating that rubromycins arenot telomerase specific.The search for more biologically effective telomerase

inhibitors has encouraged innovative yeast-based screens.Natural products chrolactomycin (10) and UCS1025A (11)were identified as direct telomerase inhibitors using a forwardchemical genetics screen in a yeast system (Figure 6).74 A yeast

strain with artificially shortened telomeres was used to screen amicrobial metabolite library. Since telomeres were shortened,the strain was more sensitive to telomerase inhibition than wild

type. Therefore, compounds with selective activity in thetelomere shortened strain were more likely to be telomeraseinhibitors. With this assay, two new telomerase inhibitors 10(IC50 = 0.5 μM) and 11 (IC50 = 1.3 μM) were identified. Inaddition, the Hsp90 inhibitor radicicol was also identified as atelomerase maintenance inhibitor in screen.74 The synthesis of11 has been accomplished, but structure−activity relationshipsof analogues have not been reported.75,76 More recently, analternative yeast based assay was developed using recombinanthuman telomerase. Expression of human telomerase activity inyeast results in a growth delay; therefore, potential inhibitors ofhuman telomerase could rescue the transfected cells. By use ofthis screen, several potential telomerase inhibitors withmoderate potency (IC50 values of ∼1−10 μM) were identifiedto confirm the screening platform.77 The most potentcompound identified in the screen was the dichlorothiophene12 (CD11359).One promising class of small molecule telomerase inhibitors

is the potent enoylaminobenzoic acids represented by 13(BIBR1532, IC50 = 0.093 μM) and 14 (BIBR1591, IC50 = 0.47μM) that function as selective mixed-type, noncompetitivetelomerase inhibitors and were developed from systematicstructure−activity correlations (Figure 7).37 Limited structure−activity relationship data are publically available and show thatthe α−β unsaturated amide is essential and modifications to thenaphthyl group are not well tolerated.78 The mechanism ofinhibition by 13 is distinct from those by other non-nucleosidecompounds or antisense oligonucleotides, as it does not causechain termination events but rather inhibits the formation oflong reaction products. Compound 13 seems to act allosteri-cally to impair the unique ability of telomerase to catalyzeaddition of multiple telomeric repeats onto a DNA primer.79

Acute treatment with high concentrations of 13 caused directcytotoxic effects on malignant cells of the hematopoieticsystem, resulting in end to end fusions, phosphorylated p53,and decreased expression of TRF2, consistent with a telomere-associated effect.80 Compound 13 is also capable of sensitizingdrug-resistant cells to chemotherapeutic agents.81 Chronic

Figure 6. Small molecules identified as telomerase inhibitors utilizingyeast-based assays.

Figure 7. Treatment with 14 reversibly inhibits telomere length maintenance. (A) Structures of 13 and 14. (B) Chronic treatment of NCI-H460cells with 14 results in growth arrest that can be reversed after ending the treatment. Open circles are untreated cells. Filled triangles are cells treatedwith 14. Treatment was halted at day 220; note the return to proliferative growth. (C) Southern blot analysis of average telomere length oftelomerase inhibitor treated cells and cells released from telomerase inhibition. Treatment with 14 resulted in loss of telomere length and exhibited∼1.5 kb of telomeric DNA (lanes 1 and 2). Removal of 14 resulted in telomere elongation to ∼3 kb of telomeric DNA (lane 3). Untreated cells areshown in lane 4 for comparison and have 3 kb of telomeric DNA. Parts B and C are reprinted by permission from Macmillan Publishers Ltd.: TheEMBO Journal (http://www.nature.com/emboj/index.html) (Damm, K.; Hemmann, U.; Garin-Chesa, P.; Hauel, N.; Kauffmann, I.; Priepke, H.;Niestroj, C.; Daiber, C.; Enenkel, B.; Guilliard, B.; Lauritsch, I.; Muller, E.; Pascolo, E.; Sauter, G.; Pantic, M.; Martens, U. M.; Wenz, C.; Lingner, J.;Kraut, N.; Rettig, W. J.; Schnapp, A. EMBO J. 2001, 20, 6958−6968),37 copyright 2001.

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exposure to lower doses of 14 resulted in progressive erosion oftelomeric DNA leading to senescence.37 However, the lagphase between telomerase inhibition and the impact onproliferative capacity under the chronic treatment condition isa major concern,37 as is the apparently high protein bindingevidenced by the significantly decreased potency in crude cellextracts compared to purified telomerase.78 More than 120days, approximately 135 population doublings, was necessaryfor treatment with 13 to inhibit cell proliferation of NCI-H460lung cancer.82 Interestingly, washout of 14 in telomerasepositive cells allowed recovery of telomere length (Figure 7).37

■ SMALL MOLECULES THAT TARGET hTER AND THEhTER-TELOMERIC DNA HETERODUPLEX

In addition to targeting the enzymatic activity of telomerase,exploratory efforts in identifying ligands that target hTERsuggest that RNA-binding ligands may be a productive platformfor targeting telomerase. In two separate reports, oligonucleo-tides were used to identify regions of hTER that can bespecifically targeted to prevent proper telomerase assem-blage.83,84 Specifically, the CR4-CR5 and pseudoknot domainsof hTER are susceptible. Detailed analysis demonstrated thatdiscrete domains of hTER are blocked without losing the abilityof hTERT to associate with hTER.84 This suggests that theCR4-CR5 and pseudoknot domain targeting oligonucleotideseffectively cause formation of a dysfunctional telomerasecomplex. Subsequently, small molecules that bind hTER havebeen identified,85−87 and several of these including amino-glycosides and the bisbenzimide Heochst 33258 have beenshown to inhibit telomerase,86 presumably by binding hTERand perturbing proper assemblage of the holoenzyme complex(Figure 8). Efforts to target the telomerase template RNA−

DNA primer/nascent product heteroduplex have also beenreported.88,89 In these studies, several intercalators wereidentified that inhibit telomerase and bind preferentially tothe RNA−DNA heteroduplex as opposed to G-quadruplexDNA. Advances in this area include second generationintercalators and bis-intercalators that are fairly potenttelomerase inhibitors in cell free assays. A small library of bis-ethidium derivatives with varying linkers was screened.Surprisingly, the library exhibited a narrow range of moderatelypotent telomerase inhibition without significant improvementover the parent monointercalator.88 Data for cell based assaysusing small molecule targeting of hTER or the hTER/DNAheteroduplex have not yet been reported, so it is still too earlyto tell if this approach will be clinical viable.

■ TELOMERASE INHIBITION BY INDIRECT MEANS

hTERT Transcription Regulators. Cellular telomeraseactivity is regulated at various levels including transcription,mRNA splicing, post-transcriptional and post-translationalmodifications of hTER and hTERT, transport and subcellularlocalization, assembly of active telomerase ribonucleoprotein,and telomeric DNA accessibility. Transcriptional regulation ofhTERT is thought to be the major mechanism of telomeraseregulation.90 The hTERT promoter contains binding sites forseveral transcription factors, suggesting that the regulation ofhTERT expression may be subject to multiple levels of controlby various factors and dependent on cellular contexts.91 Aprimary activator of hTERT is the c-Myc oncogene, a knownregulator of cell proliferation, growth, and apoptosis.92 Targetsof the Myc family are involved in various aspects of cellularfunctions, including cell cycle, growth, differentiation, and lifespan. c-Myc expression correlates with hTERT, and both are

Figure 8. Ligands that inhibit telomerase by binding to the RNA subunit hTER (Hoechst 33258 and neomycin) and the telomerase DNA/RNAheteroduplex (bis-ethidium compounds).

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up-regulated in highly proliferative and immortal cells butdown-regulated during terminal differentiation and in non-proliferative cells. Therefore, it is not surprising that inhibitorsof c-Myc, butein,93 gamboic acid,94 and genistein,95 also inhibittelomerase. Additionally, genistein treatment resulted in down-regulation of the protein kinase Akt and thus hTERTphosphorylation, indicating that genistein can inhibit telomer-ase by at least two mechansisms: decreased hTERT tran-scription and decreased posttranslational activation ofhTERT.95

Second generation tyrosine kinase inhibitors and the lipid-derived signaling molecule ceramide have been shown to inhibithTERT promoter activity by altering the functions of Sp1 andSp3.96,97 Sp1 has been shown to collaborate with c-Myc inorder to initiate hTERT transcription.98 Imatinib, dasatinib, andnilotininb decrease telomerase activity and hTERT expressionby decreasing Sp1 nuclear levels and activation and reducingthe nuclear translocation of hTERT via suppression of Aktphosphorylation.96 Agents that act on hormonal pathways havealso been linked to telomerase activity regulation. For example,estrogen activates telomerase in hormone-sensitive tissues suchas mammary and ovary epithelial cells,99,100 while the selectiveestrogen receptor modulator tamoxifen reduces telomeraseactivity in cells where it functions as an estrogen antago-nist.101,102 Progesterone also has an antagonistic effect onestrogen-induced hTERT expression.103 These observationshighlight the fact that the polypharmacological effects of manyanticancer drugs are anticipated to include inhibition oftelomere maintenance as a mechanism of their anticancereffects.Negative Regulators of hTERT Transcription. The lack

of telomerase activity in somatic cells appears to be aconsequence of transcriptional repression of the hTERTexpression. The loss of transcriptional repression results inthe up-regulation of hTERT expression and telomerase activityas seen during carcinogenesis and cellular immortalization.Several transcriptional repressors of hTERT have beenidentified. One such negative regulator of hTERT transcriptionis Mad1. Mad1, c-Myc, and Max are part of a family oftranscription factors that bind to E-box-containing promotersto either down-regulate or up-regulate gene expression. Mad1 isup-regulated in telomerase negative somatic cells, c-Myc is up-regulated in cancer cell lines, and Max is expressedubiquitously.104 Consistent with its role in regulatingtelomerase, overexpression of Mad1 resulted in decreasedhTERT promoter activity in gene reporter assays.105

Another negative regulator of hTERT transcription is thetumor suppressor protein p53, which induces cell cycle arrest orapoptosis in response to DNA damage and other cellularstresses to inhibit tumor formation. p53 has been shown toinhibit telomerase activity through the repression of hTERTtranscription.106 Repression of telomerase activity was alsoobserved by overexpressing pRB in human squamous cellcarcinomas107 and E2F1 in head and neck squamous cellcarcinomas.108 pRB and E2F1 have the potential to transcrip-tionally repress hTERT activity either independently or intandem with one another. Another tumor suppressor involvedin the transcriptional repression of hTERT is Wilm’s tumorprotein 1 (WT1). WT1 has also been shown to significantlydecrease hTERT mRNA and telomerase activity in HEK293cells.109 The role of WT1 in hTERT repression is cell typedependent, as the WT1 gene is expressed only in specific cellssuch as the kidney, gonad, and spleen.

Other negative regulators of hTERT transcription includethe myeloid cell-specific zinc finger protein 2, interferon-α, andTGF-β. Interferon-α- and -γ-induced sensitization to apoptosisis associated with repressed transcriptional activity of thehTERT promoter in multiple myeloma.110 Transforminggrowth factor β, a potent tumor suppressor, inhibits telomerasethrough the transcription factors SMAD3 and E2F.111,112 TGF-β activated kinase 1 (TAK1) has been shown to represstranscription of the human telomerase reverse transcriptasegene. TAK1-induced repression was caused by the recruitmentof histone deacetylase to Sp1 at the hTERT promoter.113 T-cellacute lymphoblastic leukemia protein 1 (TAL1) is also anegative regulator of the hTERT promoter. Overexpression ofthe proto-oncogenic protein TAL1 leads to a decrease inhTERT mRNA levels and consequently reduced telomeraseactivity.114 Histone deacetylase (HDAC) inhibitors, such astrichostatin A, have been shown to induce apoptosis and down-regulate telomerase activity via suppression of hTERTexpression in leukemic U937 cells.115−117 Dynamic assemblyof the E2F−pocket protein−HDAC complex plays a centralrole in the regulation of hTERT. For example, a small molecule,CGK 1026, inhibits the recruitment of HDAC into E2F-pocketprotein complexes assembled on the hTERT promoter.118

These data underscore the fact that several anticancer treatmentstrategies are likely to result in telomerase inhibition indirectly.Several reports demonstrate the indirect effects on

telomerase activity that are possible with small molecules.Tea catechins such as (−)-epigallocatechin 3-gallate (15)prevent growth of a variety of cancer cell lines and inhibittelomerase activity in a concentration dependent manner bydecreasing hTERT mRNA levels.110,119 The changes in hTERTmRNA levels can be attributed to chromatin remodeling due tothe hypoacetylation of histones at the hTERT gene promoterand decreased methylation of the hTERT promoter at the E2F-1 binding site.119 Consistent with the observed decrease activityof the hTERT promoter, 15-treated U937 monoblastoidleukemia and HT29 colon adenocarcinoma cells experienceddecreased proliferation potential commensurate with telomereshortening. In addition, 15 was also shown to induce a severereduction in cell growth with enhanced membrane blebbing,cell swelling, apoptosis, and necrosis in laryngeal squamous cell,small-cell lung, and breast cancers.119−121 Further studiesshowed that the tea catechin derivative MST-312 (N,N′-bis(2,3-dihydroxybenzoyl)-1,2-phenylenediamine) appears toinhibit telomere maintenance by directly targeting telomerase(IC50 = 0.67 μM), resulting in telomere erosion at a 20-foldlower dose than 15 in the monoblastic leukemia cell lineU937.122

GSK3 inhibition by 6-bromoindirubin-3′-oxime causedsuppression of hTERT expression, telomerase activity, andtelomere length in various cancer cell lines and decreasedhTERT expression in ovarian cancer xenografts.123 Otherrepressors of hTERT transcription are retinoids, derivatives ofvitamin A, which have been shown to down-regulate telomeraseleading to telomere erosion.124 Retinoic acid receptor α andretinoid-X receptor-specific agonists synergistically down-regulated hTERT and induced tumor cell death.125

Posttranslational Regulators of hTERT. Perturbingposttranslational modifications represents another mechanismfor targeting telomerase. Because telomerase activity andcellular location is controlled by its phosphorylation state,phosphatase and kinase inhibitors can have direct effects ontelomerase. For example, the phosphatase 2A inhibitor okadaic

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acid prevents telomerase inhibition by phosphatase 2A in breastcancer cells.126 Protein kinase C (PKC) has been shown toenhance telomerase activity in certain cell types, and PKCinhibitors have been shown to inactivate telomerase innasopharyngeal127,128 and cervical129 cancer cells among others.Imatinib mesylate (Gleevec), a tyrosine kinase inhibitor, alsodown-regulates telomerase activity and inhibits proliferation intelomerase expressing cells.130 The protein kinase Akt has well-established roles in telomerase activation. Akt phosphorylateshTERT and activates telomerase in vitro, while the nonspecificAkt inhibitor wortmannin causes loss of telomerase activity.127

The tyrosine kinase c-Abl is also capable of phosphorylatinghTERT. The SH3 domain of c-Abl interacts with andphosphorylates hTERT resulting in a decrease in telomeraseactivity.131 These observations demonstrate that telomeraseactivity can be directly regulated by protein phosphorylationindependent of transcriptional regulation and highlight thattelomerase inhibition may contribute to the observedanticancer effects of several drugs. In addition to regulationby kinases, it has recently been determined that hTERT is asubstrate of caspases-6 and −7.132 Interestingly, the caspasesites are unique and the cleavage products appear remarkablystable.Hsp90 Inhibitors. The telomerase ribonucleoprotein

complex requires the Hsp90 chaperone complex, includingp23, Hsp70, p60, and Hsp40/γdl for proper assemblage. Hsp90associates directly with hTERT and is required to maintaintelomerase in an active conformation at least under someconditions.133,134 Hsp90 inhibitors cause inactivation, destabi-lization, and degradation of Hsp90 target proteins includinghTERT.46,133Since several oncogenic proteins including c-Myc,Akt, and mutated p53 require Hsp90 for their activity,inhibition of this chaperone should block multiple oncogenicpathways. Hsp90 inhibitors such as novobiocin, radicicol,geldanamycin, and 17-AAG were also shown to inhibittelomerase activity.134−137 Recently, curcumin has beenshown to inhibit nuclear localization of telomerase by

dissociating the Hsp90 co-chaperone p23 from hTERT.93

Curcumin treatment limited the association between p23 andhTERT while not affecting Hsp90 interaction with telomerase.

■ G-QUADRUPLEX STABILIZERSThe most advanced area of small-molecule drug discoverydirected at the telomere is G-quadruplex-stabilizing ligands. G-quadruplexes are unique structures formed by folding G-richnucleic acid sequences. These structures are heterogeneouswith a variety of structural topologies.138 Folding topologies areinfluenced by sequence, buffer conditions including identity ofcations present, molecular crowding, and other parameters.Significant effort toward determining the structures of humantelomeric DNA138,139 and the biological functions of G-quadruplex DNA at the telomere and in other G-rich sequencesembedded in the chromosome140 has been reported. Becausetelomerase must hybridize to its DNA primer to affordtelomeric DNA extension, it was proposed that G-quadruplexformation would render a primer inert to telomerase.141

Seminal studies by Zahler et al. showing that telomerasecould not extend a folded G-quadruplex were consistent withthis hypothesis and initiated the field of G-quadruplex bindingligands as a platform for telomerase inhibition.141 This reportwas quickly followed by the cardinal work of the Hurley andNeidle labs providing the first G-quadruplex ligands thateffectively inhibited telomerase.142,143 Since these reports, awide variety of pharmacophores have been explored as G-quadruplex ligands, and most of these are polycationic aromatichydrocarbons (Figure 9).144 Since these initial studiesdemonstrating a relationship between telomeres and G-quadruplexes, these structures have become much morecomplex as drug targets than originally assumed, as they arepredicted to be present in a wide variety of genomic locationswith increased density in promoter regions.145 Several of thesepromoters, including those for hTERT,146 c-kit,147,148 and c-myc,149,150 have verified abilities to fold into G-quadruplexstructures. Much of the work on G-quadruplex structure,

Figure 9. Representative G-quadruplex ligands that inhibit telomerase. (A) 16 (BRACO19), a 3,6,9 trisubstituted acridine derivative. (B) RHPS4, apentacyclic acridine derivative. (C) and (D) are models of 16 bound to a G-quadruplex formed by a dimer of TTAGGGTTAGGG. The structurewas rendered from PDB 3CE5. 16 is shown in red. dG residues are shown in cyan, and dA and dT residues are shown in gray.

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biological relevance, and small-molecule targeting has beenrecently and extensively reviewed, and readers are directed tothese.144,151,152 Here, we will simply highlight the majorconcerns with targeting G-quadruplexes as a specificallytelomere-directed anticancer drug modality and highlight recentapproaches toward advancing telomere-binding ligands to theclinic.The first G-quadruplex ligands explored as telomerase

inhibitors were disubstituted anthraquinone derivatives.153

Since that time, several other polycyclic heteroaromaticscaffolds and macrocycles have been explored allowing thedevelopment of potent telomerase inhibitors with increasingspecificity for G-quadruplex DNA compared to duplex DNA,and these ligands are available at the G-quadruplex ligandsdatabase (http://www.g4ldb.org/ci2/index.php).154 Impor-tantly, high resolution crystal and NMR structures of humantelomeric G-quadruplexes can inform rational design toimprove G-quadruplex affinity and specificity.152 Fromstructural studies, it is clear that critical features known toenhance G-quadruplex binding include a delocalized π-electronsystem capable of stacking onto G-tetrad a positive charge thatcan rest in the center of the G-quartets to interact with thecarbonyl oxygens from guanine that form the core of the G-quartets, and positively charged substituents that can interactwith the grooves of the G-quadruplex (Figure 9c and Figure9d).138 From a purely biochemical view, the most significantcurrent issues in G-quadruplex ligand design are specificity forG-quadruplex DNA compared to duplex DNA and specificityfor G-quadruplex type, i.e., telomeric versus other DNA andRNA quadruplexes that may be present.155 From apharmaceutical standpoint, the major issue concerning trans-lation of G-quadruplex ligands is the poor pharmacokineticprofile associated with the typically hydrophilic and positivelycharged molecules.156

One of the most significant recent advances toward selectiveG-quadruplex targeting is the ability to rationally designmolecules using structure-based approaches. Structures of G-quadruplexes formed by the human telomeric DNA sequenceby X-ray crystallography and NMR spectroscopy have providedtemplates for rational design of G-quaduplex ligands.157,158

These advances have allowed for the development of rationallydesigned molecules with increased.159,160 These promisingexamples suggest that G-quadruplex specificity is achievable.The more challenging hurdle that still remains is the poorpharmacokinetic and pharmacodynamic properties of promis-ing G-quadruplex compounds. These hurdles may not beinsurmountable, as evidenced by the G-quadruplex ligand andfluoroquinolone derivative quarfloxin, which binds to nucleo-lin/rDNA and inhibits Pol 1 transcription, thus inducingapoptosis in adenocarcinoma cells, and which has had someclinical success.161

■ DISRUPTION OF TELOMERE MAINTENANCEUSING ANTISENSE AND RELATED APPROACHES

Ribozymes. Hammerhead ribozymes are small catalyticRNA molecules that have the capacity to specifically cleavetarget RNA after NUH sequences, where N is any nucleotide,U is uridine, and H is any nucleotide except G.162 Design ofhammerhead ribozymes is relatively simple, and specificity canbe achieved by incorporating sequences complementary to thetarget. Because of this nature, ribozymes have been investigatedas a platform for telomerase inhibition via targeted RNAcleavage of hTER or hTERT mRNA.

The template region of hTER is an ideal target for ribozymecleavage because it has the requisite target sequence forHammerhead or HDV ribozymes, is essential for telomeraseactivity, and it may be solvent exposed even in the telomeraseholoenzyme. Studies using melanoma,163 hepatoma, coloncancer,164 and endometrial cancer cells165 have correlatedribozyme expression with a marked reduction in telomeraseactivity resulting from diminished telomerase RNA levels.While telomere lengths were greatly attenuated in somecarcinomas, other types did not display this phenotype.163,165

Treatment with template-targeting ribozymes also resulted insignificant growth retardation with proliferation rates reducedcompared to parent cells.166 Furthermore, treated cellsexperienced telomere shortening in concert with altered ordendritic morphologies, which are indicative of senescence,166

G0/G1 growth arrest, and increased apoptotic rate.164

Circularization and addition of a six nucleotide poly(A)sequence to these ribozymes increased target RNA cleavagewhile conferring protection from cellular exoribonucleasedegradation.167

TERT mRNA levels have been targeted by a Hammerheadribozyme targeting the initial nucleotides in hTERT mRNA,which may be essential for the proper binding of the translationinitiation complex to the 5′ mRNA cap structure.168 Ribozymecleavage in the middle of hTERT mRNA has also demonstrateddecreased telomerase activity while also influencing rapid celldeath (4 days) even without diminished telomere length,consistent with potentially nontelomeric roles of hTERT insome cancer cells.169

Oligonucleotides and Template Antagonists. Antisense(AS) oligonucleotides (ODNs) can inhibit telomerase byseveral mechanisms including blocking hTERT translation,reduction of hTERT mRNA and hTER levels, and stericallyblocking the association of telomerase with the telomere(template antagonists).42 However, oligonucleotides presentmany challenges toward achieving efficient intratumoraltelomerase inhibition. Nonetheless, studies have demonstratedthat unmodified antisense oligonucleotides can in fact potentlyinhibit telomerase activity under in vitro conditions.170−172 Oneof these was a genetic approach using a plasmid borne 125nucleotide hTR-antisense RNA, which inhibited tumor growthin mouse xenografts.170

To make up for the shortcomings of unmodified ODNs, aspectrum of ODNs with chemically modified backbones havebeen utilized to enhance the therapeutic potential of theseantisense strategies. Phosphorothioate (PS) ODNs composedof telomere repeat sequences ranging from 6 to 24 bases andtargeting the template of hTER showed significant telomeraseinhibition in various cancer cell lines.173,174 While longer PS-ODNs did not have any effect on tumor growth, a 6-mer PS-ODN decreased cellular growth in Burkett’s lymphoma,increased cell population doubling times, induced dramaticlevels of apoptosis, and reduced human xenograft tumors inmice to undetectable sizes.174 2′-O-Methyl-PS chimeric ODNstargeting the template sequence of hTER demonstrated potenttelomerase inhibition with IC50 values in the nanomolarrange.175,176 Similar ODNs blocked up to 95% of telomeraseactivity after 1 day of treatment when delivered with a cationiclipid delivery system and induced telomere erosion andsubsequent limited proliferation commensurate with markedapoptosis in long-term studies with chronic treatment.177,178

Cellular delivery with chitosan-coated PLGA nanoparticlesafforded even higher cellular uptake and the similar phenotype

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of progressive telomere shortening with an EC50 value of 10nM.179 Direct addition of 2′-O-methoxyethyl-PS chimericODNs decreased telomere length and proliferation and growthrates in MCF-7 (breast)180 and DU145181 (prostate) cancercells in culture and xenograft tumor models.180,181 Synergisticeffects were seen in DU145 prostate cancer cells upon additionof chemotherapeutic agents such as cisplatin or carboplatin.181

Peptide nucleic acids (PNAs) completely covering thetemplate region of hTER were 10−50 times more potentthan similar PS-ODNs with IC50 values in the nanomolarrange.182 The potent telomerase inhibition is due to highaffinity of the PNA to the single stranded RNA template andsuggests that affinity is not limited to base pairing but may alsoinclude interactions between hTERT and the peptide back-bone.182 Both cellular permeabilization and lipid-mediateddelivery demonstrated similar IC50 values in DU145 (prostatecancer)83 and HME50-5 (immortal breast epithelial)182 cells.Conjugation of short peptide sequences to PNAs targeting thehTER template displayed increased telomerase inhibition withIC50 values 10-fold lower than PNAs alone.183 Targeting ofhTER by 2-5A ODNs has led to a remarkable inhibition oftelomerase activity concomitant with rapid hTER degradationand a greater than 50% decrease in cell viability in a timedependent manner.41,184−186 2-5A ODNs targeting hTER canalso arrest cells in G2/M phase187 and induce apoptosis viaactivation of the caspase signaling cascade.186 Direct admin-istration of hTER-targeting 2-5A ODNs and combinationtherapy with interferon-β has demonstrated antiproliferativeeffects including a reduction in colony formation and asignificant retardation in tumor growth and volume overtime.184,185,188

In 2003, Asai et al. described a novel class of chemicallymodified N3′→P5′-thiophosphoramidates (NPS), which con-ferred high stability and sequence specific potency.42,189 Theefforts led to the development of 17 (GRN163), a 13-nucleotide long NPS-ODN with sequence 5′-TAGGGTT-AGACAA-3′ that targets the template region of hTER byoverlapping and extending four nucleotides beyond the 5′template boundary (IC50 = 0.14 μM).189 Treatment of variouscancer cell lines with 17 significantly reduced telomeraseactivity, promoted critical telomere loss, and instigatedapoptosis while concomitantly up-regulating caspase activity.189

These results combined with impressive data from long-termexperiments and its high potency and specificity indicated that17 may be an effective anticancer agent.189

In order to improve its bioavailability, lipophilicity, andcellular uptake, a 5′ terminal palmitoyl group was attachedthrough an amide bond to α-aminoglycerol of the thiophos-phoramidate 17 to produce the clinical candidate 1 (Figure10).190 Comparison studies with 17 proved that 1 was just aspotent at inhibiting telomerase in various cancers, could inhibittelomerase activity effectively without the use of a transfectionagent, and demonstrated greater tumor uptake.190−192 Since

then, many studies have been published revealing the extensiveanticancer potential of this drug in a variety of cancers. A singletreatment of 1 μM 1 yielded complete inhibition of telomeraseactivity in various cancers within hours of dosing and nearcomplete inhibition could be maintained for up to 6 days, whilechronic treatment allowed sustain inhibition with undetectabletelomerase activity.193−198 Continued treatment for 2 weekselicited morphological changes such as rounding and increasedextension of filliopodia-like structures,193,198 reduced colonyformation,194,196 and attenuated metastasis invasion.194 Longerperiods of treatment with 1 triggered decreased proliferation,196

decline in cell viability,197,199 disruption of focal adhesions andactin filament organization,193 substantial telomere short-ening,194−196 reduced capacity to form neurospheres andmammospheres,191,199 down-regulation of cell cycle regula-tors,192 G0/G1 cell cycle arrest,191,198 up-regulation of APG5and DAPK2 expression (genes implicated in the induction ofapoptosis),197 disrupted membrane intergrity,191 and promo-tion of apoptotic cell death.191,197 1 also promoted a higherpercentage of anaphase bridges, hallmarks of telomeredysfunction, and increased the frequency of γ-H2AX and53bp1 foci, known molecular markers that localize to sites ofDNA strand breaks and dysfunctional telomeres.200 In tumormodels, 1 prevented tumor formation and metastasis fromxenografts,194,195 significantly reduced growth and tumorvolume,191,200,201 and enhanced antitumor effects in combina-tion therapy with doxorubicin, temozolomide, and irradia-tion.191,200 The successful preclinical experiments demonstratedthe antitumor effectiveness of 1, and it remains the onlytelomerase inhibitor to have been tested in clinical trials.Lessons learned from clinical trials of 1 are essential for thefield, as this represents the first telomerase inhibitor to reachthe clinic. Currently, six phase I and phase I/II trials of 1 as asingle agent and three combination trials are ongoing. Majorobjectives of these studies are safety, tolerability, and maximumtolerated dose. Early reports indicate that 1 is well tolerated,but nothing further has been reported.

RNAi. RNA interference or silencing RNA (RNAi or siRNA)is double stranded RNAs that take advantage of a sequence-specific and natural mechanism that mediates gene silenc-ing.42,202 As an RNA that is complicit for telomerase activity,hTER is an excellent target for siRNA therapies. Transfectionand intravenous injection of partially double-stranded PS-ODNs targeting the template region of hTER reduced cellproliferation in human cervical carcinoma and, moreimportantly, strongly reduced the number of metastases inmurine malignant melanoma cells.203 siRNAs targetingnucleotides 11−19 of hTER expressed in various cancer celllines including HCT116 colon cancer, LOX melanoma, HeLacervical cancer, T24 bladder cancer, MCF-7 and BT424 breastcancers, and LNCaP prostate cancer, via lentiviral-vectorexpression, caused rapid growth inhibition and apoptosiswhile displaying negligible cellular cytotoxicity independent of

Figure 10. Clinical tested telomerase inhibitor. (A) 1 is a modified oligonucleotide that contains a 5′-palmotyl group. The sequence iscomplementary to the hTER template domain. (B) The N3′→P5′-thiophosphoramidate linkage present in 1.

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telomere length.204 Cancer cells exhibited knockdown ofendogenous hTER as well as down-regulation of cell cycleprogression genes (cyclin G2 and Cdc27) and specific genesthat play pivotal roles in tumor growth, angiogenesis, andmetastasis (integrin αV and Met proto-oncogene), suggesting arole for these genes in the hTER-mediated cell growthinhibition.204

Expression of siRNAs targeting hTERT mRNA in variouscancers has caused decreased hTERT mRNA levels, reducedhTERT expression, and notable telomerase inhibition within 24h of dosing and lasting up to 4 days.120,176,205−209 Stabletransfection of these siRNAs led to complete suppression oftelomerase activity in 3 days concomitant with the absence ofhTERT mRNA expression.205,207 Cancers treated with siRNAsthat deplete hTERT experienced decreased cell viability,120

prolonged population doubling times,209 suppression ofanchorage-dependent proliferation,206 significant growth inhib-ition,208 and morphological changes indicative of senescence.205

Esophageal and colon adenocarcinomas displayed extensivetelomere shortening,205,207 leading to chromosomes withtelomere free ends suggestive of complete telomere erosion.205

Genetic silencing of hTERT led to an up-regulation of genesinvolved in DNA damage recognition and repair, p53 familyproteins and their regulators, cell cycle inhibitors, proapoptoticproteins, and caspases.199,205,210 At the same time, hTERTdepletion significantly attenuated the expression of integrin αVand β3 subunits and c-Met, proteins that are known to playimportant roles in cell differentiation, adhesion, motility, andtumor metastasis.206 In tumor models, hTERT siRNAsdisrupted cell adhesion, migration, and invasion, promotedslower tumor growth,206 decreased cell viability and colonyformation, induced cell cycle arrest,199 reduced tumor sizes andvolumes in xenografts,206,208 and demonstrated additive effectsupon combination therapy with doxorubicin and intereferon-γ.120,199 Interestingly, while expression of siRNAs targetingboth hTER and hTERT concomitantly did decrease mRNA,RNA, and protein levels of telomerase components, cellproliferation, and xenograft tumor growth and promotepyknotic nuclear chromatin, the effect of silencing hTERalone was more pronounced than silencing either hTERT orboth together.211

■ IMMUNOTHERAPYThe realization that hTERT is overexpressed in the vastmajority of cancers prompted the investigation of hTERT as atumor antigen. Soon after the discovery of widespread hTERTexpression in tumors, hTERT peptide fragments were identifiedas tumor-associated antigens functioning through the majorhistocompatibility complex class I and class II pathways. Inseveral cancer types, CD8+ cytotoxic T-cells specific forhTERT peptides, primarily hTERT-I540 (ILAKFLHWL), aredetectable suggesting active hTERT-directed immune surveil-lance for these cancers.212 Subsequently, over 20 hTERT-derived peptides have been employed to boost antitumorimmunity.2 The majority of these elicit a CD8+ cytotoxic T-cellresponse, while the others activate a CD4+ response. Severalclinical trials of hTERT-based cancer vaccines have demon-strated the safety of this approach.213,214 Unfortunately, theimpact on disease progression reported to date is modest. Somepatients respond with significant tumor regression, but moregenerally only a low percentage of patients respond andresponses tend to be transient. This is despite the fact thatvaccination with hTERT-derived peptides generally does result

in hTERT-specific T cell production, indicative of animmunological response. Current efforts involve understandingthe differences between responsive and nonresponsive patients,optimization of antigenic hTERT epitopes, and optimization ofspecific immunotherapy platforms for telomerase-targetedtreatment strategies.

■ GENE DELIVERY

One of the main concerns with cancer gene therapy is theselective expression of target genes in tumor cells whileavoiding expression in normal tissues. Because activation oftelomerase activity in cancer cells is largely driven by increasedexpression of hTERT, it is reasonable to speculate that hTERTpromoter activity is commensurately increased. Takingadvantage of this increased promoter activity, several groupshave investigated the potential of the hTERT promoter forcancer-selective gene therapy. The hTERT promoter isextremely attractive for the selective expression of transgenesfor anticancer gene therapy because of the near universalexpression of hTERT in cancers and the much greater activityof the promoter in cancer cells compared to normal cells, eventhose that express telomerase. Most reports on the use of thehTERT promoter for cancer gene therapy attempt to controlexpression of anticancer genes in tumor cells. For example,proapoptotic genes (FADD and TRAIL) and a suicide gene(HSVtk) have been tested.215−217 In cell-based assays and inmouse models, tumor-specific transgene expression has beenachieved and promising results in mouse studies highlight earlysuccesses. Because these approaches utilize replication-deficientvirus in order to decrease harmful side effects, these approachesmay suffer from limited tumor-mass distribution. A differentand very promising approach that overcomes this limitation isto utilize the hTERT promoter not for transgene expressionbut instead viral replication, in essence establishing ananticancer virus. One hTERT-promoter driven adenovirus,telomelysin, has entered clinical trials in a small, 16 patient,phase I trial in which the patients had a wide variety of cancerdiagnoses.35 Early data suggest that telomelysin was welltolerated, but clinical response was limited, owing perhaps tothe single viral infection, which may have limited the viral titer.Most promising was the response of one patient with asignificant, 56%, reduction in metastatic tumor size.35

■ PERSPECTIVE AND FUTURE DIRECTIONS

Much progress has been made toward translating telomereresearch to the clinic since the seminal studies, demonstratingthe importance of telomeres, the identification of telomeraseand telomere-binding proteins, and the validation of telomerelength maintenance as a requirement of cancer cells. Earlyclinical trials with telomerase inhibitors have shown that theapproach is well tolerated and does reduce telomerase activity.However, the absence of reported clinical efficacy suggestslimited progress. Future trials examining which patients mightbe best suited for telomerase inhibitor therapy and current trialstesting telomerase inhibitors in combination with othertraditional chemotherapeutics can be expected to advance thefield. We can also expect continued advances in gene therapy bytaking advantage of the robust activity of the hTERT promoterin cancer cells as viral vectors are optimized. Likewise,immunotherapy targeting hTERT antigens is expected tocontinue its progress through clinical trials and arguably holds

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the most promise currently for telomerase-targeting therapeu-tics.Surprisingly, small-molecule inhibitors of telomerase have

not fared well. This may change as advances in telomerasestructural biology; for example, the crystal structure of an insectTERT,62 structures of several hTER domains,218,219 models ofhTERT based on the beetle crystal structure,220 and the recentthree-dimensional model of human telomerase based onelectron microscopy images221 increase our understanding oftelomerase structure and provide templates for rational small-molecule drug design. Rationally designed telomerase inhibitorsmay allow increased specificity, bioavailability, and inhibitionmechanisms that have greater effects in a clinical setting thanthose small molecules that are currently available. In addition,the observation that G-quadruplex ligands can directly disrupttelomere structure evidently by blocking binding of telomere-binding proteins such as TRF2 and POT1 suggests that thereare more telomere-associated targets to be exploited, and wecan expect that near future studies will determine theeffectiveness of directly targeting the telomere as an anticancerdrug platform. Targeting telomere biology as a means toimprove human health remains an important objective thatdrives discoveries in telomere biology and telomerasebiochemistry. The interdisciplinary approaches toward targetingtelomere biology have led to three discrete areas of clinicaltrials: inhibition of telomerase, hTERT-directed immunother-apy, and hTERT-promoter gene/viral therapy. It seems that thefuture of pharmaceutical benefit from telomere-targeting agentsremains promising, and our increased understanding oftelomere biochemistry and biology is sure to inform moreeffective approaches toward developing telomere biologytargeted agents.

■ AUTHOR INFORMATION

Corresponding Author*Phone: 919-966-6422. E-mail: jarstfer@unc.edu.

NotesThe authors declare no competing financial interest.Biographies

Vijay Sekaran is a postdoctoral fellow at the University of Colorado atBoulder working with Drs. James Goodrich and Jennifer Kugel. Hereceived his B.S. in Biomedical Engineering from the Georgia Instituteof Technology in 2005 and his Ph.D. in Pharmaceutical Sciences fromthe University of North Carolina at Chapel Hill under the guidance ofMichael Jarstfer in 2012. His dissertation focused on understandingthe structure and function of telomerase RNAs as well ascharacterizing the effects of small molecules that bind to humantelomerase RNA and inhibit telomerase activity.

Joana Soares is a research scientist at Genezyme. She received her B.S.in Chemistry from The College of William and Mary, VA, in 2004 andher Ph.D. in Pharmaceutical Sciences from the University of NorthCarolina at Chapel Hill in 2010 under the supervision of MichaelJarstfer. Her dissertation focused on elucidating the mechanism oftelomerase activation or repression and its impact on cellular pathwaysand elucidating the mechanism of action of new potential telomeraseinhibitors. Her research interrogated the relationship betweentelomerase, aging, and cancer progression.

Michael B. Jarstfer is an Associate Professor of Chemical Biology andMedicinal Chemistry in the Eshelman School of Pharmacy at theUniversity of North Carolina at Chapel Hill. He received his B.A. inBiochemistry from Trinity University, San Antonio TX, and his Ph.D.

from the University of Utah under the guidance of Dale Poulter. Heconducted postdoctoral research in the laboratory of Thomas Cech atthe University of Colorado in Boulder. His research interests includestructure and function of telomerase, development of telomeretargeting agents, the roles of telomere biology in human health, andthe molecular basis for social motivation.

■ ABBREVIATIONS USED

ALT, alternative lengthening of telomeres; AS, antisense;hTERT, human telomerase reverse transcriptase; HDAC,histone deacetylase; hTER, human telomerase RNA; NPS,N3′→P5′-thiophosphoramidates; ODN, oligonucleotide; PKC,protein kinase C; PNA, peptide nucleic acid; POT1, Protectionof Telomeres 1; PS, phosphorothioate; QSAR, quantitativestructure−activity relationship; RNAi, RNA interference; SAR,structure−activity relationship; siRNA, silencing RNA; TIF,telomere dysfunction induced focus; TAK1, TGF-β activatedkinase 1; TAL1, T-cell acute lymphoblastic leukemia protein 1;WT1, Wilm’s tumor protein 1

■ REFERENCES(1) Shay, J. W.; Bacchetti, S. A survey of telomerase activity in humancancer. Eur. J. Cancer 1997, 33, 787−791.(2) Beatty, G. L.; Vonderheide, R. H. Telomerase as a universaltumor antigen for cancer vaccines. Expert Rev. Vaccines 2008, 7, 881−887.(3) Fujiwara, T.; Urata, Y.; Tanaka, N. Telomerase-specific oncolyticvirotherapy for human cancer with the hTERT promoter. Curr. CancerDrug Targets 2007, 7, 191−201.(4) Jakupciak, J. P. Real-time telomerase activity measurements fordetection of cancer. Expert Rev. Mol. Diagn. 2005, 5, 745−753.(5) Deng, Y.; Chan, S. S.; Chang, S. Telomere dysfunction andtumour suppression: the senescence connection. Nat. Rev. Cancer2008, 8, 450−458.(6) Palm, W.; de Lange, T. How shelterin protects mammaliantelomeres. Annu. Rev. Genet. 2008, 42, 301−334.(7) Sperka, T.; Wang, J.; Rudolph, K. L. DNA damage checkpoints instem cells, ageing and cancer. Nat. Rev. Mol. Cell Biol. 2012, 13, 579−590.(8) Moon, I. K.; Jarstfer, M. B. The human telomere and itsrelationship to human disease, therapy, and tissue engineering. Front.Biosci. 2007, 12, 4595−4620.(9) Blasco, M. A. Telomeres and human disease: ageing, cancer andbeyond. Nat. Rev. Genet. 2005, 6, 611−622.(10) Collins, K. The biogenesis and regulation of telomeraseholoenzymes. Nat. Rev. Mol. Cell Biol. 2006, 7, 484−494.(11) Sekaran, V. G.; Soares, J.; Jarstfer, M. B. Structures of telomerasesubunits provide functional insights. Biochim. Biophys. Acta 2010, 1804,1190−1201.(12) Feng, J.; Funk, W. D.; Wang, S. S.; Weinrich, S. L.; Avilion, A.A.; Chiu, C. P.; Adams, R. R.; Chang, E.; Allsopp, R. C.; Yu, J.; Le, S.;West, M. D.; Harley, C.; Andrews, W. H.; Greider, C. W.; Villeponteu,B. The RNA component of human telomerase. Science 1995, 269,1236−1241.(13) Nakamura, T. M.; Morin, G. B.; Chapman, K. B.; Weinrich, S.L.; Andrews, W. H.; Lingner, J.; Harley, C. B.; Cech, T. R. Telomerasecatalytic subunit homologs from fission yeast and human. Science 1997,277, 955−959.(14) Huang, F. W.; Hodis, E.; Xu, M. J.; Kryukov, G. V.; Chin, L.;Garraway, L. A. Highly recurrent TERT promoter mutations in humanmelanoma. Science 2013, 339, 957−959.(15) Karlseder, J.; Smogorzewska, A.; de Lange, T. Senescenceinduced by altered telomere state, not telomere loss. Science 2002, 295,2446−2449.

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(16) Griffith, J. D.; Comeau, L.; Rosenfield, S.; Stansel, R. M.;Bianchi, A.; Moss, H.; de Lange, T. Mammalian telomeres end in alarge duplex loop. Cell 1999, 97, 503−514.(17) Cao, Y.; Bryan, T. M.; Reddel, R. R. Increased copy number ofthe TERT and TERC telomerase subunit genes in cancer cells. CancerSci. 2008, 99, 1092−1099.(18) Cesare, A. J.; Reddel, R. R. Telomere uncapping and alternativelengthening of telomeres. Mech. Ageing Dev. 2008, 129, 99−108.(19) Muntoni, A.; Reddel, R. R. The first molecular details of ALT inhuman tumor cells. Hum. Mol. Genet. 2005, 14, R191−R196.(20) Hahn, W. C.; Counter, C. M.; Lundbeg, A. S.; Beijersbergen, R.L.; Brooks, M. W.; Weinberg, R. A. Creation of human tumour cellswith defined genetic elements. Nature 1999, 400, 464−468.(21) Aubert, G.; Lansdorp, P. M. Telomeres and aging. Physiol. Rev.2008, 88, 557−579.(22) Bollmann, F. M. The many faces of telomerase: emergingextratelomeric effects. BioEssays 2008, 30, 728−732.(23) Artandi, S. E.; DePinho, R. A. Telomeres and telomerase incancer. Carcinogenesis 2010, 31, 9−18.(24) Xu, L.; Li, S.; Stohr, B. A. The role of telomere biology incancer. Annu. Rev. Pathol. 2013, 8, 49−78.(25) Ju, Z.; Rudolph, K. L. Telomeres and telomerase in cancer stemcells. Eur. J. Cancer 2006, 42, 1197−1203.(26) Armanios, M.; Greider, C. W. Telomerase and cancer stem cells.Cold Spring Harbor Symp. Quant. Biol. 2005, 70, 205−208.(27) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West,M. D.; Ho, P. L.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay,J. W. Specific association of human telomerase activity with immortalcells and cancer. Science 1994, 266, 2011−2015.(28) Svenson, U.; Roos, G. Telomere length as a biological marker inmalignancy. Biochim. Biophys. Acta 2009, 1792, 317−323.(29) Hahn, W. C.; Stewart, S. A.; Brooks, M. W.; York, S. G.; Eaton,E.; Kurachi, A.; Beijersbergen, R. L.; Knoll, J. H. M.; Maeyerson, M.;Weinberg, R. A. Inhibition of telomerase limits the growth of humancancer cells. Nat. Med. 1999, 5, 1164−1170.(30) Zhang, X.; Mar, V.; Zhou, W.; Harrington, L.; Robinson, M. O.Telomere shortening and apoptosis in telomerase-inhibited humantumor cells. Genes Dev. 1999, 13, 2388−2399.(31) Roth, A.; Harley, C. B.; Baerlocher, G. M. Imetelstat(GRN163L)telomerase-based cancer therapy. Recent Results CancerRes. 2010, 184, 221−234.(32) Wong, H. M.; Payet, L.; Huppert, J. L. Function and targeting ofG-quadruplexes. Curr. Opin. Mol. Ther. 2009, 11, 146−155.(33) Monchaud, D.; Teulade-Fichou, M. P. A hitchhiker’s guide to G-quadruplex ligands. Org. Biomol. Chem. 2008, 6, 627−636.(34) Kyo, S.; Takakura, M.; Fujiwara, T.; Inoue, M. Understandingand exploiting hTERT promoter regulation for diagnosis andtreatment of human cancers. Cancer Sci. 2008, 99, 1528−1538.(35) Nemunaitis, J.; Tong, A. W.; Nemunaitis, M.; Senzer, N.;Phadke, A. P.; Bedell, C.; Adams, N.; Zhang, Y. A.; Maples, P. B.;Chen, S.; Pappen, B.; Burke, J.; Ichimaru, D.; Urata, Y.; Fujiwara, T. Aphase I study of telomerase-specific replication competent oncolyticadenovirus (telomelysin) for various solid tumors. Mol. Ther. 2010, 18,429−434.(36) Liu, J. P.; Chen, W.; Schwarer, A. P.; Li, H. Telomerase incancer immunotherapy. Biochim. Biophys. Acta, Rev. Cancer 2010, 1805,35−42.(37) Damm, K.; Hemmann, U.; Garin-Chesa, P.; Hauel, N.;Kauffmann, I.; Priepke, H.; Niestroj, C.; Daiber, C.; Enenkel, B.;Guilliard, B.; Lauritsch, I.; Muller, E.; Pascolo, E.; Sauter, G.; Pantic,M.; Martens, U. M.; Wenz, C.; Lingner, J.; Kraut, N.; Rettig, W. J.;Schnapp, A. A highly selective telomerase inhibitor limiting humancancer cell proliferation. EMBO J. 2001, 20, 6958−6968.(38) Huffman, K. E.; Levene, S. D.; Tesmer, V. M.; Shay, J. W.;Wright, W. E. Telomere shortening is proportional to the size of theG-rich telomeric 3′-overhang. J. Biol. Chem. 2000, 275, 19719−19722.(39) Zou, Y.; Sfeir, A.; Gryaznov, S. M.; Shay, J. W.; Wright, W. E.Does a sentinel or a subset of short telomeres determine replicativesenescence? Mol. Biol. Cell 2004, 15, 3709−3718.

(40) de Lange, T. Shelterin: the protein complex that shapes andsafeguards human telomeres. Genes Dev. 2005, 19, 2100−2110.(41) Hu, J.; Hwang, S. S.; Liesa, M.; Gan, B.; Sahin, E.; Jaskelioff, M.;Ding, Z.; Ying, H.; Boutin, A. T.; Zhang, H.; Johnson, S.; Ivanova, E.;Kost-Alimova, M.; Protopopov, A.; Wang, Y. A.; Shirihai, O. S.; Chin,L.; DePinho, R. A. Antitelomerase therapy provokes ALT andmitochondrial adaptive mechanisms in cancer. Cell 2012, 148, 651−663.(42) Folini, M.; Zaffaroni, N. Targeting telomerase by antisense-based approaches: perspectives for new anti-cancer therapies. Curr.Pharm. Des. 2005, 11, 1105−1117.(43) Cong, Y.; Shay, J. W. Actions of human telomerase beyondtelomeres. Cell Res. 2008, 18, 725−732.(44) Park, J. I.; Venteicher, A. S.; Hong, J. Y.; Choi, J.; Jun, S.; Shkreli,M.; Chang, W.; Meng, Z.; Cheung, P.; Ji, H.; McLaughlin, M.;Veenstra, T. D.; Nusse, R.; McCrea, P. D.; Artandi, S. E. Telomerasemodulates Wnt signalling by association with target gene chromatin.Nature 2009, 460, 66−72.(45) Ghosh, A.; Saginc, G.; Leow, S. C.; Khattar, E.; Shin, E. M.; Yan,T. D.; Wong, M.; Zhang, Z.; Li, G.; Sung, W. K.; Zhou, J.; Chng, W. J.;Li, S.; Liu, E.; Tergaonkar, V. Telomerase directly regulates NF-kappaB-dependent transcription. Nat. Cell Biol. 2012, 14, 1270−1281.(46) De Cian, A.; Lacroix, L.; Douarre, C.; Temime-Smaali, N.;Trentesaux, C.; Riou, J. F.; Mergny, J. L. Targeting telomeres andtelomerase. Biochimie 2008, 90, 131−155.(47) Salvati, E.; Leonetti, C.; Rizzo, A.; Scarsella, M.; Mottolese, M.;Galati, R.; Sperduti, I.; Stevens, M. F.; D’Incalci, M.; Blasco, M.;Chiorino, G.; Bauwens, S.; Horard, B.; Gilson, E.; Stoppacciaro, A.;Zupi, G.; Biroccio, A. Telomere damage induced by the G-quadruplexligand RHPS4 has an antitumor effect. J. Clin. Invest. 2007, 117, 3236−3247.(48) Miyazaki, T.; Pan, Y.; Joshi, K.; Purohit, D.; Hu, B.; Demir, H.;Mazumder, S.; Okabe, S.; Yamori, T.; Viapiano, M.; Shin-ya, K.;Seimiya, H.; Nakano, I. Telomestatin impairs glioma stem cell survivaland growth through the disruption of telomeric G-quadruplex andinhibition of the proto-oncogene, c-Myb. Clin. Cancer Res. 2012, 18,1268−1280.(49) Gong, Y.; de Lange, T. A Shld1-controlled POT1a providessupport for repression of ATR signaling at telomeres through RPAexclusion. Mol. Cell 2010, 40, 377−387.(50) Melana, S. M.; Holland, J. F.; Pogo, B. G. Inhibition of cellgrowth and telomerase activity of breast cancer cells in vitro by 3′-azido-3′-deoxythymidine. Clin. Cancer Res. 1998, 4, 693−696.(51) Strahl, C.; Blackburn, E. H. Effects of reverse transcriptaseinhibitors on telomere length and telomerase activity in twoimmortalized human cell lines. Mol. Cell. Biol. 1996, 16, 53−65.(52) Datta, A.; Bellon, M.; Sinha-Datta, U.; Bazarbachi, A.;Lepelletier, Y.; Canioni, D.; Waldmann, T. A.; Hermine, O.; Nicot,C. Persistent inhibition of telomerase reprograms adult T-cell leukemiato p53-dependent senescence. Blood 2006, 108, 1021−1029.(53) Johnston, J. S.; Johnson, A.; Gan, Y.; Wientjes, M. G.; Au, J. L.Synergy between 3′-azido-3′-deoxythymidine and paclitaxel in humanpharynx FaDu cells. Pharm. Res. 2003, 20, 957−961.(54) Yasuda, C.; Kato, M.; Kuroda, D.; Ohyanagi, H. Experimentalstudies on potentiation of the antitumor activity of 5-fluorouracil with3′-azido-3′-deoxythymidine for the gastric cancer cell line MKN28 invivo. Jpn. J. Cancer Res 1997, 88, 97−102.(55) Chen, C.; Zhang, Y.; Wang, Y.; Huang, D.; Xi, Y.; Qi, Y.Synergic effect of 3′-azido-3′-deoxythymidine and arsenic trioxide insuppressing hepatoma cells. Anti-Cancer Drugs 2011, 22, 435−443.(56) Hukezalie, K. R.; Thumati, N. R.; Cote, H. C.; Wong, J. M. Invitro and ex vivo inhibition of human telomerase by anti-HIVnucleoside reverse transcriptase inhibitors (NRTIs) but not by non-NRTIs. PLoS One 2012, 7, e47505.(57) Leeansyah, E.; Cameron, P. U.; Solomon, A.; Tennakoon, S.;Velayudham, P.; Gouillou, M.; Spelman, T.; Hearps, A.; Fairley, C.;Smit de, V.; Pierce, A. B.; Armishaw, J.; Crowe, S. M.; Cooper, D. A.;Koelsch, K. K.; Liu, J. P.; Chuah, J.; Lewin, S. R. Inhibition oftelomerase activity by human immunodeficiency virus (HIV) nucleos-

Journal of Medicinal Chemistry Perspective

dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXM

(t)ide reverse transcriptase inhibitors: a potential factor contributing toHIV-associated accelerated aging. J. Infect. Dis. 2013, 207, 1157−1165.(58) Fletcher, T. M.; Cathers, B. E.; Ravikumar, K. S.; Mamiya, B. M.;Kerwin, S. M. Inhibition of human telomerase by 7-deaza-2′-deoxyguanosine nucleoside triphosphate analogs: potent inhibitionby 6-thio-7-deaza-2′-deoxyguanosine 5′-triphosphate. Bioorg. Chem.2001, 29, 36−55.(59) Hayakawa, N.; Nozawa, K.; Ogawa, A.; Kato, N.; Yoshida, K.;Akamatsu, K.; Tsuchiya, M.; Nagasaka, A.; Yoshida, S. Isothiazolonederivatives selectively inhibit telomerase from human and rat cancercells in vitro. Biochemistry 1999, 38, 11501−11507.(60) Kim, J. H.; Lee, G. E.; Lee, J. E.; Chung, I. K. Potent inhibitionof human telomerase by nitrostyrene derivatives. Mol. Pharmacol.2003, 63, 1117−1124.(61) Kim, J. H.; Lee, G. E.; Kim, S. W.; Chung, I. K. Identification ofa quinoxaline derivative that is a potent telomerase inhibitor leading tocellular senescence of human cancer cells. Biochem. J. 2003, 373, 523−529.(62) Gillis, A. J.; Schuller, A. P.; Skordalakes, E. Structure of theTribolium castaneum telomerase catalytic subunit TERT. Nature 2008,455, 633−637.(63) Liu, X. H.; Li, J.; Shi, J. B.; Song, B. A.; Qi, X. B. Design andsynthesis of novel 5-phenyl-N-piperidine ethanone containing 4,5-dihydropyrazole derivatives as potential antitumor agents. Eur. J. Med.Chem. 2012, 51, 294−299.(64) Liu, X. H.; Ruan, B. F.; Liu, J. X.; Song, B. A.; Jing, L. H.; Li, J.;Yang, Y.; Zhu, H. L.; Qi, X. B. Design and synthesis of N-phenylacetyl(sulfonyl) 4,5-dihydropyrazole derivatives as potential antitumoragents. Bioorg. Med. Chem. Lett. 2011, 21, 2916−2920.(65) Luo, Y.; Zhang, S.; Qiu, K. M.; Liu, Z. J.; Yang, Y. S.; Fu, J.;Zhong, W. Q.; Zhu, H. L. Synthesis, biological evaluation, 3D-QSARstudies of novel aryl-2H-pyrazole derivatives as telomerase inhibitors.Bioorg. Med. Chem. Lett. 2013, 23, 1091−1095.(66) Liu, X. H.; Liu, H. F.; Chen, J.; Yang, Y.; Song, B. A.; Bai, L. S.;Liu, J. X.; Zhu, H. L.; Qi, X. B. Synthesis and molecular docking studyof novel coumarin derivatives containing 4,5-dihydropyrazole moietyas potential antitumor agents. Bioorg. Med. Chem. Lett. 2010, 20,5705−5708.(67) Zheng, Q. Z.; Zhang, X. M.; Xu, Y.; Cheng, K.; Jiao, Q. C.; Zhu,H. L. Synthesis, biological evaluation, and molecular docking studies of2-chloropyridine derivatives possessing 1,3,4-oxadiazole moiety aspotential antitumor agents. Bioorg. Med. Chem. 2010, 18, 7836−7841.(68) Sun, J.; Zhu, H.; Yang, Z. M.; Zhu, H. L. Synthesis, molecularmodeling and biological evaluation of 2-aminomethyl-5-(quinolin-2-yl)-1,3,4-oxadiazole-2(3H)-thione quinolone derivatives as novelanticancer agent. Eur. J. Med. Chem. 2013, 60, 23−28.(69) Huang, P. R.; Yeh, Y. M.; Wang, T. C. Potent inhibition ofhuman telomerase by helenalin. Cancer Lett. 2005, 227, 169−174.(70) Soares, J.; Keppler, B. R.; Wang, X.; Lee, K. H.; Jarstfer, M. B.Ortho-quinone tanshinones directly inhibit telomerase through anoxidative mechanism mediated by hydrogen peroxide. Bioorg. Med.Chem. Lett. 2011, 21, 7474−7478.(71) Chen, Y. J.; Sheng, W. Y.; Huang, P. R.; Wang, T. C. Potentinhibition of human telomerase by U-73122. J. Biomed. Sci. 2006, 13,667−674.(72) Ueno, T.; Takahashi, H.; Oda, M.; Mizunuma, M.; Yokoyama,A.; Goto, Y.; Mizushina, Y.; Sakaguchi, K.; Hayashi, H. Inhibition ofhuman telomerase by rubromycins: implication of spiroketal system ofthe compounds as an active moiety. Biochemistry 2000, 39, 5995−6002.(73) Mizushina, Y.; Ueno, T.; Oda, M.; Yamaguchi, T.; Saneyoshi,M.; Sakaguchi, K. The biochemical mode of inhibition of DNApolymerase beta by alpha-rubromycin. Biochim. Biophys. Acta 2000,1523, 172−181.(74) Nakai, R.; Ishida, H.; Asai, A.; Ogawa, H.; Yamamoto, Y.;Kawasaki, H.; Akinaga, S.; Mizukami, T.; Yamashita, Y. Telomeraseinhibitors identified by a forward chemical genetics approach using ayeast strain with shortened telomere length. Chem. Biol. 2006, 13,183−190.

(75) Uchida, K.; Ogawa, T.; Yasuda, Y.; Mimura, H.; Fujimoto, T.;Fukuyama, T.; Wakimoto, T.; Asakawa, T.; Hamashima, Y.; Kan, T.Stereocontrolled total synthesis of (+)-UCS1025A. Angew. Chem.2012, 51, 12850−12853.(76) Lambert, T. H.; Danishefsky, S. J. Total synthesis of UCS1025A.J. Am. Chem. Soc. 2006, 128, 426−427.(77) Wong, L. H.; Unciti-Broceta, A.; Spitzer, M.; White, R.; Tyers,M.; Harrington, L. A yeast chemical genetic screen identifies inhibitorsof human telomerase. Chem. Biol. 2013, 20, 333−340.(78) Barma, D. K.; Elayadi, A.; Falck, J. R.; Corey, D. R. Inhibition oftelomerase by BIBR 1532 and related analogues. Bioorg. Med. Chem.Lett. 2003, 13, 1333−1336.(79) Pascolo, E.; Wenz, C.; Lingner, J.; Hauel, N.; Priepke, H.;Kauffmann, I.; Garin-Chesa, P.; Rettig, W. J.; Damm, K.; Schnapp, A.Mechanism of human telomerase inhibition by BIBR1532, a synthetic,non-nucleosidic drug candidate. J. Biol. Chem. 2002, 277, 15566−15572.(80) El-Daly, H.; Kull, M.; Zimmermann, S.; Pantic, M.; Waller, C.F.; Martens, U. M. Selective cytotoxicity and telomere damage inleukemia cells using the telomerase inhibitor BIBR1532. Blood 2005,105, 1742−1749.(81) Ward, R. J.; Autexier, C. Pharmacological telomerase inhibitioncan sensitize drug-resistant and drug-sensitive cells to chemo-therapeutic treatment. Mol. Pharmacol. 2005, 68, 779−786.(82) Pantic, M.; Zimmermann, S.; Waller, C. F.; Martens, U. M. Thelevel of telomere dysfunction determines the efficacy of telomerase-based therapeutics in a lung cancer cell line. Int. J. Cancer 2005, 26,1227−1232.(83) Hamilton, S. E.; Simmons, C. G.; Kathiriya, I. S.; Corey, D. R.Cellular delivery of peptide nucleic acids and inhibition of humantelomerase. Chem. Biol. 1999, 6, 343−351.(84) Keppler, B. R.; Jarstfer, M. B. Inhibition of telomerase activity bypreventing proper assemblage. Biochemistry 2004, 43, 334−343.(85) Pinto, I. G.; Guilbert, C.; Ulyanov, N. B.; Stearns, J.; James, T. L.Discovery of ligands for a novel target, the human telomerase RNA,based on flexible-target virtual screening and NMR. J. Med. Chem.2008, 51, 7205−7215.(86) Dominick, P. K.; Keppler, B. R.; Legassie, J. D.; Moon, I. K.;Jarstfer, M. B. Nucleic acid-binding ligands identify new mechanismsto inhibit telomerase. Bioorg. Med. Chem. Lett. 2004, 14, 3467−3471.(87) Kaiser, M.; Sainlos, M.; Lehn, J. M.; Bombard, S.; Teulade-Fichou, M. P. Aminoglycoside-quinacridine conjugates: towardsrecognition of the P6.1 element of telomerase RNA. ChemBioChem2006, 7, 321−329.(88) Jain, N.; Francis, S.; Friedman, S. H. Inhibition of therapeuticallyimportant polymerases with high affinity bis-intercalators. Bioorg. Med.Chem. Lett. 2012, 22, 4844−4848.(89) Rangarajan, S.; Friedman, S. H. Design, synthesis, andevaluation of phenanthridine derivatives targeting the telomeraseRNA/DNA heteroduplex. Bioorg. Med. Chem. Lett. 2007, 17, 2267−2273.(90) Meyerson, M.; Counter, C. M.; Eaton, E. N.; Ellisen, L. W.;Steiner, P.; Caddle, S. D.; Ziaugra, L.; Beijersbergen, R. L.; Davidoff,M. J.; Liu, Q.; Bacchetti, S.; Haber, D. A.; Weinberg, R. A. hEST2, theputative human telomerase catalytic subunit gene, is up-regulated intumor cells and during immortalization. Cell 1997, 90, 785−795.(91) Cong, Y. S.; Wen, J.; Bacchetti, S. The human telomerasecatalytic subunit hTERT: organization of the gene and characterizationof the promoter. Hum. Mol. Genet. 1999, 8, 137−142.(92) Grandori, C.; Cowley, S. M.; James, L. P.; Eisenman, R. N. TheMyc/Max/Mad network and the transcriptional control of cellbehavior. Annu. Rev. Cell Dev. Biol. 2000, 16, 653−699.(93) Moon, D. O.; Kim, M. O.; Lee, J. D.; Choi, Y. H.; Kim, G. Y.Butein suppresses c-Myc-dependent transcription and Akt-dependentphosphorylation of hTERT in human leukemia cells. Cancer Lett.2009, 286, 172−179.(94) Guo, Q. L.; Lin, S. S.; You, Q. D.; Gu, H. Y.; Yu, J.; Zhao, L.; Qi,Q.; Liang, F.; Tan, Z.; Wang, X. Inhibition of human telomerase

Journal of Medicinal Chemistry Perspective

dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXN

reverse transcriptase gene expression by gambogic acid in humanhepatoma SMMC-7721 cells. Life Sci. 2006, 78, 1238−1245.(95) Jagadeesh, S.; Kyo, S.; Banerjee, P. P. Genistein repressestelomerase activity via both transcriptional and posttranslationalmechanisms in human prostate cancer cells. Cancer Res. 2006, 66,2107−2115.(96) Shapira, S.; Granot, G.; Mor-Tzuntz, R.; Raanani, P.; Uziel, O.;Lahav, M.; Shpilberg, O. Second-generation tyrosine kinase inhibitorsreduce telomerase activity in K562 cells. Cancer Lett. 2012, 323, 223−231.(97) Wooten, L. G.; Ogretmen, B. Sp1/Sp3-dependent regulation ofhuman telomerase reverse transcriptase promoter activity by thebioactive sphingolipid ceramide. J. Biol. Chem. 2005, 280, 28867−28876.(98) Kyo, S.; Takakura, M.; Taira, T.; Kanaya, T.; Itoh, H.; Yutsudo,M.; Ariga, H.; Inoue, M. Sp1 cooperates with c-Myc to activatetranscription of the human telomerase reverse transcriptase gene(hTERT). Nucleic Acids Res. 2000, 28, 669−677.(99) Misiti, S.; Nanni, S.; Fontemaggi, G.; Cong, Y. S.; Wen, J.; Hirte,H. W.; Piaggio, G.; Sacchi, A.; Pontecorvi, A.; Bacchetti, S.; Farsetti, A.Induction of hTERT expression and telomerase activity by estrogensin human ovary epithelium cells. Mol. Cell. Biol. 2000, 20, 3764−3771.(100) Kyo, S.; Takakura, M.; Kanaya, T.; Zhuo, W.; Fujimoto, K.;Nishio, Y.; Orimo, A.; Inoue, M. Estrogen activates telomerase. CancerRes. 1999, 59, 5917−5921.(101) Nanni, S.; Narducci, M.; Della Pietra, L.; Moretti, F.; Grasselli,A.; De Carli, P.; Sacchi, A.; Pontecorvi, A.; Farsetti, A. Signalingthrough estrogen receptors modulates telomerase activity in humanprostate cancer. J. Clin. Invest. 2002, 110, 219−227.(102) Aldous, W. K.; Marean, A. J.; DeHart, M. J.; Matej, L. A.;Moore, K. H. Effects of tamoxifen on telomerase activity in breastcarcinoma cell lines. Cancer 1999, 85, 1523−1529.(103) Wang, Z.; Kyo, S.; Takakura, M.; Tanaka, M.; Yatabe, N.;Maida, Y.; Fujiwara, M.; Hayakawa, J.; Ohmichi, M.; Koike, K.; Inoue,M. Progesterone regulates human telomerase reverse transcriptasegene expression via activation of mitogen-activated protein kinasesignaling pathway. Cancer Res. 2000, 60, 5376−5381.(104) Xu, D.; Popov, N.; Hou, M.; Wang, Q.; Bjorkholm, M.;Gruber, A.; Menkel, A. R.; Henriksson, M. Switch from Myc/Max toMad1/Max binding and decrease in histone acetylation at thetelomerase reverse transcriptase promoter during differentiation ofHL60 cells. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3826−3831.(105) Gunes, C.; Lichtsteiner, S.; Vasserot, A. P.; Englert, C.Expression of the hTERT gene is regulated at the level oftranscriptional initiation and repressed by Mad1. Cancer Res. 2000,60, 2116−2121.(106) Kanaya, T.; Kyo, S.; Hamada, K.; Takakura, M.; Kitagawa, Y.;Harada, H.; Inoue, M. Adenoviral expression of p53 repressestelomerase activity through down-regulation of human telomerasereverse transcriptase transcription. Clin. Cancer Res. 2000, 6, 1239−1247.(107) Crowe, D. L.; Nguyen, D. C. Rb and E2F-1 regulate telomeraseactivity in human cancer cells. Biochim. Biophys. Acta 2001, 1518, 1−6.(108) Henderson, Y. C.; Breau, R. L.; Liu, T. J.; Clayman, G. L.Telomerase activity in head and neck tumors after introduction ofwild-type p53, p21, p16, and E2F-1 genes by means of recombinantadenovirus. Head Neck 2000, 22, 347−354.(109) Oh, S.; Song, Y.; Yim, J.; Kim, T. K. The Wilms’ tumor 1tumor suppressor gene represses transcription of the humantelomerase reverse transcriptase gene. J. Biol. Chem. 1999, 274,37473−37478.(110) Lindkvist, A.; Ivarsson, K.; Jernberg-Wiklund, H.; Paulsson-Karlsson, Y. Interferon-induced sensitization to apoptosis is associatedwith repressed transcriptional activity of the hTERT promoter inmultiple myeloma. Biochem. Biophys. Res. Commun. 2006, 341, 1141−1148.(111) Lacerte, A.; Korah, J.; Roy, M.; Yang, X. J.; Lemay, S.; Lebrun,J. J. Transforming growth factor-beta inhibits telomerase through

SMAD3 and E2F transcription factors. Cell. Signalling 2008, 20, 50−59.(112) Li, H.; Xu, D.; Li, J.; Berndt, M. C.; Liu, J. P. Transforminggrowth factor beta suppresses human telomerase reverse transcriptase(hTERT) by Smad3 interactions with c-Myc and the hTERT gene. J.Biol. Chem. 2006, 281, 25588−25600.(113) Fujiki, T.; Miura, T.; Maura, M.; Shiraishi, H.; Nishimura, S.;Imada, Y.; Uehara, N.; Tashiro, K.; Shirahata, S.; Katakura, Y. TAK1represses transcription of the human telomerase reverse transcriptasegene. Oncogene 2007, 26, 5258−5266.(114) Terme, J. M.; Mocquet, V.; Kuhlmann, A. S.; Zane, L.;Mortreux, F.; Wattel, E.; Duc Dodon, M.; Jalinot, P. Inhibition of thehTERT promoter by the proto-oncogenic protein TAL1. Leukemia2009, 23, 2081−2089.(115) Wu, P.; Meng, L.; Wang, H.; Zhou, J.; Xu, G.; Wang, S.; Xi, L.;Chen, G.; Wang, B.; Zhu, T.; Lu, Y.; Ma, D. Role of hTERT inapoptosis of cervical cancer induced by histone deacetylase inhibitor.Biochem. Biophys. Res. Commun. 2005, 335, 36−44.(116) Woo, H. J.; Lee, S. J.; Choi, B. T.; Park, Y. M.; Choi, Y. H.Induction of apoptosis and inhibition of telomerase activity bytrichostatin A, a histone deacetylase inhibitor, in human leukemicU937 cells. Exp. Mol. Pathol. 2007, 82, 77−84.(117) Choi, Y. H. Apoptosis of U937 human leukemic cells bysodium butyrate is associated with inhibition of telomerase activity. Int.J. Oncol. 2006, 29, 1207−1213.(118) Won, J.; Chang, S.; Oh, S.; Kim, T. K. Small-molecule-basedidentification of dynamic assembly of E2F-pocket protein-histonedeacetylase complex for telomerase regulation in human cells. Proc.Natl. Acad. Sci. U.S.A. 2004, 101, 11328−11333.(119) Berletch, J. B.; Liu, C.; Love, W. K.; Andrews, L. G.; Katiyar, S.K.; Tollefsbol, T. O. Epigenetic and genetic mechanisms contribute totelomerase inhibition by EGCG. J. Cell. Biochem. 2008, 103, 509−519.(120) Wang, X.; Hao, M. W.; Dong, K.; Lin, F.; Ren, J. H.; Zhang, H.Z. Apoptosis induction effects of EGCG in laryngeal squamous cellcarcinoma cells through telomerase repression. Arch. Pharmacal Res.2009, 32, 1263−1269.(121) Sadava, D.; Whitlock, E.; Kane, S. E. The green tea polyphenol,epigallocatechin-3-gallate inhibits telomerase and induces apoptosis indrug-resistant lung cancer cells. Biochem. Biophys. Res. Commun. 2007,360, 233−237.(122) Seimiya, H.; Oh-hara, T.; Suzuki, T.; Naasani, I.; Shimazaki, T.;Tsuchiya, K.; Tsuruo, T. Telomere shortening and growth inhibitionof human cancer cells by novel synthetic telomerase inhibitors MST-312, MST-295, and MST-1991. Mol. Cancer Ther. 2002, 1, 657−665.(123) Bilsland, A. E.; Hoare, S.; Stevenson, K.; Plumb, J.; Gomez-Roman, N.; Cairney, C.; Burns, S.; Lafferty-Whyte, K.; Roffey, J.;Hammonds, T.; Keith, W. N. Dynamic telomerase gene suppressionvia network effects of GSK3 inhibition. PLoS One 2009, 4, e6459.(124) Pendino, F.; Flexor, M.; Delhommeau, F.; Buet, D.; Lanotte,M.; Segal-Bendirdjian, E. Retinoids down-regulate telomerase andtelomere length in a pathway distinct from leukemia cell differ-entiation. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6662−6667.(125) Pendino, F.; Dudognon, C.; Delhommeau, F.; Sahraoui, T.;Flexor, M.; Bennaceur-Griscelli, A.; Lanotte, M.; Segal-Bendirdjian, E.Retinoic acid receptor alpha and retinoid-X receptor-specific agonistssynergistically target telomerase expression and induce tumor celldeath. Oncogene 2003, 22, 9142−9150.(126) Li, H.; Zhao, L. L.; Funder, J. W.; Liu, J. P. Protein phosphatase2A inhibits nuclear telomerase activity in human breast cancer cells. J.Biol. Chem. 1997, 272, 16729−16732.(127) Kang, S. S.; Kwon, T.; Kwon, D. Y.; Do, S. I. Akt protein kinaseenhances human telomerase activity through phosphorylation oftelomerase reverse transcriptase subunit. J. Biol. Chem. 1999, 274,13085−13090.(128) Yu, C. C.; Lo, S. C.; Wang, T. C. Telomerase is regulated byprotein kinase C-zeta in human nasopharyngeal cancer cells. Biochem.J. 2001, 355, 459−464.

Journal of Medicinal Chemistry Perspective

dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXO

(129) Kim, Y. W.; Hur, S. Y.; Kim, T. E.; Lee, J. M.; Namkoong, S. E.;Ki, I. K.; Kim, J. W. Protein kinase C modulates telomerase activity inhuman cervical cancer cells. Exp. Mol. Med. 2001, 33, 156−163.(130) Uziel, O.; Fenig, E.; Nordenberg, J.; Beery, E.; Reshef, H.;Sandbank, J.; Birenbaum, M.; Bakhanashvili, M.; Yerushalmi, R.; Luria,D.; Lahav, M. Imatinib mesylate (Gleevec) downregulates telomeraseactivity and inhibits proliferation in telomerase-expressing cell lines. Br.J. Cancer 2005, 92, 1881−1891.(131) Kharbanda, S.; Kumar, V.; Dhar, S.; Pandey, P.; Chen, C.;Majumder, P.; Yuan, Z. M.; Whang, Y.; Strauss, W.; Pandita, T. K.;Weaver, D.; Kufe, D. Regulation of the hTERT telomerase catalyticsubunit by the c-Abl tyrosine kinase. Curr. Biol. 2000, 10, 568−575.(132) Soares, J.; Lowe, M. M.; Jarstfer, M. B. The catalytic subunit ofhuman telomerase is a unique caspase-6 and caspase-7 substrate.Biochemistry 2011, 50, 9046−9055.(133) Holt, S. E.; Aisner, D. L.; Baur, J.; Tesmer, V. M.; Dy, M.;Ouellette, M.; Trager, J. B.; Morin, G. B.; Toft, D. O.; Shay, J. W.;Wright, W. E.; White, M. A. Functional requirement of p23 and Hsp90in telomerase complexes. Genes Dev. 1999, 13, 817−826.(134) Keppler, B. R.; Grady, A. T.; Jarstfer, M. B. The biochemicalrole of the heat shock protein 90 chaperone complex in establishinghuman telomerase activity. J. Biol. Chem. 2006, 281, 19840−19848.(135) Chang, J. T.; Lu, Y. C.; Chen, Y. J.; Tseng, C. P.; Chen, Y. L.;Fang, C. W.; Cheng, A. J. hTERT phosphorylation by PKC is essentialfor telomerase holoprotein integrity and enzyme activity in head neckcancer cells. Br. J. Cancer 2006, 94, 870−878.(136) Compton, S. A.; Elmore, L. W.; Haydu, K.; Jackson-Cook, C.K.; Holt, S. E. Induction of nitric oxide synthase-dependent telomereshortening after functional inhibition of Hsp90 in human tumor cells.Mol. Cell. Biol. 2006, 26, 1452−1462.(137) Villa, R.; Folini, M.; Porta, C. D.; Valentini, A.; Pennati, M.;Daidone, M. G.; Zaffaroni, N. Inhibition of telomerase activity bygeldanamycin and 17-allylamino, 17-demethoxygeldanamycin inhuman melanoma cells. Carcinogenesis 2003, 24, 851−859.(138) Phan, A. T. Human telomeric G-quadruplex: structures ofDNA and RNA sequences. FEBS J. 2010, 277, 1107−1117.(139) Neidle, S. Human telomeric G-quadruplex: the current statusof telomeric G-quadruplexes as therapeutic targets in human cancer.FEBS J 2010, 277, 1118−1125.(140) Bochman, M. L.; Paeschke, K.; Zakian, V. A. DNA secondarystructures: stability and function of G-quadruplex structures. Nat. Rev.Genet. 2012, 13, 770−780.(141) Zahler, A. M.; Williamson, J. R.; Cech, T. R.; Prescott, D. M.Inhibition of telomerase by G-quartet DNA structures. Nature 1991,350, 718−720.(142) Sun, D.; Thompson, B.; Cathers, B. E.; Salazar, M.; Kerwin, S.M.; Trent, J. O.; Jenkins, T. C.; Neidle, S.; Hurley, L. H. Inhibition ofhuman telomerase by a G-quadruplex-interactive compound. J. Med.Chem. 1997, 40, 2113−2116.(143) Harrison, R. J.; Gowan, S. M.; Kelland, L. R.; Neidle, S. Humantelomerase inhibition by substituted acridine derivatives. Bioorg. Med.Chem. Lett. 1999, 9, 2463−2468.(144) Monchaud, D.; Granzhan, A.; Saettel, N.; Guedin, A.; Mergny,J. L.; Teulade-Fichou, M. P. “One ring to bind them all”-part I: theefficiency of the macrocyclic scaffold for G-quadruplex DNArecognition. J. Nucleic Acids 2010, 2010, 1−19.(145) Lam, E. Y.; Beraldi, D.; Tannahill, D.; Balasubramanian, S. G-quadruplex structures are stable and detectable in human genomicDNA. Nat. Commun. 2013, 4, 1796.(146) Palumbo, S. L.; Ebbinghaus, S. W.; Hurley, L. H. Formation ofa unique end-to-end stacked pair of G-quadruplexes in the hTERTcore promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands. J. Am. Chem. Soc. 2009, 131, 10878−10891.(147) Fernando, H.; Reszka, A. P.; Huppert, J.; Ladame, S.; Rankin,S.; Venkitaraman, A. R.; Neidle, S.; Balasubramanian, S. A conservedquadruplex motif located in a transcription activation site of the humanc-kit oncogene. Biochemistry 2006, 45, 7854−7860.

(148) Rankin, S.; Reszka, A. P.; Huppert, J.; Zloh, M.; Parkinson, G.N.; Todd, A. K.; Ladame, S.; Balasubramanian, S.; Neidle, S. PutativeDNA quadruplex formation within the human c-kit oncogene. J. Am.Chem. Soc. 2005, 127, 10584−10589.(149) Ambrus, A.; Chen, D.; Dai, J.; Jones, R. A.; Yang, D. Solutionstructure of the biologically relevant G-quadruplex element in thehuman c-MYC promoter. Implications for G-quadruplex stabilization.Biochemistry 2005, 44, 2048−2058.(150) Siddiqui-Jain, A.; Grand, C. L.; Bearss, D. J.; Hurley, L. H.Direct evidence for a G-quadruplex in a promoter region and itstargeting with a small molecule to repress c-MYC transcription. Proc.Natl. Acad. Sci. U.S.A. 2002, 99, 11593−11598.(151) Chen, Y.; Yang, D. Sequence, Stability, and Structure of G-Quadruplexes and Their Interactions with Drugs. Curr. Protoc. NucleicAcid Chem. 2012, 50, 17.5.1−17.5.17.(152) Vy Thi Le, T.; Han, S.; Chae, J.; Park, H. J. G-quadruplexbinding ligands: from naturally occurring to rationally designedmolecules. Curr. Pharm. Des. 2012, 18, 1948−1972.(153) Perry, P. J.; Read, M. A.; Davies, R. T.; Gowan, S. M.; Reszka,A. P.; Wood, A. A.; Kelland, L. R.; Neidle, S. 2,7-Disubstitutedamidofluorenone derivatives as inhibitors of human telomerase. J. Med.Chem. 1999, 42, 2679−2684.(154) Li, Q.; Xiang, J. F.; Yang, Q. F.; Sun, H. X.; Guan, A. J.; Tang,Y. L. G4LDB: a database for discovering and studying G-quadruplexligands. Nucleic Acids Res. 2013, 41, D1115−D1123.(155) Nicoludis, J. M.; Miller, S. T.; Jeffrey, P. D.; Barrett, S. P.;Rablen, P. R.; Lawton, T. J.; Yatsunyk, L. A. Optimized end-stackingprovides specificity of N-methyl mesoporphyrin IX for humantelomeric G-quadruplex DNA. J. Am. Chem. Soc. 2012, 134, 20446−20456.(156) Li, Q.; Xiang, J. F.; Zhang, H.; Tang, Y. L. Searching drug-likeanti-cancer compound(s) based on G-quadruplex ligands. Curr. Pharm.Des. 2012, 18, 1973−1983.(157) Bazzicalupi, C.; Ferraroni, M.; Bilia, A. R.; Scheggi, F.; Gratteri,P. The crystal structure of human telomeric DNA complexed withberberine: an interesting case of stacked ligand to G-tetrad ratio higherthan 1:1. Nucleic Acids Res. 2013, 41, 632−638.(158) Patel, D. J.; Phan, A. T.; Kuryavyi, V. Human telomere,oncogenic promoter and 5′-UTR G-quadruplexes: diverse higher orderDNA and RNA targets for cancer therapeutics. Nucleic Acids Res. 2007,35, 7429−7455.(159) Collie, G. W.; Promontorio, R.; Hampel, S. M.; Micco, M.;Neidle, S.; Parkinson, G. N. Structural basis for telomeric G-quadruplex targeting by naphthalene diimide ligands. J. Am. Chem.Soc. 2012, 134, 2723−2731.(160) Sparapani, S.; Haider, S. M.; Doria, F.; Gunaratnam, M.;Neidle, S. Rational design of acridine-based ligands with selectivity forhuman telomeric quadruplexes. J. Am. Chem. Soc. 2010, 132, 12263−12272.(161) Drygin, D.; Siddiqui-Jain, A.; O’Brien, S.; Schwaebe, M.; Lin,A.; Bliesath, J.; Ho, C. B.; Proffitt, C.; Trent, K.; Whitten, J. P.; Lim, J.K.; Von Hoff, D.; Anderes, K.; Rice, W. G. Anticancer activity of CX-3543: a direct inhibitor of rRNA biogenesis. Cancer Res. 2009, 69,7653−7661.(162) Citti, L.; Rainaldi, G. Synthetic hammerhead ribozymes astherapeutic tools to control disease genes. Curr. Gene Ther. 2005, 5,11−24.(163) Folini, M.; Colella, G.; Villa, R.; Lualdi, S.; Daidone, M. G.;Zaffaroni, N. Inhibition of telomerase activity by a hammerheadribozyme targeting the RNA component of telomerase in humanmelanoma cells. J. Invest. Dermatol. 2000, 114, 259−267.(164) Lu, Y.; Gu, J.; Jin, D.; Gao, Y.; Yuan, M. Inhibition oftelomerase activity by HDV ribozyme in cancers. J. Exp. Clin. CancerRes. 2011, 30, 1.(165) Yokoyama, Y.; Takahashi, Y.; Shinohara, A.; Lian, Z.; Wan, X.;Niwa, K.; Tamaya, T. Attenuation of telomerase activity by ahammerhead ribozyme targeting the template region of telomeraseRNA in endometrial carcinoma cells. Cancer Res. 1998, 58, 5406−5410.

Journal of Medicinal Chemistry Perspective

dx.doi.org/10.1021/jm400528t | J. Med. Chem. XXXX, XXX, XXX−XXXP

(166) Yeo, M.; Rha, S. Y.; Jeung, H. C.; Hu, S. X.; Yang, S. H.; Kim,Y. S.; An, S. W.; Chung, H. C. Attenuation of telomerase activity byhammerhead ribozyme targeting human telomerase RNA inducesgrowth retardation and apoptosis in human breast tumor cells. Int. J.Cancer 2005, 114, 484−489.(167) Ochi, A.; Umekage, S.; Kikuchi, Y. Non-enzymatic in vitroproduction of circular hammerhead ribozyme targeting the templateregion of human telomerase RNA. Nucleic Acids Symp. Ser. 2009, 275−276.(168) Yokoyama, Y.; Takahashi, Y.; Shinohara, A.; Wan, X.;Takahashi, S.; Niwa, K.; Tamaya, T. The 5′-end of hTERT mRNAis a good target for hammerhead ribozyme to suppress telomeraseactivity. Biochem. Biophys. Res. Commun. 2000, 273, 316−321.(169) Hao, Z. M.; Luo, J. Y.; Cheng, J.; Li, L.; He, D.; Wang, Q. Y.;Yang, G. X. Intensive inhibition of hTERT expression by a ribozymeinduces rapid apoptosis of cancer cells through a telomere length-independent pathway. Cancer Biol. Ther. 2005, 4, 1098−1103.(170) You, Y.; Pu, P.; Huang, Q.; Xia, Z.; Wang, C.; Wang, G.; Yu,C.; Yu, J. J.; Reed, E.; Li, Q. Q. Antisense telomerase RNA inhibits thegrowth of human glioma cells in vitro and in vivo. Int. J. Cancer 2006,28, 1225−1232.(171) Glukhov, A. I.; Zimnik, O. V.; Gordeev, S. A.; Severin, S. E.Inhibition of telomerase activity of melanoma cells in vitro byantisense oligonucleotides. Biochem. Biophys. Res. Commun. 1998, 248,368−371.(172) Tarkanyi, I.; Horvath, A.; Szatmari, I.; Eizert, H.; Vamosi, G.;Damjanovich, S.; Segal-Bendirdjian, E.; Aradi, J. Inhibition of humantelomerase by oligonucleotide chimeras, composed of an antisensemoiety and a chemically modified homo-oligonucleotide. FEBS Lett.2005, 579, 1411−1416.(173) Ohnuma, T.; Li, F. L.; Holland, J. F. Inhibitory effects oftelomere-mimic phosphorothioate oligonucleotides on various humantumor cells in vitro. Anticancer Res. 1997, 17, 2455−2458.(174) Mata, J. E.; Joshi, S. S.; Palen, B.; Pirruccello, S. J.; Jackson, J.D.; Elias, N.; Page, T. J.; Medlin, K. L.; Iversen, P. L. A hexamericphosphorothioate oligonucleotide telomerase inhibitor arrests growthof Burkitt’s lymphoma cells in vitro and in vivo. Toxicol. Appl.Pharmacol. 1997, 144, 189−197.(175) Pitts, A. E.; Corey, D. R. Inhibition of human telomerase by 2′-O-methyl-RNA. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11549−11554.(176) Natarajan, S.; Chen, Z.; Wancewicz, E. V.; Monia, B. P.; Corey,D. R. Telomerase reverse transcriptase (hTERT) mRNA andtelomerase RNA (hTR) as targets for downregulation of telomeraseactivity. Oligonucleotides 2004, 14, 263−273.(177) Herbert, B.; Pitts, A. E.; Baker, S. I.; Hamilton, S. E.; Wright,W. E.; Shay, J. W.; Corey, D. R. Inhibition of human telomerase inimmortal human cells leads to progressive telomere shortening andcell death. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14276−14281.(178) Beisner, J.; Dong, M.; Taetz, S.; Piotrowska, K.; Kleideiter, E.;Friedel, G.; Schaefer, U.; Lehr, C. M.; Klotz, U.; Murdter, T. E.Efficient telomerase inhibition in human non-small cell lung cancercells by liposomal delivery of 2′-O-methyl-RNA. J. Pharm. Sci. 2009,98, 1765−1774.(179) Beisner, J.; Dong, M.; Taetz, S.; Nafee, N.; Griese, E. U.;Schaefer, U.; Lehr, C. M.; Klotz, U.; Murdter, T. E. Nanoparticlemediated delivery of 2′-O-methyl-RNA leads to efficient telomeraseinhibition and telomere shortening in human lung cancer cells. LungCancer 2010, 68, 346−354.(180) Chen, Z.; Monia, B. P.; Corey, D. R. Telomerase inhibition,telomere shortening, and decreased cell proliferation by cell permeable2′-O-methoxyethyl oligonucleotides. J. Med. Chem. 2002, 45, 5423−5425.(181) Chen, Z.; Koeneman, K. S.; Corey, D. R. Consequences oftelomerase inhibition and combination treatments for the proliferationof cancer cells. Cancer Res. 2003, 63, 5917−5925.(182) Norton, J. C.; Piatyszek, M. A.; Wright, W. E.; Shay, J. W.;Corey, D. R. Inhibition of human telomerase activity by peptidenucleic acids. Nat. Biotechnol. 1996, 14, 615−619.

(183) Harrison, J. G.; Frier, C.; Laurant, R.; Dennis, R.; Raney, K. D.;Balasubramanian, S. Inhibition of human telomerase by PNA-cationicpeptide conjugates. Bioorg. Med. Chem. Lett. 1999, 9, 1273−1278.(184) Paranjape, J. M.; Xu, D.; Kushner, D. M.; Okicki, J.; Lindner,D. J.; Cramer, H.; Silverman, R. H.; Leaman, D. W. Human telomeraseRNA degradation by 2′-5′-linked oligoadenylate antisense chimeras ina cell-free system, cultured tumor cells, and murine xenograft models.Oligonucleotides 2006, 16, 225−238.(185) Kondo, S.; Kondo, Y.; Li, G.; Silverman, R. H.; Cowell, J. K.Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA. Oncogene 1998, 16,3323−3330.(186) Yatabe, N.; Kyo, S.; Kondo, S.; Kanaya, T.; Wang, Z.; Maida,Y.; Takakura, M.; Nakamura, M.; Tanaka, M.; Inoue, M. 2-5Aantisense therapy directed against human telomerase RNA inhibitstelomerase activity and induces apoptosis without telomere impair-ment in cervical cancer cells. Cancer Gene Ther. 2002, 9, 624−630.(187) Wong, S. C.; Yu, H.; Moochhala, S. M.; So, J. B. Antisensetelomerase induced cell growth inhibition, cell cycle arrest andtelomerase activity down-regulation in gastric and colon cancer cells.Anticancer Res. 2003, 23, 465−469.(188) Mukai, S.; Kondo, Y.; Koga, S.; Komata, T.; Barna, B. P.;Kondo, S. 2-5A antisense telomerase RNA therapy for intracranialmalignant gliomas. Cancer Res. 2000, 60, 4461−4467.(189) Asai, A.; Oshima, Y.; Yamamoto, Y.; Uochi, T. A.; Kusaka, H.;Akinaga, S.; Yamashita, Y.; Pongracz, K.; Pruzan, R.; Wunder, E.;Piatyszek, M.; Li, S.; Chin, A. C.; Harley, C. B.; Gryaznov, S. A noveltelomerase template antagonist (GRN163) as a potential anticanceragent. Cancer Res. 2003, 63, 3931−3939.(190) Herbert, B. S.; Gellert, G. C.; Hochreiter, A.; Pongracz, K.;Wright, W. E.; Zielinska, D.; Chin, A. C.; Harley, C. B.; Shay, J. W.;Gryaznov, S. M. Lipid modification of GRN163, an N3′→P5′ thio-phosphoramidate oligonucleotide, enhances the potency of telomeraseinhibition. Oncogene 2005, 24, 5262−5268.(191) Marian, C. O.; Cho, S. K.; McEllin, B. M.; Maher, E. A.;Hatanpaa, K. J.; Madden, C. J.; Mickey, B. E.; Wright, W. E.; Shay, J.W.; Bachoo, R. M. The telomerase antagonist, imetelstat, efficientlytargets glioblastoma tumor-initiating cells leading to decreasedproliferation and tumor growth. Clin. Cancer Res. 2010, 16, 154−163.(192) Tokcaer-Keskin, Z.; Dikmen, Z. G.; Ayaloglu-Butun, F.;Gultekin, S.; Gryaznov, S. M.; Akcali, K. C. The effect of telomerasetemplate antagonist GRN163L on bone-marrow-derived rat mesen-chymal stem cells is reversible and associated with altered expressionof cyclin d1, cdk4 and cdk6. Stem Cell Rev. 2010, 6, 224−233.(193) Goldblatt, E. M.; Gentry, E. R.; Fox, M. J.; Gryaznov, S. M.;Shen, C.; Herbert, B. S. The telomerase template antagonistGRN163L alters MDA-MB-231 breast cancer cell morphology,inhibits growth, and augments the effects of paclitaxel. Mol. CancerTher. 2009, 8, 2027−2035.(194) Hochreiter, A. E.; Xiao, H.; Goldblatt, E. M.; Gryaznov, S. M.;Miller, K. D.; Badve, S.; Sledge, G. W.; Herbert, B. S. Telomerasetemplate antagonist GRN163L disrupts telomere maintenance, tumorgrowth, and metastasis of breast cancer. Clin. Cancer Res. 2006, 12,3184−3192.(195) Dikmen, Z. G.; Gellert, G. C.; Jackson, S.; Gryaznov, S.;Tressler, R.; Dogan, P.; Wright, W. E.; Shay, J. W. In vivo inhibition oflung cancer by GRN163L: a novel human telomerase inhibitor. CancerRes. 2005, 65, 7866−7873.(196) Gellert, G. C.; Dikmen, Z. G.; Wright, W. E.; Gryaznov, S.;Shay, J. W. Effects of a novel telomerase inhibitor, GRN163L, inhuman breast cancer. Breast Cancer Res. Treat. 2006, 96, 73−81.(197) Shammas, M. A.; Koley, H.; Bertheau, R. C.; Neri, P.; Fulciniti,M.; Tassone, P.; Blotta, S.; Protopopov, A.; Mitsiades, C.; Batchu, R.B.; Anderson, K. C.; Chin, A.; Gryaznov, S.; Munshi, N. C. Telomeraseinhibitor GRN163L inhibits myeloma cell growth in vitro and in vivo.Leukemia 2008, 22, 1410−1418.(198) Dikmen, Z. G.; Wright, W. E.; Shay, J. W.; Gryaznov, S. M.Telomerase targeted oligonucleotide thio-phosphoramidates in T24-luc bladder cancer cells. J. Cell. Biochem. 2008, 104, 444−452.

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(199) George, J.; Banik, N. L.; Ray, S. K. Knockdown of hTERT andconcurrent treatment with interferon-gamma inhibited proliferationand invasion of human glioblastoma cell lines. Int. J. Biochem. Cell Biol.2010, 42, 1164−1173.(200) Djojosubroto, M. W.; Chin, A. C.; Go, N.; Schaetzlein, S.;Manns, M. P.; Gryaznov, S.; Harley, C. B.; Rudolph, K. L. Telomeraseantagonists GRN163 and GRN163L inhibit tumor growth andincrease chemosensitivity of human hepatoma. Hepatology 2005, 42,1127−1136.(201) Joseph, I.; Tressler, R.; Bassett, E.; Harley, C.; Buseman, C. M.;Pattamatta, P.; Wright, W. E.; Shay, J. W.; Go, N. F. The telomeraseinhibitor imetelstat depletes cancer stem cells in breast and pancreaticcancer cell lines. Cancer Res. 2010, 70, 9494−9504.(202) Siomi, H.; Siomi, M. C. On the road to reading the RNA-interference code. Nature 2009, 457, 396−404.(203) Noreen, F.; Heinrich, J.; Moelling, K. Antitumor activity ofsmall double-stranded oligodeoxynucleotides targeting telomeraseRNA in malignant melanoma cells. Oligonucleotides 2009, 19, 169−178.(204) Li, S.; Crothers, J.; Haqq, C. M.; Blackburn, E. H. Cellular andgene expression responses involved in the rapid growth inhibition ofhuman cancer cells by RNA interference-mediated depletion oftelomerase RNA. J. Biol. Chem. 2005, 280, 23709−23717.(205) Shammas, M. A.; Koley, H.; Batchu, R. B.; Bertheau, R. C.;Protopopov, A.; Munshi, N. C.; Goyal, R. K. Telomerase inhibition bysiRNA causes senescence and apoptosis in Barrett’s adenocarcinomacells: mechanism and therapeutic potential. Mol. Cancer 2005, 4, 24.(206) Shen, Y.; Zhang, Y. W.; Zhang, Z. X.; Miao, Z. H.; Ding, J.hTERT-targeted RNA interference inhibits tumorigenicity andmotility of HCT116 cells. Cancer Biol. Ther. 2008, 7, 228−236.(207) de Souza Nascimento, P.; Alves, G.; Fiedler, W. Telomeraseinhibition by an siRNA directed against hTERT leads to telomereattrition in HT29 cells. Oncol. Rep. 2006, 16, 423−428.(208) Gandellini, P.; Folini, M.; Bandiera, R.; De Cesare, M.; Binda,M.; Veronese, S.; Daidone, M. G.; Zunino, F.; Zaffaroni, N. Down-regulation of human telomerase reverse transcriptase through specificactivation of RNAi pathway quickly results in cancer cell growthimpairment. Biochem. Pharmacol. 2007, 73, 1703−1714.(209) Miri-Moghaddam, E.; Deezagi, A.; Soheili, Z. S. Down-regulation of telomerase activity in human promyelocytic cell lineusing RNA interference. Ann. Hematol. 2009, 88, 1169−1176.(210) Liu, X.; Huang, H.; Wang, J.; Wang, C.; Wang, M.; Zhang, B.;Pan, C. Dendrimers-delivered short hairpin RNA targeting hTERTinhibits oral cancer cell growth in vitro and in vivo. Biochem.Pharmacol. 2011, 82, 17−23.(211) Li, Y.; Li, M.; Yao, G.; Geng, N.; Xie, Y.; Feng, Y.; Zhang, P.;Kong, X.; Xue, J.; Cheng, S.; Zhou, J.; Xiao, L. Telomerase inhibitionstrategies by siRNAs against either hTR or hTERT in oral squamouscell carcinoma. Cancer Gene Ther. 2011, 18, 318−325.(212) Vonderheide, R. H.; Domchek, S. M.; Schultze, J. L.; George,D. J.; Hoar, K. M.; Chen, D. Y.; Stephans, K. F.; Masutomi, K.; Loda,M.; Xia, Z.; Anderson, K. S.; Hahn, W. C.; Nadler, L. M. Vaccinationof cancer patients against telomerase induces functional antitumorCD8+ T lymphocytes. Clin. Cancer Res. 2004, 10, 828−839.(213) Mizukoshi, E.; Nakamoto, Y.; Arai, K.; Yamashita, T.; Sakai, A.;Sakai, Y.; Kagaya, T.; Honda, M.; Kaneko, S. Comparative analysis ofvarious tumor-associated antigen-specific t-cell responses in patientswith hepatocellular carcinoma. Hepatology 2011, 53, 1206−1216.(214) Hunger, R. E.; Kernland Lang, K.; Markowski, C. J.; Trachsel,S.; Moller, M.; Eriksen, J. A.; Rasmussen, A. M.; Braathen, L. R.;Gaudernack, G. Vaccination of patients with cutaneous melanoma withtelomerase-specific peptides. Cancer Immunol. Immunother. 2011, 60,1553−1564.(215) Painter, R. G.; Lanson, N. A., Jr.; Jin, Z.; Park, F.; Wang, G.Conditional expression of a suicide gene by the telomere reversetranscriptase promoter for potential post-therapeutic deletion oftumorigenesis. Cancer Sci. 2005, 96, 607−613.(216) Koga, S.; Hirohata, S.; Kondo, Y.; Komata, T.; Takakura, M.;Inoue, M.; Kyo, S.; Kondo, S. FADD gene therapy using the human

telomerase catalytic subunit (hTERT) gene promoter to restrictinduction of apoptosis to tumors in vitro and in vivo. Anticancer Res.2001, 21, 1937−1943.(217) Kazhdan, I.; Marciniak, R. A. Death receptor 4 (DR4)efficiently kills breast cancer cells irrespective of their sensitivity totumor necrosis factor-related apoptosis-inducing ligand (TRAIL).Cancer Gene Ther. 2004, 11, 691−698.(218) Zhang, Q.; Kim, N. K.; Peterson, R. D.; Wang, Z.; Feigon, J.Structurally conserved five nucleotide bulge determines the overalltopology of the core domain of human telomerase RNA. Proc. Natl.Acad. Sci. U.S.A. 2010, 107, 18761−18768.(219) Leeper, T. C.; Varani, G. The structure of an enzyme-activatingfragment of human telomerase RNA. RNA 2005, 11, 394−403.(220) Steczkiewicz, K.; Zimmermann, M. T.; Kurcinski, M.; Lewis, B.A.; Dobbs, D.; Kloczkowski, A.; Jernigan, R. L.; Kolinski, A.; Ginalski,K. Human telomerase model shows the role of the TEN domain inadvancing the double helix for the next polymerization step. Proc. Natl.Acad. Sci. U.S.A. 2011, 108, 9443−9448.(221) Sauerwald, A.; Sandin, S.; Cristofari, G.; Scheres, S. H.;Lingner, J.; Rhodes, D. Structure of active dimeric human telomerase.Nat. Struct. Mol. Biol. 2013, 20, 454−460.

■ NOTE ADDED AFTER ASAP PUBLICATIONAfter this paper was published ASAP on October 15, 2013, acorrection was made to Figure 9. The corrected version wasreposted October 18, 2013.

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