Telomere Maintenance as a Target for Drug Discovery - Journal of

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Telomere Maintenance as a Target for Drug Discovery Vijay G Sekaran, Joana Soares, and Michael Bruce Jarstfer J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400528t • Publication Date (Web): 20 Sep 2013 Downloaded from http://pubs.acs.org on September 21, 2013

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Journal of Medicinal Chemistry

Telomere Maintenance as a Target for Drug Discovery Vijay 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. AUTHOR EMAIL ADDRESS [email protected] RECEIVED DATE TITLE RUNNING HEAD Targeting telomere biology CORRESPONDING AUTHOR FOOTNOTE Michael Jarstfer, UNC, CB 7568, Chapel Hill, NC 27599; phone: +1 919 966-6422; email: [email protected]

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Abstract The observation that the enzyme telomerase is upregulated in 80-90% of cancer cells isolated from primary human tumors but is absent in neighboring cells of healthy tissue has resulted in significant efforts to validate telomerase as an anticancer drug target and to develop effective approaches towards its inhibition. In addition to inhibitors that target the enzymatic function of telomerase, efforts towards immunotherapy using peptides derived from its catalytic subunit hTERT and hTERT-promoter driven gene therapy have made significant advances. The increased level of telomerase in cancer cells also provides a potential platform for cancer diagnostics. Telomerase inhibition leads to disruption of a cells ability to maintain the very ends of the chromosomes, which are called telomeres. Thus, the telomere itself has also attracted attention as an anticancer drug target. In this perspective, interdisciplinary efforts to realize the therapeutic potential of targeting telomere maintenance with a focus on telomerase are discussed.


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Introduction

Pathways exhibiting differential activity in cancer cells when compared to healthy cells are attractive targets for therapeutic intervention. Accordingly, the observation that the enzyme telomerase is up-regulated in 80-90% of all cancer cells isolated from primary human tumors but is absent in neighboring cells of healthy tissue has resulted in significant efforts to validate telomerase as an anticancer drug target and to develop effective approaches towards its inhibition.1 In addition to inhibitors that target the enzymatic function of telomerase, efforts towards immunotherapy using peptides derived from catalytic telomerase subunit hTERT, for human TElomerase Reverse Transcriptase)2 and hTERT-promoter driven gene therapy3 have made significant advances. The increased level of telomerase activity in cancer cells also provides a potential platform for cancer diagnostics.4 Telomerase inhibition leads to disruption of a cell’s ability to maintain the very ends of the chromosomes, which are called telomeres. Thus, the telomere itself has also attracted attention as an anticancer drug target. Here, we discuss the interdisciplinary efforts to realize the therapeutic potential of targeting telomere maintenance with a specific focus on telomerase.

Telomere Maintenance and Telomerase in Human Cancer Cells

Telomeres are the physical ends of linear chromosomes and their importance to human health is underscored by the Nobel Prize in Physiology or Medicine awarded to Elizabeth Blackburn, Jack Szostak, and Carol Grieder in 2009 for their work on telomere



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biology and biochemistry. Telomeres are so important because proper telomere maintenance is an absolute requirement for continued cellular proliferation. Functional telomeres require a specific repetitive telomeric DNA sequence and several telomerespecific proteins.5, 6 These telomere-associated proteins function in two primary capacities: maintaining the structural and functional integrity of the telomere and ensuring complete replication of telomeric DNA. In the absence of functional telomeres, chromosome ends are recognized as DNA damage, and the ensuing DNA damage response generally results in either senescence or apoptosis, depending on the cell type.7 Mammalian telomeric DNA contains several thousand base pairs of repetitive DNA with the sequence 5′-TTAGGG/3′-AATCCC and a single stranded 3′ overhang of the G-rich sequence.8, 9 The canonical DNA polymerases responsible for DNA replication cannot efficiently replicate the very ends of linear DNA templates. As a result, proliferative cells must employ additional pathways to overcome this end-replication problem. In human cells, this additional activity resides primarily in the telomerase complex. Telomerase is a ribonucleoprotein complex that contains several protein subunits and one RNA subunit.10, 11 Human Telomerase RNA (hTER) is a highly structured 451 nucleotide long RNA that contains several functional domains in addition to a templating sequence used to code for the G-rich telomeric DNA strand.12 The protein subunit hTERT functions as a reverse transcriptase by using the templating portion of hTER to synthesize telomeric DNA.13 In the majority of cancer cells, hTERT is up-regulated, increasing the telomerase activity required for continuous proliferation.8, 14 In contrast, telomerase activity is down-regulated in most normal human diploid cells, with the


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exception of germline and stem cells, primarily owing to inhibition of hTERT expression during 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 mitotic hourglass that regulates cellular replication and arguably functions as a tumor suppressor mechanism.5 Cells lacking telomerase activity experience erosion of their telomeric DNA when they divide. At a critical length, the shortened telomeres are recognized as DNA damage and form a telomere dysfunction induced focus (TIF). Typically, cells respond to TIFs through senescence or apoptosis. Importantly, the telomere-length dependence of proliferation appears to be a result of the structure and not the length of the telomeric DNA per se. This is evidenced in part by experiments in the de Lange lab showing that the negative consequences of short telomeres can be overcome by the overexpression of the telomere-binding protein TRF2.15 It is likely that as telomeres erode in cells lacking an active telomere maintenance pathway, the telomere structure, predicted to be a lasso-like fold back structure termed a t-loop,16 becomes increasingly difficult to sustain eventually leading to TIFs and inevitably senescence or apoptosis if telomere function is not reestablished.6

Telomere maintenance directly impacts cancer in two specific ways. The most obvious is the absolute requirement for telomeric DNA maintenance to offset telomere loss from the end-replication problem, processing, and degradation. Most cancer cells up-regulate expression of hTERT, the catalytic subunit of telomerase, in order to increase



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telomerase activity.17 In 10-15% of cancers, a recombination based telomere lengthening pathway called alternative lengthening of telomeres (ALT) supplants the need for telomerase.18 The ALT pathway is particularly prevalent in astrocytic brain tumors and osteosarcomas.19 A second connection between telomere biology and neoplasias is the effect of overall length of telomeric DNA and the actions of telomere-associated proteins on genetic stability. Telomere erosion has been argued to serve as a tumor suppression mechanism because it can activate the senescence pathway, which suppresses neoplastic transformation.5 However, even in the presence of telomere erosion, senescence is not activated in the absence of the Rb/p16 or p53 pathways, leading to genetic instability and increased potential for transformation. These pivotal roles of telomere maintenance in tumorigenicity are evidenced by the ability to inhibit tumor growth by inhibiting telomerase in cancer cells and by the ability to convert primary human cells to cancer cells by combining constitutive telomerase expression with oncogene expression and/or loss of a tumor repressor gene. For example, the simian virus 40 large-T oncoprotein together with an oncogenic allele of H-ras and ectopic expression of hTERT has been demonstrated to transform a variety of cell types.20 For more comprehensive discussions on the roles of telomerase and telomere maintenance on tumor biology, several recent reviews are recommended.21-24

Targeting Telomere Maintenance for Cancer Therapy

When considering anti-cancer drug targets, several advantages for targeting telomerase and telomere maintenance exist when compared to other targets and


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pathways. Most important is the apparently universal requirement for telomere maintenance in cancer cells including putative cancer stem cells.25, 26 In addition, normal human cells including stem cells express lower telomerase activity and generally maintain telomeres at longer lengths when compared to cancer cells.27, 28 These features provide a level of specificity with telomerase inhibition. Telomerase activity itself was validated as an anti-cancer drug target in 1999 when two independent studies reported that overexpressing dominant-negative-hTERT mutants resulted in telomerase inhibition, telomere shortening, morphological changes, growth arrest, and apoptosis in a variety of tumor cell lines.29, 30 Since that time, several strategies for targeting telomere maintenance and telomerase-positive cancer cells have been explored (Figure 1). The initial approach explored was the direct inhibition of telomerase’s enzymatic activity. As validation of the approach, the telomerase inhibitor Imetelstat (1, GRN163L) has been explored in anti-cancer therapy in several clinical trials.31 Direct targeting of the telomere itself has also been accomplished with specific telomeric DNA binding agents that bind to and stabilize the G-quadruplex structures formed by telomeric DNA.32, 33 The high activity of the hTERT promoter in cancer cells has resulted in the exploration of hTERT-promoter driven expression of cancer killing genes and viruses,34 and one clinical trial for hTERT-driven replication of an oncolytic virus is underway.35 Immunotherapy targeting hTERT-derived peptides has also been explored leading to several clinical trials.36 Each of these platforms will be discussed further below.

Consequence of telomerase inhibition and telomere disruption



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If telomere maintenance is to be considered a viable anti-cancer drug discovery platform, it is paramount to understand the consequences of disrupting telomere maintenance for both healthy and cancerous cells. The simplest and best explored telomere-targeted anticancer drug platform is the inhibition of telomerase enzymatic activity. When telomerase activity is inhibited, DNA replication attending cellular proliferation results in telomeric DNA erosion.29, 37 Erosion of telomeric DNA eventually results in dysfunctional telomeres that initiate TIF formation.5, 6 Generally, proliferating human cells lose 50-200 base pairs of telomeric DNA during each cell cycle in the absence of telomerase activity.38 Therefore a lag between the phenotypic consequences of telomerase inhibition and the initiation of telomerase inhibition therapy is likely to occur. The duration of this phenotypic lag depends on both the initial length of telomeric DNA, which ranges from 5,000 to 15,000 base pairs in human cancer cells, and the rate of erosion. Importantly, it is suspected that the length of a few telomeres, not the average length of all telomeres, dictates the cellular response to telomerase inhibition.39 This suggests that some tumor types may be especially sensitive to telomerase inhibition. Once telomeres erodes to a critically short length, they cannot function properly,40 perhaps because they cannot be folded into the proper protected state, presumably a tloop.16 In most cancer cells, the loss of telomere function results in apoptosis, whereas in normal cells, telomere loss results in cell cycle arrest. Notably, telomerase inhibition has also been observed to promote the ALT phenotype in tumors concomitant with upregulation of the PGC -1β, a master regulator of mitochondrial biogenesis.41 This report offers critical insight into the mechanisms of adaptation that may be in play when tumors are subjected to telomerase inhibition.


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Inhibition of telomerase by some methods, for example antisense oligonucleotides targeting the hTERT mRNA,42 can result in immediate effects that are not preceded by the phenotypic lag characteristic of telomerase activity inhibitors and is not coupled to erosion of telomeric DNA. While a precise explanation remains missing, one intriguing possibility is that hTERT has several non-canonical properties that are separate from its enzymatic activity and inhibition of these may have immediate effects.43 Proposed additional activities of telomerase include telomere capping, inhibition of apoptosis, activation of the Wnt pathway, and activation of the NFκB pathway.44, 45 Thus, downregulation of hTERT can potentially lead to immediate anti-proliferative effects. Understanding why some cells are immediately impacted by the loss of hTERT and other are not remains to be determined. Perhaps, some cancer cells become oncogenically addicted to hTERT and require hTERT to maintain elevated growth signaling. Currently, one of the most studied small-molecule telomerase inhibitors are Gquadruplex stabilizers.46 This class of molecules can impact telomere functions in at least two primary mechanisms. One is by inhibiting the enzymatic activity of telomerase by preventing telomerase from binding to its primer or by interacting with the nascent DNA telomerase product. The second is to dissociate or block telomere-binding proteins from the telomere disrupting the formation of the protective cap. As expected, the former mechanism results in a delayed but highly telomerase-expression specific effect while the second mechanism is more immediate but may suffer from reduced specificity as formation of proper telomere structure is required in all cells including normal somatic and stem cells. Early evidence suggests that the therapeutic index for telomere disrupters is surprisingly high at least in a mouse model.47, 48 One potential reason that telomere



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disruption may be well tolerated in normal cells is that the DNA damage response at the telomere may be transient and reversible. Evidence for this hypothesis comes from experiments using a ligand-stabilized version of the telomere binding protein Protection of Telomeres 1 (POT1). POT1 is essential for telomere protection, and the ligandstabilized 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 by reactivating POT1.49

Small-Molecule Telomerase Inhibitors Once the importance of telomerase activity for cancer cell growth was recognized, several academic and pharmaceutical groups initiated programs to discover small molecule telomerase inhibitors. Approaches towards the identification of effective telomerase inhibitors have included repurposing of viral reverse transcriptase inhibitors, screening of natural products, high throughput screens of diverse compound libraries, and more recently, the beginnings of structure-based discovery and optimization. Despite these efforts, small molecule telomerase inhibitors have yet to reach the clinic. In this section, the varieties of approaches towards direct telomerase activity inhibition with small molecules will be discussed.

Nucleoside analogs

Nucleoside analogs developed as viral reverse transcriptase inhibitors have been investigated as telomerase inhibitors (Figure 2). Nucleoside analogs can hypothetically affect the telomere through several mechanisms, including telomerase inhibition and


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chain termination that can lead to telomere shortening and perturbation of telomere sequence that can lead to disruption of telomere function. The chain terminator 3′-azido2′,3′-dideoxythymidine (2, AZT), the first nucleoside reverse transcriptase inhibitor approved for the treatment of HIV-1, inhibits telomerase in vitro and in vivo giving rise to telomere shortening.50, 51 In cultured cells, treatment with 2 resulted in telomere shortening, cell-cycle perturbation, and increased p14ARF expression,52 and 2 exhibited synergistic effects when combined with other chemotherapeutic drugs.53-55 Several nucleoside HIV reverse transcriptase inhibitors including stavudine, tenofovir, didanosine, and abacavir also inhibit telomerase activity and lead to telomere erosion in cultured cells, but non-nucleoside HIV RT inhibitors did not inhibit telomerase in these studies.56 HIV patients have been taking these and other nucleoside reverse transcriptase inhibitors for decades, leading to possible effects from long term telomerase inhibition. This potential side effect has been investigated in a small patient population, and it was found that telomere length was inversely correlated with total time patients were exposed to nucleoside reverse transcriptase inhibitors.57 Although other nucleoside triphosphate analogs, including arabinofuranyl-guanosine and 6-thio-7-deaza-2′-deoxyguanosine 5′ triphosphate, ddGTP, ddATP, and ddTTP, produced substantial inhibition of telomerase and telomere shortening, they have not been further tested in cell cultures or in animal models and are likely to be nonspecific, thus limiting their clinical utility.51, 58 As telomerase structural biology increases, it may be possible to rationally design nucleoside analogs with the requisite telomerase specificity for this platform to be viable.

Non- nucleoside analogs



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Non-nucleoside, small molecule telomerase inhibitors have been identified by a variety of approaches. High throughput screening of chemical libraries has been used to identify telomerase inhibitors (Figure 3). The isothiazolone derivative 2-[3(trifluoromethyl) phenyl]isothiazolin-3-one (3, IC50 = 1 µM) was identified from a screen of a 16,000-member, chemical library using rat telomerase (Figure 3).59 The reducing agent dithiothreitol was found to protect telomerase from inhibition by 3 suggesting that inhibition resulted from interaction between the isothiazolone moiety of 3 with the sulfhydryl group of key cysteine residue(s).59 The nitrostyrene derivative 3-(3,5dichlorophenoxy)-nitrostyrene (4, IC50 = 0.4 µM)60 and the quinoxaline derivative 2,3,7trichloro-5-nitroquinoxaline (5)61, two noncompetitive telomerase inhibitors, were also identified by screening chemical libraries. At low concentrations (1 µM), 4 was not acutely cytotoxic to HeLa cells, but chronic exposure led to progressive telomere erosion followed by the induction of senescence consistent with telomerase inhibition.60 Longterm treatment of the breast cancer cell line MCF7 with 5 (1 µM ) also resulted in telomere erosion, chromosome abnormalities, and senescence.61 The crystal structure of TERT from the beetle Tribolium castaneum62 has been used in limited structure based design of telomerase inhibitors. Because 2H-pyrazole and dihydropyrazole derivatives have been shown to have strong anticancer, antmalarial, antiviral, and anti-inflammatory activity, they were selected as a starting scaffold for telomere inhibition.63-65 Aryl-2H-pyrazoles with 1,3-benzodioxoles and electron withdrawing aryls groups and sulfonyl-4,5-dihydropyrazoles displayed telomerase inhibition with IC50 values ranging from 0.8 to 10 µM in a variety of cancer cells (Figure 4).63-65 Since previous studies have shown that tetracyclic coumarins have notable anti-


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HIV activities and that the HIV and telomerase reverse transcriptase proteins display structural homology, dihydropyrazole piperidine derivatives containing coumarin moieties were also synthesized.63, 66 These molecules showed anticancer activity against gastric cancer and the most potent inhibited telomerase in crude cell extracts with IC50’s in the 1-2 µM range.63, 66 Results from docking studies were used to suggest that these molecules bind the hTERT active site through key intermolecular hydrogen bonding and π-cation interactions with invariant residues Lys189, Asp254, and Gln308 and by projecting aryl and dihyrdopyrazole rings into the hydrophobic dNTP-binding pocket.63, 65, 66

The π-cation interaction is consistent with the structure activity relationship (SAR)

trend in which electron withdrawing groups on the aromatic ring attenuate inhibition, whereas electron donating groups increased inhibition. 3D quantitative SAR (QSAR) was used to rationalize the proposed binding mechanism and a correlation coefficient of r2 = 0.966 was found for predicted and observed IC50 for a small, chemically similar test set.65 Other small molecules with notable telomerase inhibition and anticancer activities were also proposed to bind telomerase using docking studies including 2-chloropyridines and 2-aminomethyl-5-(quinolin-2-yl)-1,3,4-oxadiazole-2(3H)-thionequinoline derivatives, though these studies did not offer strong validation of the binding models.67, 68

Several natural products have been identified as telomerase inhibitors as well (Figure 5). Helenalin (6), a natural sesquiterpene lactone, was found to inhibit telomerase apparently by reaction with a cysteine residue similar to 3.69 Ortho-Quinone containing compounds for example tanshinone 2A (7), a plant diterpene known to exhibit diverse pharmacological activities, also inhibit telomerase (IC50 = 3 µM). The quinone was fully



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responsible for inhibition that was time- and DTT-dependent. It was also found that 7 undergoes redox cycling to produce hydrogen peroxide. The data suggest that quinone redox cycling inhibits telomerase, perhaps by oxidation of conserved cysteine residues.70 Another apparently reversible inhibitor is 8 (U-73122), commonly used as an inhibitor of phospholipase C, which was reported to be a potent (IC50 = 0.2 µM) telomerase inhibitor.71 Inhibition was shown to be dependent on the pyrrole-2,5-dione functional group, suggesting potential alkylation of telomerase. β-rubromycin (9), a quinone antibiotic, inhibited telomerase with an IC50 of 3 µM, whereas 9 showed substantially decreased inhibitory potency toward telomerase.72 Although, 9 appears to be a competitive inhibitor versus the telomerase primer, it also inhibits retroviral reverse transcriptases, mammalian DNA polymerases, and terminal deoxynucleotidyl transferase,73 indicating that rubromycins are not 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 forward chemical genetics screen in a yeast system (Figure 6).74 A yeast strain with artificially shortened telomeres was used to screen a microbial metabolite library. Since telomeres were shortened, the strain was more sensitive to telomerase inhibition than wild type. Therefore, compounds with selective activity in the telomere shortened strain were more likely to be telomerase inhibitors. With this assay, two new telomerase inhibitors: 10 (IC50 = 0.5 µM) and 11 (IC50 = 1.3 µM) were identified. In addition, the hsp90 inhibitor radicicol was also identified as a telomerase maintenance inhibitor in screen.74 The synthesis of 11 has been accomplished, but reports on structure activity relationships of analogs have not been


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reported.75, 76 More recently, an alternative yeast based assay was developed using recombinant human telomerase. Expression of human telomerase activity in yeast results in a growth delay, therefore potential inhibitors of human telomerase could rescue the transfected cells. Using this screen, several potential telomerase inhibitors with moderate potency (IC50’s ~1-10 µM) were identified to confirm the screening platform.77 The most potent compound identified in the screen was the dichlorothiophene 12 (CD11359). One promising class of small molecule telomerase inhibitors are the potent enoylamino-benzoic acids represented by 13 (BIBR1532, IC50 = 0.093 µM) and 14 (BIBR1591, IC50 = 0.47 µM) that function as selective mixed-type, non-competitive telomerase inhibitors and were developed from systematic structure-activity correlations (Figure 7).37 Limited structure activity relationship data are publically available, but show that alpha-beta unsaturated amide is essential and modifications to the naphthyl group are not well tolerated.78 The mechanism of inhibition by 13 is distinct from other non-nucleoside compounds or antisense oligonucleotides as it does not cause chain termination events but rather inhibits the formation of long reaction products. Compound 13 seems to act allosterically impair the unique ability of telomerase to catalyze addition of multiple telomeric repeats onto a DNA primer.79 Acute treatment with high concentrations of 13 caused direct cytotoxic effects on malignant cells of the hematopoietic system 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 sensitizing drug-resistant cells to chemotherapeutic agents.81 Chronic exposure to lower doses of 14 resulted in progressive erosion of telomeric DNA leading to senescence.37 However, the lag phase between telomerase inhibition and the impact on



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proliferative capacity under the chronic treatment condition is a major concern37 as is the apparently high protein binding evidenced by the significantly decreased potency in crude cell extracts compared to purified telomerase.78 More than 120 days, approximately 135 population doublings, was necessary for treatment with 13 to inhibit cell proliferation of NCI-H460 lung cancer.82 Interestingly, washout of 14 in telomerase positive cells allowed recovery of telomere length (Figure 7).37

Small-molecules that target hTER and the hTER-telomeric DNA heteroduplex In addition to targeting the enzymatic activity of telomerase, exploratory efforts in identifying ligands that target hTER suggest RNA-binding ligands may be a productive platform for targeting telomerase. In two separate reports, oligonucleotides were used to identify regions of hTER that can be specifically targeted to prevent proper telomerase assemblage.83, 84 Specifically, the CR4-CR5 and pseudoknot domains of hTER are susceptible. Detailed analysis demonstrated that discrete domains of hTER are blocked without losing the ability of hTERT to associate with hTER.84 This suggests that the CR4-CR5 and pseudoknot domain targeting oligonucleotides effectively cause formation of a dysfunctional telomerase complex. Subsequently, small-molecules that bind hTER have been identified,85-87 and several of these including aminoglycosides and the bisbenzimide Heochst 33258 have been shown to inhibit telomerase,86 presumably by binding hTER and perturbing proper assemblage of the holoenzyme complex (Figure 8). Efforts to target the telomerase template RNA-DNA primer/nascent product heteroduplex have also been reported.88, 89 In these studies, several intercalators were identified that inhibit telomerase and bind preferentially to the RNA/DNA heteroduplex as opposed to


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G-quadruplex DNA. Advances in this area include second generation intercalators and bis-intercalators that have results in fairly potent telomerase 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 moderately potent telomerase inhibition without significant improvement over the parent mono-intercalator.88 Data for cell based assays using small molecule targeting of hTER or the hTER/DNA heteroduplex have not yet been reported, so it is still too early to tell if this approach will be clinical viable.

Telomerase inhibition by indirect means

hTERT transcription regulators Cellular telomerase activity is regulated at various levels including transcription, mRNA splicing, post-transcriptional and post-translational modifications of hTER and hTERT, transport and subcellular localization, assembly of active telomerase ribonucleoprotein, and telomeric DNA accessibility. Transcriptional regulation of hTERT is thought to be the major mechanism of telomerase regulation.90 The hTERT promoter contains binding sites for several transcription factors, suggesting that the regulation of hTERT expression may be subject to multiple levels of control by various factors and dependent on cellular contexts.91 A primary activator of hTERT is the c-Myc oncogene, a known regulator of cell proliferation, growth, and apoptosis.92 Targets of the Myc family are involved in various aspects of cellular functions, including cell cycle, growth, differentiation, and life span. c-Myc expression correlates with hTERT and both are up-



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regulated in highly proliferative and immortal cells but down-regulated during terminal differentiation and in non-proliferative cells. Therefore, it is not surprising that inhibitors of c-Myc: butein93, gamboic acid94, and genistein,95 also inhibit telomerase. Additionally, genistein treatment resulted in down-regulation of the protein kinase Akt and thus hTERT phosphorylation, indicating that genistein can inhibit telomerase by at least two mechansisms: decreased hTERT transcription and decreased posttranslational activation of hTERT.95 Second generation tyrosine kinase inhibitors and the lipid-derived signaling molecule ceramide have been shown to inhibit hTERT promoter activity by altering the functions of Sp1 and Sp3.96, 97 Sp1 has been shown to collaborate with c-Myc in order to initiate hTERT transcription.98 Imatinib, dasatinib, and nilotininb decrease telomerase activity and hTERT expression by decreasing Sp1 nuclear levels and activation and reducing the nuclear translocation of hTERT via suppression of Akt phosphorylation.96 Agents that act on hormonal pathways have also been linked to telomerase activity regulation. For example, estrogen activates telomerase in hormone-sensitive tissues such as mammary and ovary epithelial cells99, 100, while the selective estrogen receptor modulator tamoxifen reduces telomerase activity in cells where it functions as an estrogen antagonist.101, 102 Progesterone also has an antagonistic effect on estrogeninduced hTERT expression.103 These observations highlight the fact that the polypharmacological effects of many anti-cancer drugs are anticipated to include inhibition of telomere maintenance as a mechanism of their anti-cancer effects.

Negative regulators of hTERT transcription


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The lack of telomerase activity in somatic cells appears to be a consequence of transcriptional repression of the hTERT expression. The loss of transcriptional repression results in the up-regulation of hTERT expression and telomerase activity as seen during carcinogenesis and cellular immortalization. Several transcriptional repressors of hTERT have been identified. One such negative regulator of hTERT transcription is Mad1. Mad1, c-Myc, and Max are part of a family of transcription factors that bind to E-boxcontaining promoters to either down-regulate or up-regulate gene expression. Mad1 is upregulated in telomerase negative somatic cells, c-Myc is up-regulated in cancer cell lines, and Max is expressed ubiquitously.104 Consistent with its role of Mad1 in regulating telomerase, overexpression of Mad1 resulted in decreased hTERT promoter activity in gene reporter assays.105 Another negative regulator of hTERT transcription is the tumor suppressor protein p53, which induces cell cycle arrest or apoptosis in response to DNA damage and other cellular stresses to inhibit tumor formation. p53 has been shown to inhibit telomerase activity through the repression of hTERT transcription.106 Repression of telomerase activity was also observed by overexpressing pRB in human squamous cell carcinomas107 and E2F1 in head and neck squamous cell carcinomas.108 pRB and E2F1 have the potential to transcriptionally repress hTERT activity either independently or in tandem with one another. Another tumor suppressor involved in the transcriptional repression of hTERT is Wilm’s tumor protein 1 (WT1). WT1 has also been shown to significantly decrease hTERT mRNA and telomerase activity in HEK293 cells.109 The role of WT1 in hTERT repression is cell type dependent as the WT1 gene is expressed only in specific cells such as the kidney, gonad, and the spleen.



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Other negative regulators of hTERT transcription include the myeloid cellspecific zinc finger protein 2, interferon- α, and TGF-β. Interferon-α and –γ-induced sensitization to apoptosis is associated with repressed transcriptional activity of the hTERT promoter in multiple myeloma.110 Transforming growth factor-β, a potent tumor suppressor, inhibits telomerase through the transcription factors SMAD3 and E2F.111, 112 TGF-β activated kinase 1 (TAK1) has been shown to repress transcription of the human telomerase reverse transcriptase gene. TAK1-induced repression was caused by the recruitment of histone deacetylase to Sp1 at the hTERT promoter.113 T-cell acute lymphoblastic leukemia protein 1 (TAL1) is also a negative regulator of the hTERT promoter. Over-expression of the proto-oncogenic protein TAL1 leads to a decrease in hTERT mRNA levels and consequently, reduced telomerase activity.114 Histone deacetylase (HDAC) inhibitors, such as trichostatin A, have been shown to induce apoptosis and down-regulate telomerase activity via suppression of hTERT expression in leukemic U937 cells.115-117 Dynamic assembly of E2F-pocket protein-HDAC complex plays a central role in the regulation of hTERT. For example, a small molecule, CGK 1026, inhibits the recruitment of HDAC into E2F-pocket protein complexes assembled on the hTERT promoter.118 These data underscore the fact that several anti-cancer treatment strategies 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 inhibit telomerase activity in a concentration dependent manner by decreasing hTERT mRNA levels.110, 119 The changes in hTERT mRNA levels can be attributed to chromatin remodeling due to the


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hypoacetylation of histones at the hTERT gene promoter and decreased methylation directly of the hTERT promoter at the E2F-1 binding site.119 Consistent with the observed decrease activity of the hTERT promoter, 15-treated U937 monoblastoid leukemia and HT29 colon adenocarcinoma cells experienced decreased proliferation potential commensurate with telomere shortening. In addition, 15 was also shown to induce a severe reduction 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 studies showed that the tea catechin derivative MST-312 (N,N′bis(2,3-dihydroxybenzoyl)-1,2-phenylenediamine) appears inhibit telomere maintenance by directly targeting telomerase (IC50 = 0.67 µM) resulting in telomere erosion at a 20 fold lower dose than 15 in the monoblastic leukemia cell line U937.122 GSK3 inhibition by 6-bromoindirubin-3′-oxime caused suppression of hTERT expression, telomerase activity, and telomere length in various cancer cell lines and decreased hTERT expression in ovarian cancer xenografts.123 Other repressors of hTERT transcription are retinoids, derivatives of vitamin A, which have been shown to downregulate telomerase leading to telomere erosion.124 Retinoic acid receptor alpha and retinoid-X receptor-specific agonists synergistically down-regulated hTERT and induced tumor cell death.125

Posttranslational regulators of hTERT Perturbing posttranslational modifications represents another mechanism for targeting telomerase. Because telomerase activity and cellular location is controlled by its phosphorylation state, phosphatase and kinase inhibitors can have direct effects on



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telomerase. For example, the phosphatase 2A inhibitor okadaic acid prevents telomerase inhibition by phosphatase 2A activation in breast cancer cells.126 Protein kinase C (PKC) has been shown to enhance telomerase activity in certain cell types, and the PKC inhibitors have been shown to inactivate telomerase in nasopharyngeal127, 128 and cervical129 cancer cells among others. Imatinib mesylate (Gleevec), a tyrosine kinase inhibitor, also down-regulates telomerase activity and inhibits proliferation in telomerase expressing cells.130 The protein kinase Akt has well-established roles in telomerase activation. Akt kinase phosphorylates hTERT and activate telomerase in vitro, while the none specific Akt inhibitor wortmannin causes loss of telomerase activity.127 The tyrosine kinase c-Abl is also capable of phosphorylating hTERT. The SH3 domain of c-Abl interacts with and phosphorylates hTERT resulting in a decrease in telomerase activity.131 These observations demonstrate that telomerase activity can be directly regulated by protein phosphorylation independent of transcriptional regulation and highlight that telomerase inhibition may contribute to the observed anti-cancer effects of several drugs. In addition to regulation by kinases, it has recently been determined that hTERT is a substrate of caspases-6 and -7.132 Interestingly, the caspase sites are unique and the cleavage products appear remarkably stable.

Hsp90 inhibitors The telomerase ribonucleoprotein complex requires the Hsp90 chaperone complex, including p23, Hsp70, p60, and Hsp40/γdl for proper assemblage. Hsp90 associates directly with hTERT and is required in order to maintain telomerase in an active conformation at least under some conditions.133, 134 Hsp90 inhibitors cause


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inactivation, destabilization, and degradation of Hsp90 target proteins including hTERT.46, 133 Since several oncogenic proteins including c-Myc, Akt, and mutated p53 require Hsp90 for their activity, inhibition of this chaperone should block multiple oncogenic pathways. Hsp90 inhibitors such as novobiocin, radicicol, geldanamycin, and 17-AAG were also shown to inhibit telomerase activity.134-137 Recently, curcumin has been shown to inhibit nuclear localization of telomerase by dissociating the Hsp90 cochaperone p23 from hTERT.93 Curcumin treatment limited the association between p23 and hTERT while not affecting Hsp90 interaction with the telomerase.

G-Quadruplex Stabilizers

The most advanced area of small-molecule drug discovery directed at the telomere is G-quadruplex-stabilizing ligands. G-quadruplexes are unique structures formed by folding G-rich nucleic acid sequences. These structures are heterogeneous with a variety of structural topologies.138 Folding topologies are influenced by sequence, buffer conditions including identity of cations present, molecular crowding, and other parameters. Significant effort towards determining the structures of human telomeric DNA138, 139 and the biological functions of G-quadruplex DNA at the telomere and in other G-rich sequences embedded in the chromosome140 have been reported. Because telomerase must hybridize to its DNA primer to afford telomeric DNA extension, it was proposed that G-quadruplex formation would render a primer inert to telomerase.141 Seminal studies by Zahler et al. showing that telomerase could not extend a folded G-



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quadruplex were consistent with this hypothesis and initiated the field of G-quadruplex binding ligands as a platform for telomerase inhibition.141 This report was quickly followed by the cardinal work of the Hurley and Neidle labs providing the first Gquadruplex ligands that effectively inhibited telomerase.142, 143 Since these reports, a wide variety of pharmacophores have been explored as G-quadruplex ligands, and most of these are polycationic aromatic hydrocarbons (Figure 9).144 Since these initial studies demonstrating a relationship between telomeres and G-quadruplexes, these structures have become much more complex as drug targets than originally assumed as they are predicted to be present in a wide variety of genomic locations with increased density in promoter regions.145 Several of these promoters, including those for hTERT,146 c-kit,147, 148

and c-myc,149, 150 have verified abilities to fold into G-quadruplex structures. Much of

the work on G-quadruplex structure, biological relevance, and small-molecule targeting has been recently and extensively reviewed and readers are directed to these. Specific Gquadruplex ligands explored have been extensively reviewed and this class of ligands has been expanded to many non-telomeric targets.144, 151, 152 Here, we will simply highlight the major concerns with targeting G-quadruplexes as a specifically telomere-directed anticancer drug modality and highlight recent approaches towards advancing telomerebinding ligands to the clinic. The first G-quadruplex ligands explored as telomerase inhibitors were disubstituted anthraquinone derivatives.153 Since that time several other polycyclic heteroaromatic scaffolds and macrocycles have been explored allowing the development of potent telomerase inhibitors with increasing specificity for G-quadruplex DNA compared to duplex DNA and these ligands are available at the G-quadruplex ligands


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database (http://www.g4ldb.org/ci2/index.php).154 Importantly, high resolution crystal and NMR structures of human telomeric G-quadruplexes can inform rational design to improve G-quadruplex affinity and specificity.152 From structural studies, it is clear that critical features known to enhance G-quadruplex binding include a delocalized π-electron system capable of stacking onto G-quartet face, a positive charge that can rest in the center of the G-quartets to interact with the carbonyl oxygens from guanine that form the core of the G-quartets, and positively charged substituents that can interact with the grooves of the G-quadruplex (Figure 9c and 9d).138 From a purely biochemical view, the most significant current issues in G-quadruplex ligand design are specificity for Gquadruplex DNA compared to duplex DNA and specificity for G-quadruplex type, i.e telomeric versus other DNA and RNA quadruplexes that may be present in the cell.155 From a pharmaceutical standpoint, the major issue concerning translation of Gquadruplex ligands is the poor pharmacokinetic profile associated with the typically hydrophilic and positively charged molecules.156 One of the most significant recent advances towards selective G-quadruplex targeting is the ability to rationally design molecules using structure-based approaches. Structures of G-quadruplexes formed by the human telomeric DNA sequence by X-ray crystallography and NMR spectroscopy have provided templates for rational design of Gquaduplex ligands.157, 158 These advances have allowed for the development of rationally designed molecules with increased sequence and topological folding specificity.159, 160 These promising examples suggest that G-quadruplex specificity is achievable. The more challenging hurdle that still remains is the poor pharmacokinetic and pharmacodynamic properties of promising G-quadruplex compounds. These hurdles may not be



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insurmountable as evidenced by the G-quadruplex ligand and fluoroquinolone derivative quarfloxin, that binds to nucleolin/rDNA and inhibits Pol 1 transcription thus inducing apoptosis in adenocarcinoma cells, which has had some clinical success.161

Disruption of Telomere-Maintenance using Antisense and Related Approaches

Ribozymes

Hammerhead ribozymes are small catalytic RNA molecules that have the capacity to specifically cleave target RNA after NUH sequences, where N is any nucleotide, U is uridine, and H is any nucleotide except G.162 Design of hammerhead ribozymes is relatively simple and specificity can be achieved by incorporating sequences complementary to the target. Due to this nature, ribozymes have been investigated as a platform for telomerase inhibition via targeted RNA cleavage of hTER or hTERT mRNA. The template region of hTER is an ideal target for ribozyme cleavage because it has the requisite target sequence for Hammerhead or HDV ribozymes, is essential for telomerase activity, and it may be solvent exposed even in the telomerase holoenzyme. Studies using melanoma,163 hepatoma, colon cancer,164 and endometrial cancer cells165 have correlated ribozyme expression with a marked reduction in telomerase activity resulting from diminished telomerase RNA levels. While telomere lengths were greatly attenuated in some carcinomas, other types did not display this phenotype.163, 165 Treatment with template-targeting ribozymes also resulted in significant growth


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retardation with proliferation rates reduced compared to parent cells.166 Furthermore, treated cells experienced telomere shortening in concert with altered or dendritic morphologies, which are indicative of senescence phenotypes,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 cleavage while conferring protection from cellular exoribonuclease degradation, which has been shown to increase the efficiency of functional RNAs.167 TERT mRNA levels has been targeted by a Hammerhead ribozyme targeting the initial nucleotides in hTERT mRNA, which may be essential for the proper binding of the translation initiation complex to the 5′ mRNA cap structure.168 Ribozyme cleavage in the middle of hTERT mRNA has also demonstrated decreased telomerase activity while also influencing rapid cell death (4 days) even without diminished telomere length, consistent with potentially nontelomeric roles of hTERT in some cancer cells.169

Oligonucleotides and Template Antagonists

Antisense (AS) oligonucleotides (ODNs) can inhibit telomerase by several mechanisms including blocking hTERT translation, reduction of hTERT mRNA and hTER levels, and sterically blocking the association of telomerase with the telomere (template antagonists).42 However, oligonucleotides present many challenges towards achieving efficient intratumoral telomerase inhibition. Nonetheless, studies have demonstrated that unmodified antisense oligonucleotides can in fact potently inhibit telomerase activity under in vitro conditions.170, 171 Additionally, coupling of standard



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AS-ODNs with Oligofectamine lipid carriers has ensured proper nuclear delivery of chimeric AS-ODNs and decreased tumor growth in in vitro and in vivo tumor models.170, 172

Due to the effectiveness of initial antisense ODNs experiments, a variety of studies have been completed that use antisense therapies to target and inhibit telomerase. To make up for the shortcomings of unmodified ODNs, a spectrum of ODNs with chemically modified backbones have been utilized to enhance the therapeutic potential of these antisense strategies. Phosphorothioate (PS)-ODNs composed of telomere repeat sequences ranging from 6 to 24 bases and targeting the template of hTER showed significant telomerase inhibition in various cancer cell lines.173, 174 While longer PSODNs 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 dramatic levels of apoptosis, and reduced human xenograft tumors in mice to undetectable sizes.174 2′-O-Methyl-PS chimeric ODNs targeting the template sequence of hTER demonstrated potent telomerase inhibition with IC50 values in the nanomolar range.175, 176 Similar ODNs blocked up to 95% of telomerase activity after one day treatment when delivered with a cationic lipid delivery system and induced telomere erosion and subsequent limited proliferation commensurate with marked apoptosis in long term studies with chronic treatment.177, 178 Cellular delivery with chitosan-coated PLGA nanoparticles afforded even higher cellular uptake and the similar phenotype of progressive telomere shortening with an EC50 value of 10 nM.179 Direct addition of 2′-Omethoxyethyl-PS chimeric ODNs decreased telomere length and proliferation and growth rates in MCF-7 (breast)180 and DU145181 (prostate) cancer cells in culture and xenograft


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tumor models.180, 181 Synergistic effects were seen in DU145 prostate cancer cells upon addition of chemotherapeutic agents such as cisplatin or carboplatin.181 Peptide nucleic acids (PNAs) completely covering the template region of hTER were 10-50 times more potent than similar PS-ODNs with IC50 values in the nanomolar range.182 The potent telomerase inhibition is due to high affinity of the PNA to the single stranded RNA template and suggests that affinity is not limited to base pairing but may also include interactions between hTERT and the peptide backbone.182 Both cellular permeabilization and lipid-mediated delivery demonstrated similar IC50 values in DU145 (prostate cancer)83 and HME50-5 (immortal breast epithelial)182 cells. Conjugation of short peptide sequences to PNAs targeting the hTER template displayed increased telomerase inhibition with IC50 values 10 fold lower than PNAs alone.183 Targeting of hTER by 2-5A ODNs has led a remarkable inhibition of telomerase activity concomitant with rapid hTER degradation and a greater that 50% decrease in cell viability in a time dependent manner (see recent reviews for more details).41,184-186 2-5A ODNs targeting hTER can also arrest cells in G2/M phase187 and induce apoptosis via activation of the caspase signaling cascade.186 Direct administration of hTER- targeting 2-5A ODNs and combination therapy with interferon-β has demonstrated anti-proliferative effects including a reduction in colony formation and a significant retardation in tumor growth and volume over time.184, 185, 188 In 2003, Asai et al. described a novel class of chemically modified N3′P5′thiophosphoramidates (NPS) which incorporated a nucleophilic sulfur atom, typical of PSODNs, in the internucleotide backbone and conferred enhanced stabilization between the ODN backbone and hTERT amino acids adjacent to the template region of hTER and



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greater in vitro bioavailability.42, 189 The efforts led to the development of 17 (GRN163), a 13 nucleotide long NPS-ODN with sequence 5′-TAGGGTTAGACAA-3′ that targets the template region of hTER by overlapping and extending four nucleotides beyond the 5′ template boundary as a potent telomerase inhibitor (IC50 = 0.14 µM).189 Treatment of various cancer cell lines with 17 significantly reduced telomerase activity, promoted critical telomere loss, and instigated apoptosis while concomitantly up-regulating caspase activity.189 These results combined with impressive data from long-term experiments and its high potency and specificity indicated that 17 may be an effective anticancer agent.189 In order to improve its bioavailability, lipophilicity, and cellular uptake, a 5′ terminal lipophilic palmitoyl group was attached through an amide bond to α-amino glycerol of the thio-phosphoramidate 17 to produce the clinical candidate 1 (Figure 10).190 Comparison studies with 17 proved that 1 was just as potent at inhibiting telomerase in various cancers, could inhibit telomerase activity effectively without the use of a transfection agent, demonstrated more cellular uptake in tissue and xenograft models, and has been shown to be completely reversible.190-192 Since then, many studies have been published revealing the extensive anticancer potential of this drug in a variety of cancers. A single treatment of 1 µM 1 yielded complete inhibition of telomerase activity in various cancers within hours of dosing and could be maintained at nearly undetectable levels for up to six days, while everyday treatment virtually suppressed telomerase activity.193-198 Continued treatment for two weeks elicited morphological changes such as rounding-up adherent cells and increased extention of filliopodia-like structures193, 198, reduced colony formation194, 196, and inhibited invasion194 Longer periods of treatment with 1 triggered decreased proliferation196, decline in cell viability197,


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199

, disruption of focal adhesions and actin filament organization,193 substantial telomere

shortening194-196, reduced capacity to form neurospheres and mammospheres,191, 199 down-regulation of cell cycle regulators,192 G0/G1 cell cycle arrest191, 198, up-regulation of APG5 and DAPK2 expression (genes implicated in the induction of apoptosis)197, disrupted membrane intergrity,191 and promotion of apoptotic cell death.191, 197 1 also promoted a higher percentage of anaphase bridges, hallmarks of telomere dysfunction, and increased the frequency of γ-H2AX and 53bp1 foci, known molecular markers that localize to sites of DNA strand breaks and dysfunctional telomeres.200 In tumor models, 1 prevented tumor formation and metastasis from xenografts194, 195, significantly reduced growth and tumor volume191, 200, 201, and enhanced antitumor effects in combination therapy with doxorubicin, temozolomide, and irradiation.191, 200 The successful preclinical experiments demonstrated the antitumor effectiveness of 1, and it remains the only telomerase inhibitor to have been tested in clinical trials. Lessons learned from 1 clinical trials are essential for the field as this represents the first telomerase inhibitor to reach the clinic. Currently, six phase I and phase I/II trials of 1 as a single agent and three combination trials are ongoing. Major objectives of these studies are safety, tolerability, and maximum tolerated 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 silencing.42, 202 As an RNA that is complicit for telomerase activity, hTER is an excellent



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target for siRNA therapies. Transfection and intravenous injection of siRNAs conjugated to PS-ODNs targeting the template region of hTER reduced cell proliferation in human cervical carcinoma and more importantly, strongly reduced the number of metastases in murine malignant melanoma cells.203 siRNAs targeting nucleotides 11-19 of hTER expressed in various cancer cell lines incluing HCT116 colon cancer, LOX melanoma, HeLa cervical cancer, T24 bladder cancer, MCF-7 and BT424 breast cancers, LNCaP prostate cancer, via lentiviral-vector expression caused rapid growth inhibition and apoptosis meanwhile displaying negligible cellular cytotoxicity independent of affecting telomere length.204 Cancer cells exhibited knockdown of endogenous hTER as well as down-regulation of cell cycle progression genes (cyclin G2 and Cdc27) and specific genes that play pivotal roles in tumor growth, angiogenesis, and metastasis (integrin αV and Met proto-oncogene) suggesting a role for these genes in the hTER-mediated cell growth inhibition.204 Expression of siRNAs targeting hTERT mRNA in various cancers has caused decreased hTERT mRNA levels, knockdown of hTERT protein expression and notable telomerase inhibition within 24 hours of dosing and lasting up to four days.120, 176, 205-209 Stable transfection of these siRNAs led to complete suppression of telomerase activity in three days concomitant with the absence of hTERT mRNA expression.205, 207 Cancers treated with siRNAs to hTERT experienced decreased cell viability,120 prolonged population doubling times,209 suppression of anchorage-dependent proliferation,206 significant growth inhibition,208 and morphological changes indicative of senescence.205 Esophageal and colon adenocarcinomas displayed extensive telomere shortening and attrition,205, 207 supported by chromosomes with telomere free ends suggestive of


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complete telomere erosion, which suppressed proliferation and induced apoptotic cell death.205 Genetic silencing of hTERT led to an up-regulation of genes involved in DNA damage recognition and repair, p53 family proteins and their regulators, cell cycle inhibitors, pro-apoptotic proteins, and caspases.199, 205, 210 At the same time, hTERT depletion significantly attenuated the expression of integrin αV and β3 subunits and cMet, proteins that are known to play important roles in cell differentiation, adhesion, motility, and tumor metastasis.206 In tumor models, hTERT siRNAs disrupted cell adhesion, migration, and invasion, promoted slower tumor growth,206 decreased cell viability and colony formation, induced cell cycle arrest,199 reduced tumor sizes and volumes in xenografts,206, 208 and demonstrated additive effects upon combination therapy with doxorubicin and intereferon-γ.120, 199 Interestingly, while expression of siRNAs targeting both hTER and hTERT concomitantly did decrease mRNA, RNA, and protein levels of telomerase components, cell proliferation, and xenograft tumor growth and promote pyknotic nuclear chromatin, the effect of silencing hTER alone was more pronounced than silencing either hTERT or both together.211

Immunotherapy The realization that hTERT is overexpressed in the vast majority of cancers prompted the investigation of hTERT as a tumor antigen. Soon after the discovery of widespread hTERT expression in tumors, hTERT peptide fragments were identified as tumor-associated antigens functioning through the major histocompatibility complex class I and class II pathways. In several cancer types, CD8+ cytotoxic T-cells specific for hTERT peptides, primarily hTERT-I540 (ILAKFLHWL), are detectable suggesting



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active hTERT-directed immune-surveillance for these cancers.212 Subsequently, over 20 hTERT-derived peptides have been employed to boost anti-tumor immunity.2 The majority of these elicit a CD8+ cytotoxic T-cell response, while the others activate a CD4+ response. Several clinical trials of hTERT-based cancer vaccines have demonstrated the safety of this approach.213, 214 Unfortunately, the impact on disease progression reported to date is modest. Some patients respond with significant tumor regression but more generally only a low percentage of patients respond and responses tend to be transient. This is despite the fact that vaccination with hTERT-derived peptides generally does result in hTERT-specific T cell production, indicative of an immunological response. Current efforts involve understanding the differences between responsive and non-responsive patients, optimization of antigenic hTERT epitopes, and optimization of specific immunotherapy platforms for telomerase-targeted treatment strategies.

Gene Delivery One of the main concerns with cancer gene therapy is the selective expression of target genes in tumor cells while avoiding expression in normal tissues. Because activation of telomerase activity in cancer cells is largely driven by increased expression of hTERT, it is reasonable to speculate that hTERT promoter activity is commensurately increased. Taking advantage of this increased promoter activity, several groups have investigated the potential of the hTERT-promoter for cancer-selective gene therapy. The hTERT promoter is extremely attractive for the selective expression of transgenes for anticancer gene therapy because of the near universal expression of hTERT in cancers


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and the much greater activity of the promoter in cancer cells compared to normal cells, even those the express telomerase. Most reports on the use of the hTERT promoter for cancer gene therapy attempt to control expression of anti-cancer genes in tumor cells. For example, pro-apoptotic genes (FADD and TRAIL), and a suicide gene (HSVtk) have been tested.215-217 In cell-based assays and in mouse models, tumor-specific transgene expression has been achieved and promising results in mouse studies highlight early successes. Because these approaches utilize replication-deficient virus in order to decrease harmful side effects, these approaches may suffer from limited tumor-mass distribution. A different and very promising approach that overcomes this limitation is to utilize the hTERT promoter not for transgene expression, but instead viral replication; in essence, establishing an anti-cancer 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 cancer diagnoses.35 Early data suggest that Telomelysin was well tolerated but clinical response was limited, owing perhaps to the single viral infection, which may have limited the viral titer. Most promising was the response of one patient with a significant,56%, reduction in metastatic tumor size.35

Perspective and Future Directions

Much progress has been made towards translating telomere research to the clinic since the seminal studies demonstrating the importance of telomeres, the identification of telomerase and telomere-binding proteins, and the validation of telomere length maintenance as a requirement of cancer cells. Early clinical trials with telomerase



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inhibitors have shown that the approach is well tolerated and does reduce telomerase activity. However, the absence of reported clinical efficacy suggests limited progress. Future trials examining which patients might be best suited for telomerase inhibitor therapy and current trials testing telomerase inhibitors in combination with other traditional chemotherapeutics can be expected to advance the field. We can also expect continued advances in gene therapy by taking advantage of the robust activity of the hTERT promoter in cancer cells as viral vectors are optimized. Likewise, immunotherapy targeting hTERT antigens is expected to continue its progress through clinical trials and arguably holds the most promise currently for telomerase-targeting therapeutics. Surprisingly, small-molecule inhibitors of telomerase have not fared well. This may change as advances in telomerase structural biology; for example the crystal structure of an insect TERT,62 structures of several hTER domains,218, 219 models of hTERT based on the beetle crystal structure,220 and the recent three dimensional model of human telomerase based on electron microscopy images,221 increase our understanding of telomerase structure and provide templates for rational small-molecule drug design. Rationally designed telomerase inhibitors may allow increased specificity, bioavailability, and inhibition mechanisms that have greater effects in a clinical setting than those small molecules that are currently available. In addition, the observation that G-quadruplex ligands can directly disrupt telomere structure evidently by blocking binding of telomere-binding proteins such as TRF2 and POT1 suggests that there are more telomere-associated targets to be exploited, and we can expect that near future studies will determine the effectiveness of directly targeting the telomere as an anti-


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cancer drug platform. Targeting telomere biology as a means to improve human health remains an important objective that drives discoveries in telomere biology and telomerase biochemistry. The interdisciplinary approaches towards targeting telomere biology have led to three discrete areas of clinical trials: inhibition of telomerase, hTERT-directed immunotherapy, and hTERT-promoter gene/viral therapy. It seems that the future of pharmaceutical benefit of telomere-targeting agents remains promising and our increased understanding of telomere biochemistry and biology are sure to inform more effective approaches toward developing telomere biology targeted agents.

Biographical sketches Vijay Sekaran is a post-doctoral fellow at the University of Colorado at Boulder working with Drs. James Goodrich and Jennifer Kugel. He received his BS in Biomedical Engineering from the Georgia Institute of Technology in 2005 and his PhD in Pharmaceutical Sciences from the University of North Carolina at Chapel Hill under the guidance of Michael Jarstfer in 2012. His dissertation focused on understanding the structure and function of telomerase RNAs as well as characterizing the effects of small molecules that bind to human telomerase 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, and completed her Ph.D in Pharmaceutical Sciences from the University of North Carolina at Chapel Hill in 2010 under the supervision of Michael Jarstfer. Her dissertation focused on elucidating the mechanism of telomerase activation or repression and its impact on cellular pathways and



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elucidating the mechanism of action of new potential telomerase inhibitors. Her research interrogated the relationship between telomerase, aging, and cancer progression.

Michael Jarstfer is an Associate Professor of Chemical Biology and Medicinal Chemistry in the Eshelman School of Pharmacy at the University of North Carolina at Chapel Hill. He received his BA in Biochemistry from Trinity University, San Antonio TX and his PhD from the University of Utah under the guidance of Dale Poulter. He conducted post-doctoral research in the laboratory of Thomas Cech at the University of Colorado in Boulder. His research interests include structure and function of telomerase, development of telomere targeting agents, the roles of telomere biology in human health, and the molecular basis for social motivation.

Corresponding author information. Phone: 919 966-6422. E-mail: [email protected]. Abbreviations used: ALT, alternative lengthening of telomeres; AS, antisense; hTERT, human telomerase reverse transcriptase; HDAC, histone deacetylase; hTER, human telomerase RNA; NPS, N3′P5′thio-phosphoramidates; ODN, oligonucleotide; PKC, protein kinase C; PNA, peptide nucleic acids (PNA); POT1, protection of telomeres 1; PS, phosphorothioate; QSAR, quantitative SAR; RNAi, RNA interference, SAR, structure activity relationship; siRNA, silencing RNA; TIF, telomere dysfunction induced focus; TAK1, TGF-β activated kinase 1; TAL1, T-cell acute lymphoblastic leukemia protein 1; WT1, Wilm’s tumor protein 1.


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(192) Tokcaer-Keskin, Z.; Dikmen, Z. G.; Ayaloglu-Butun, F.; Gultekin, S.; Gryaznov, S. M.; Akcali, K. C. The effect of telomerase template antagonist GRN163L on bonemarrow-derived rat mesenchymal stem cells is reversible and associated with altered expression of 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 antagonist GRN163L alters MDA-MB-231 breast cancer cell morphology, inhibits growth, and augments the effects of paclitaxel. Mol. Cancer Ther. 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. Telomerase template antagonist GRN163L disrupts telomere maintenance, tumor growth, 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 of lung cancer by GRN163L: a novel human telomerase inhibitor. Cancer Res. 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, in human 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. Telomerase inhibitor GRN163L inhibits myeloma cell growth in vitro and in vivo. Leukemia 2008, 22, 1410-1418.



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(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. (199) George, J.; Banik, N. L.; Ray, S. K. Knockdown of hTERT and concurrent treatment with interferon-gamma inhibited proliferation and 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. Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase 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 telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer 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 of small double-stranded oligodeoxynucleotides targeting telomerase RNA in malignant melanoma cells. Oligonucleotides 2009, 19, 169-178. (204) Li, S.; Crothers, J.; Haqq, C. M.; Blackburn, E. H. Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference-mediated depletion of telomerase RNA. J. Biol. Chem. 2005, 280, 2370923717.


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(205) Shammas, M. A.; Koley, H.; Batchu, R. B.; Bertheau, R. C.; Protopopov, A.; Munshi, N. C.; Goyal, R. K. Telomerase inhibition by siRNA causes senescence and apoptosis in Barrett's adenocarcinoma cells: 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 and motility of HCT116 cells. Cancer Biol. Ther. 2008, 7, 228-236. (207) de Souza Nascimento, P.; Alves, G.; Fiedler, W. Telomerase inhibition by an siRNA directed against hTERT leads to telomere attrition 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 specific activation of RNAi pathway quickly results in cancer cell growth impairment. Biochem. Pharmacol. 2007, 73, 1703-1714. (209) Miri-Moghaddam, E.; Deezagi, A.; Soheili, Z. S. Downregulation of telomerase activity in human promyelocytic cell line using 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 hTERT inhibits 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 inhibition strategies by siRNAs against either hTR or hTERT in oral squamous cell carcinoma. Cancer Gene Ther. 2011, 18, 318-325.



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(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. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ 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 of various tumor-associated antigen-specific t-cell responses in patients with 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 with telomerase-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 reverse transcriptase promoter for potential post-therapeutic deletion of tumorigenesis. 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 restrict induction 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 to tumor necrosis factor-related apoptosisinducing ligand (TRAIL). Cancer Gene Ther. 2004, 11, 691-698.


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Figure Legends Figure 1. Many ways to target the overexpression of telomerase in cancer cells have been developed. Figure 2. Representative nucleoside analogs identified as telomerase inhibitors. Figure 3. Examples of small molecule telomerase inhibitors identified by 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 as telomerase inhibitors. Figure 6. Small molecules identified as telomerase inhibitors utilizing yeast-based assays.

Figure 7. Treatment with 14 reversibly inhibits telomere length maintenance. A. Structures of 13 and 14. B. Chronic treatment of NCI-H460 cells with 14 results in growth arrest that can be reversed after ending the treatment. Open circles are untreated cells, filled triangles are cells treated with 14. Treatment was halted at day 220 – note the return to proliferative growth. C. Southern blot analysis of average telomere length of telomerase 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


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DNA (lane 3). Untreated cells are shown in lane 4 for comparison and have 3 kb of telomeric DNA. Figures 7B and 7C are from Damm et al.36 and are used with permission.

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

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

Figure 10. The clinical tested telomerase inhibitor. A. 1 is a modified oligonucleotide that contains a 5’-palmotyl group. The sequence is complementary to the hTER template domain. B. The N3′→P5′thio-phosphoramidate linkage present in 1.



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Table of Contents Graphic


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Figure 1



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Figure 2 O HO O

N

N

O

OH

N

N H

N

O O O O P O P O P O OOO-

N

NH

-

O

N

N

NH2

N

N3 2; AZT

S

NH2

NH

N3 Abacavir

6-thio-7-deaza-2'-deoxyguanosine 5' triphosphate


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Figure 3.



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Figure 4


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Figure 5



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Figure 6.


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Figure 7.

A

N H

B

O COOH O

13 N

O

N H

COOH

14


C


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Figure 8. NH2 OH OH H N HO

O NH2

H N

N

NH2 O

N

H2N

O OH O

N N

HO

OH

O

a

HN O N+

NH2

OH

Neomycin

O

H N

H2N

OH

O H2N

Bisbenzimide Hoechst 33258

NH2

O

O

NH2

bN H

c NH O H2N

N+

H2N

NH2

Bis-ethidiumbenzyl amides: a,c = di-amino propionic acid, ornithine or lysine; b = glycine, alanine, gamma amino butyric acid, -amino hexanoic acid, or no linker


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Figure 9



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Figure 10.


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Telomere Maintenance as a Target for Drug Discovery - Journal of

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