Oncogene (2000) 19, 6632 ± 6641 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Telomere maintenance mechanisms as a target for drug development

David J Bearss1, Laurence H Hurley1,2 and Daniel D Von Ho€*,1

1The Arizona Center, The University of Arizona, 1515 N. Campbell Ave., Tucson, Arizona, AZ 85724, USA; 2College of Pharmacy, The University of Arizona, 1703 E. Mabel Ave. Tucson, Arizona, AZ 85724, USA

The shortening of the telomeric DNA sequences at the 1999; Makarow et al., 1997; Wright et al., 1997). These ends of is thought to play a critical role in fundamental new ®ndings of the structure of telomeres regulating the lifespan of cells. Since all dividing have enormous implications in the design of com- cells are subject to the loss of telomeric sequences, cells pounds that can target and disrupt the telomere with long proliferative lifespans need mechanisms to structure by providing us with more targets for new maintain telomere integrity. It appears that the activa- drug development. tion of the is the major mechanism by Telomeres are highly conserved in organisms ranging which these cells maintain their telomeres. The proposal from unicellular eukaryocytes to mammals, indicating that a critical step in the process of the malignant a strong preservation of their protective mechanisms transformation of cells is the upregulation of expression for preventing chromosomal ends from undergoing of telomerase has made this enzyme a potentially useful degradation and ligation with other chromosomes. prognostic and diagnostic marker for cancer, as well as a Without telomeric caps, human chromosomes would new target for therapeutic intervention for the treatment undergo end-to-end fusions with formation of dicentric of patients with cancer. It is now clear that simply and multicentric chromosomes (Counter et al., 1992; inhibiting telomerase may not result in the anticancer Harley, 1995; Harley et al., 1990; van Steensel et al., e€ects that were originally hypothesized. While telomer- 1998). These abnormal chromosomes would break ase may not be the universal target for cancer therapy, during , resulting in severe damage to the we certainly believe that targeting the telomere main- and the activation DNA damage checkpoints tenance mechanisms will be important in future research and leading to or the initiation of aimed toward a successful strategy for curing cancer. cell death pathways (deLange and Jacks, Oncogene (2000) 19, 6632 ± 6641. 1999). In addition to protecting from end-to-end fusion of Keywords: telomere; telomerase; G-quadruplex; drug chromosomes, telomeres are also thought to protect targets against the loss of DNA at the end of each upon the completion of DNA replication. Dividing cells have been shown to undergo a progressive loss of 25-200 DNA base pairs following Introduction each (Harley et al., 1990; Hastie et al., 1990; Mehle et al., 1994). This loss of telomeric DNA Telomere structure and function is largely due to the `end replication problem' Telomeres are specialized DNA- structures that (Olovnikov, 1973; Watson, 1972) (see Figure 1a for cap the ends of linear chromosomes. Mammalian classical (old) model of telomere ends). The `end telomeres are composed of the hexanucleotide of replication problem' refers to the inability of the TTAGGG repeated from 5 ± 25 kilobase pairs in length DNA replication machinery to copy the ®nal few base oriented 5' to 3' toward the chromosome end (Allshire pairs of the lagging strand during DNA synthesis et al., 1989; Blackburn and Gall, 1978; deLange et al., (Olovnikov, 1973; Watson, 1972). Another possible 1990; Moyzis et al., 1988). Several recent studies have cause for loss of telomeric sequence is by a 5' to 3' suggested that the structure of the ends of telomeres exonuclease activity which recesses the telomeric CA- may be more complex than originally thought. Grith rich strand (Makarow et al., 1997; Wellinger et al., and colleagues have found that telomeres do not end in 1996). Since the telomere consists of a repetitive DNA a linear manner (Figure 1a) (Grith et al., 1999). sequence, its loss is thought to be less important to the Instead the end of the telomere forms a loop structure cell than the loss of critical encoding sequences with the 3' G-rich strand (referred to as the `T' loop) that may be near the end of a chromosome; therefore, invading the duplex telomeric repeats, forming a telomeric sequences protect from the loss of more displacement or D loop (Figure 1b). Telomere- critical gene encoding sequences. associated may facilitate the formation and the maintenance of the both the T- and D-loops, Telomere maintenance by telomerase suggesting a large DNA-protein structure at the end of each chromosome. In addition, most human telomeres Normal human somatic cells have a limited replicative appear to terminate in a single-stranded 3' GT-rich life span when grown as cell cultures. After overhang, which is thought to play an important role approximately 50 cell divisions, these cells will enter in telomere structure and function (Colgin and Reddel, (mortality stage 1 or M1), char- acterized by irreversible cell cycle arrest. It is now generally accepted that there is a causal relationship *Correspondence: DD Von Ho€ between telomere shortening and M1 senescence. This Targeting telomere maintenance DJ Bearrs et al 6633

Figure 1 The Classical (a) and New (b) view of the telomeric end structures (Grith et al., 1999) acceptance is based on many studies showing that the and Reddel, 1999; Nugent and Lundblad, 1998). There telomeres of normal diploid cells decrease as they are is an additional motif, T, which is speci®c to grown in culture, as well as studies showing the telomerase reverse transcriptases (Colgin and Reddel, correlation of telomere length and age in human 1999). tissues (Harley, 1995; Harley et al., 1990). These Several variant hTERT mRNAs derived, presum- ®ndings support the hypothesis that telomere erosion ably, from alternative splicing have also been described is an important signal in the aging of a cell, leading to (Kilian et al., 1997). It is possible that alternative the induction of cellular senescence and cell death. The splicing would play a role in regulating the activity of cumulative loss of telomeric DNA sequence acts as a telomerase during development and di€erentiation biological clock, eventually limiting the replicative (Colgin and Reddel, 1999). Of importance is that potential of cells when a critical telomere length is transfection of hTERT into hTR (telomerase mRNA) reached. positive/telomerase negative cells results in telomerase Since all replicating cells are subject to cell activity, which suggests that hTERT may be rate- senescence caused by telomeric DNA loss, cells with limiting for the assembly of a catalytically active long-lived replicative life spans need a mechanism to telomerase (Beattie et al., 1998; Colgin and Reddel, counteract or prevent this loss. One way cells have to 1999; Counter et al., 1998; Nakayama et al., 1998; restore telomeric DNA is the activation of the enzyme Weinrich et al., 1997; Wen et al., 1998). telomerase. Telomerase activity was initially described Control of hTERT expression appears to be by in the , with subsequent studies transcriptional control but may also be through leading to the molecular of several of the phosphorylation (Bodnar et al., 1996; Kilian et al., components of the telomerase holoenzyme from many 1997; et al., 1997; Li et al., 1997, 1998; Meyerson et di€erent organisms (Greider and Blackburn, 1985, al., 1997). Of note is that c-myc can induce telomerase 1987; Henderson, 1995; Morin, 1989). The telomerase activity by increasing the of hTERT enzyme is a ribonucleoprotein (Wang et al., 1998). There are c-myc binding sequences enzyme complex that is responsible for maintenance of on the hTERT gene as well as binding sites for other the telomeric DNA at the end of chromosomes (Morin, transcription factors and hormone response elements, 1989). It is a very unique , as it uses its own making the transcriptional control of hTERT expres- RNA template to add repeats onto the G-rich strand sion an area of very active research (Cong et al., 1999). (Harrington et al., 1997; Nakamura et al., 1997). The In addition to the above, it has recently been complimentary C-rich strand is thought to be partially reported by Holt et al. (1999) and colleagues that the synthesized by other replication such as molecular chaperones p23 and Hsp90 bind to the polymerase a (Colgin and Reddel, 1999). catalytic subunit of telomerase. If one blocks the The basal human telomerase enzyme consists of at interaction of Hsp90 or p23 with the catalytic subunit least two components, the catalytic subunit, hTERT, of telomerase, the assembly of active telomerase is and the telomerase RNA, hTR (Collins et al., 1995; blocked in vitro. Inhibition of Hsp90 function in cells Harrington et al., 1997; Kilian et al., 1997; Meyerson with geldanamycin (an Hsp90 inhibitor) blocks the et al., 1997; Nakamura et al., 1997). The various assembly of active telomerase (Holt et al., 1999). It has components of telomerase are encoded by distinct been suggested that Hsp90 and p23 act as a (Colgin and Reddel, 1999). The catalytic subunit `foldosome' that mediates the assembly of a biologi- hTERT has seven protein domains in common with cally active protein complex (Holt et al., 1999). retroviral and reverse transcriptases. Another protein, TEP-1, is also a component of the These conserved motifs are part of a protein fold that telomerase complex. The function of TEP-1 is still forms the active site of the enzyme and includes three incompletely understood, but it may play a role in the aspartate residues that are critical for catalysis (Colgin transport and integration of telomerase into the

Oncogene Targeting telomere maintenance DJ Bearrs et al 6634 telomere. Additional proteins may be involved in the telomerase is not active in most somatic tissue but is telomerase holoenzyme and may add functionality such active in cancer cells and in tissues (Allsopp as enzyme . et al., 1992; Kim et al., 1994; Raymond et al., 1996a; Shay and Bacchetti, 1997). Table 2 summarizes our data and the data of others for the presence of Telomerase and cancer telomerase activity in tumor cells taken directly from Telomerase activity allows cells to maintain their patients (Raymond et al., 1996b). In general agreement telomeric DNA and avoid the M1 senescence check- with our data, a recent survey of the status of point. This is a necessary process for normal cells that telomerase positive cells has suggested that close to need the ability to proliferate inde®nitely such as germ 85% of human malignancies have telomerase activity, line cells. Unfortunately, telomerase activity can also while less than 0.5% of normal cells are telomerase contribute to the inde®nite replicative capacity of positive (Shay and Bacchetti, 1997). cancer cells. Shortened telomeres have been documen- These data suggest that telomere shortening may ted in multiple tumor types taken directly from patients function as a tumor suppressor mechanism and that (see Table 1) and it is thought that there is a reduction activation of telomerase is an important step in in telomere length in almost all tumor cells compared overcoming this tumor suppression. This thought is to normal cells. Many studies have reported that strengthened by recent work that implicates telomerase activity as a necessary step in the tumorigenesis process. Hahn et al. (1999) in Dr Weinberg's Table 1 Telomere length in tumors taken directly from patients laboratory have documented that expression of hTERT Telomere Telomere with SV40 large T antigen and H-ras results in direct length length conversion of normal cells to tumor cells. Expression (Relative (Relative of all three of these proteins is necessary to see this to normal) to normal) Tumor type Reduced Increased References conversion, as expression of each protein alone is not sucient to cause the conversion (Hahn et al., 1999). Colorectal Yes ± (Hastie et al., 1990); In addition, Bodnar et al. (1998) have documented that (Sciadini et al., 1994) transfection of cells with hTERT results in elongation Endometrial Yes ± (Smith and Yeh, 1992) Neuroblastoma Yes ± (Hiyama et al., 1993) of telomeres and bypassing of crisis. This series of very Wilm's Yes ± (deLange et al., 1990); important ®ndings helps validate telomerase as a very (Sciadini et al., 1994) important anticancer drug target. Hepatocellular Yes ± (Ohashi et al., 1996) ALL Yes ± (Adamson et al., 1992) Renal Yes ± (Mehle et al., 1994) Telomerase as a drug target Breast Yes ± (Odagiri et al., 1994); (deLange et al., 1990); The fact that telomerase activation appears to be a (Sciadini et al., 1994) near-universal occurrence in cancer cells suggests that Prostate Yes ± (Sommerfeld et al., 1996) telomerase may be an Achilles heel of the tumor cell. If Lung Yes ± (Sciadini et al., 1994) Ovarian Yes ± (Sciadini et al., 1994); this is indeed correct, then inhibition of the telomerase (Counter et al., 1994) enzyme would result in the erosion of telomeric DNA leading to the arrest of tumor cell growth or regression of the tumor due to the e€ects of sustained telomere erosion. Telomere shortening acts as a trigger for cells Table 2 Presence of telomerase activity on tumor cells taken directly to either senesce or die, or upregulate telomerase and from patients (adapted from Raymond et al., 1996) develop or progress into tumor cells Tested (Allsopp et al., 1992; Counter et al., 1992; Harley, Tumor type Positive Total % 1991; Levy et al., 1992; Lundblad and Szostak, 1989; Hematologic malignancies 57 85 67 Morin, 1989). Since telomerase activity has been Acute myeloid leukemia 8 12 67 described in most tumor cells but not in many normal Acute lymphoid leukemia 18 25 72 cells (see Table 3), there is the potential for relatively Chronic myeloid leukemia 12 12 100 Chronic lymphoid leukemia (early) 2 14 14 minor side e€ects (if any) of a telomerase-speci®c Chronic lymphoid leukemia (late) 5 9 55 inhibitor. This gives us some hope for selectivity of Myeloma 1 1 100 telomerase inhibitors for tumor cells versus normal Low-grade lymphoma 2 3 67 cells. High-grade lymphoma 9 9 100 Breast 28 33 85 Prostate 24 30 80 Lung 88 115 76 Non-small cell 74 101 73 Table 3 Detected telomerase activity in normal regenerative cells Small cell 14 14 100 with long proliferative life spans Colon 22 23 96 Cell type Reference Ovarian 13 14 93 Head and neck 14 16 87 Hematopoietic cells (Chiu et al., 1996; Hiyama et al., 1995) Kidney 41 55 74 Lymphocytes (Hiyama et al., 1995) Melanoma 7 7 100 Basal cells of skin (Harle-Bachor and Boukamp, 1996; Neuroblastoma 53 76 70 Yasumoto et al., 1996) Glioblastoma 6 8 75 Crypt cells of the intestine (Park et al., 1998) Hepatocellular carcinoma 28 33 85 Endometrium (Harle-Bachor and Boukamp, 1996; Gastric 56 66 85 Kyo et al., 1997) Bladder 39 40 97 Breast epithelial cells (Tsao et al., 1997) Total 476 601 79 Bronchial cells (Yashima et al., 1997)

Oncogene Targeting telomere maintenance DJ Bearrs et al 6635 Telomerase inhibitors 1999; Gilley et al., 1995; Pe€er et al., 1993; Tzfati et al., 2000). These domains include an alignment Reverse transcriptase inhibitors Because the hTERT domain that facilitates substrate binding to the protein is a reverse transcriptase, several well-known template domain for polymerization and non-template reverse transcriptase inhibitors (RTIs) have been tested domains that are probably involved with the binding as telomerase inhibitors (Strahl and Blackburn, 1994, of the RNA to the telomerase protein components 1996). Several studies have reported the e€ects of and further involved in modulating enzymatic azidothymidine triphosphate (AZT-TP), arabinofura- activity. The RNA component is critical to the nyl-guanosine triphosphate (Ara-GTP), dideoxyinosine enzymatic function of telomerase. This has motivated triphosphate (ddITP), ddTTP, and ddGTP on telomer- a number of investigators to design telomerase ase activity (Strahl and Blackburn, 1994, 1996). All of inhibitors based on the sequence and structure of these nucleoside triphosphate analogs inhibit telomer- the telomerase RNA. Since the RNA component of ase activity in vitro, but the analogs of AZT, 3'-deoxy- telomerase is an ideal target for oligonucleotides that 2',3'-didehydrothymidine (d4T), and Ara-G also caused are able to recognize and hybridize to accessible a consistent and rapid telomere shortening in vegeta- complementary sequences of the RNA, several tively growing Tetrahymena (Strahl and Blackburn, antisense strategies have been explored (Glukhov et 1994, 1996). More recently, the e€ects of AZT on al., 1998; Hamilton et al., 1999; Kushner et al., 2000; telomere length of a murine metastatic melanoma cell Pandit and Bhattacharyya, 1998; Pitts and Corey, line, K-1735 clone X-21, and a human breast cancer 1998). cell line, MCF-7, were determined (Multani et al., A number of studies have been directed toward 1998). Using ¯uorescence in situ hybridization (FISH) targeting the template domain of telomerase RNA with analysis to measure telomere length, a signi®cant antisense oligonucleotides, and e€ective inhibition of concentration-dependent decrease of telomeric signal telomerase activity has been achieved (Betts et al., intensity was seen in the AZT-treated cells, while no 1995; Hamilton et al., 1997, 1999; Pe€er et al., 1993). telomere shortening was observed in the untreated Several di€erent oligonucleotides with di€erent chemi- controls. These results suggest that analogs cal modi®cations have been tried, with a recent report may be able to disrupt the telomere maintenance of suggesting that 2'-O-alkyl-RNA oligomers are more cancer cells by targeting the hTERT reverse transcrip- selective than peptide nucleic acids (PNAs) in binding tase. Alternatives to the nucleotide analogs are the to the complementary telomerase RNA sequences growing family of potent telomerase inhibitors that (Pitts and Corey, 1998). Results from the same study have been identi®ed through screening of chemical have also shown that 2'-O-Me-RNA-based oligomers small molecule libraries using a telomerase assay. In complexed with cationic lipids can eciently inhibit one study, a group of isothiazolone-containing telo- telomerase activity upon transfection of the human merase inhibitors were identi®ed to have submicromo- prostate tumor cell line DU145 (Pitts and Corey, 1998). lar potency in a telomerase assay (Bare et al., 1998). Since isothiazolones are known to react with the Structural targeting Other suggested approaches to reduced thiols of cysteine and DTT, these compounds target telomerase have focused on structure-based are proposed to interfere with telomerase activity by design of telomerase inhibitors based upon the three- modifying a cysteine residue(s) in or near the reverse dimensional structures of functionally important do- transcriptase active site of telomerase (Bare et al., mains of the enzyme. It is clear that as more 1998). These results suggest that the use of a information becomes available about the structural biochemical screening assay system that targets the importance of di€erent RNA components in telomer- reverse transcriptase activity of telomerase may be ase function, targeting strategies using oligonucleotides useful for identifying inhibitors of telomerase. or small molecules aimed at select RNA components of hTR may prove very promising in achieving selective Antisense approaches A second approach for targeting inhibition of telomerase activity (Chen et al., 2000). hTERT protein is to use antisense oligonucleotides that are designed to hybridize with complementary se- quences of hTERT mRNA and subsequently deplete Potential problems with targeting telomerase hTERT protein levels inside cells. The recent clinical As with most biological pathways, telomere main- success of the ®rst antisense drug provides the impetus tenance may be much more complex than previously for the further development of these strategies for thought. In fact, telomere maintenance may not be selective disruption of telomerase expression (Webb et simply a result of the activation of telomerase activity. al., 1997). Since only the RNA component has been It does appear that all immortalized cell lines have a targeted in the area of telomerase-directed antisense telomere maintenance mechanism, but that mechanism strategies, in future studies it will be valuable to test can be either telomerase or an alternative mechanism the e€ect of an antisense drug designed to target called `alternative lengthening of telomeres' (ALT) hTERT. (Colgin and Reddel, 1999; Niida et al., 2000). There is a growing list of immortalized lines that have no Targeting the telomerase RNA The second essential detectable telomerase activity. Notwithstanding the component of the telomerase enzyme is the RNA absence of telomerase activity, these human cell lines template hTR. The RNA component of telomerase is maintain very long telomeres. This raises a crucial divided into several unique domains that are critical question for the future use of telomerase inhibitors as for normal enzymatic function and assembly of the anticancer agents. If telomerase is inhibited in cancer telomerase enzyme (Betts et al., 1995; Bhattacharyya cells that are maintaining their telomeres by telomer- and Blackburn, 1997; Gilley and Blackburn, 1996, ase, will these cells be able to activate the ALT

Oncogene Targeting telomere maintenance DJ Bearrs et al 6636 pathway and therefore become resistant to the anti- what mechanisms they use, cancer cells need to telomerase agents? This question has yet to be maintain telomeres to remain viable. Therefore, answered in vivo, but it raises the possibility that targeting telomere maintenance mechanisms as a whole resistance mechanisms may be available and selected (instead of just focusing on telomerase) may be a more for in cancer cells treated with telomerase inhibitors. e€ective drug development strategy. With the ongoing However, it is of note that most carcinoma-derived cell discovery of the structure and function of the telomere lines utilize upregulation of telomerase rather than an we now have many ways to target telomere main- alternative mechanism of lengthening telomeres (Bryan tenance mechanisms. et al., 1997; Colgin and Reddel, 1999). In fact, the alternative mechanism of telomere maintenance ap- New strategies to target telomere maintenance pears to be present most often in human ®broblasts mechanisms and other cells of mesenchymal origin and not in human carcinoma cells. Thus, telomerase would appear Targeting T-loops One strategy for targeting the to be an excellent target in human carcinoma cells. telomere maintenance mechanisms in the cell is to take Another confounding issue facing the use of advantage of the distinctive DNA structures that have telomerase inhibitors as anticancer agents comes from been shown to form with telomeric DNA sequences. the lessons learned from the telomerase knock-out The unique structure and dynamics of T-loops found mouse. These mice were generated through targeted at the ends of telomeres provide a number of disruption of the telomerase RNA gene (mTR) in opportunities to selectively target telomere mainte- mouse ES cells (Blasco et al., 1997). The mTR knock- nance mechanisms. The T-loop consists of a duplex 6- out mice have no detectable telomerase activity but base-pair repeat (dATTGGG)n with a G-rich single- show no obvious ill e€ects through the ®rst ®ve stranded end that invades and closes into the duplex successive generations of interbreeding. Interestingly, region to form an as yet undetermined secondary DNA ®broblast cells derived from these mice display structure (Grith et al., 1999). In addition to the progressively shortening telomeres, suggesting that nucleic acid component, a number of proteins, telomerase is necessary for maintenance of mouse including TRF1, TRF2, and probably telomerase, are telomeres, but is dispensable for the ®rst six genera- associated with the T-loop structure (see Figure 1). tions of mice (Blasco et al., 1997). After six generations TRF1 is associated with the duplex telomeric repeat of interbreeding the mice develop some phenotypes, region, and its binding is determined by post- such as male sterility, that indicate that progressive transcriptional modi®cation by the poly(ADP-ribose) telomere shortening causes disruptions in highly polymerase (Smith et al., 1998; Smith and de proliferative organs (Lee et al., 1998). Lange, 1997). TRF2 appears to be associated with the Cancer rates in the telomerase knock-out mice have junction site between the invading single-stranded been extensively examined. Interestingly, instead of telomeric sequence and duplex DNA, although it is having lower cancer rates, as one might expect, these not restricted to this site (see Figure 1) (Broccoli et al., telomerase null mice actually have slightly higher rates of 1997). Because the single-stranded telomeric DNA tumor formation compared to controls (Rudolph et al., sequence is sequestered in the junction with TRF2 1999). How cancer formation is e€ected by loss of and duplex DNA, it is unavailable for telomerase telomerase was also explored by breeding the telomerase extension. Through as yet unde®ned events involving RNA knock-out mice to the knock-out mice for the dissociation of TRF1 and TRF2 from the T-loop, the INK4A and tumor suppressor genes (Chin et al., single-stranded telomeric end can become accessible to 1999; Greenberg et al., 1999). Once again, surprising and telomerase for reverse transcriptase activity, which confounding results were generated by these interbreed- leads to telomeric extension. Thus telomere extension ings. Consistent with their roles as important tumor by telomerase involves both structural and dynamic suppressors, both the INK4A and p53 knock-out mice changes in the T-loop that go beyond the telomerase are highly tumor prone. However, breeding these mice to cycle of elongation and translocation. the telomerase knock-outs resulted in opposite e€ects in The T-loop and postulated D-loop duplex ± single- each model, with the INK4A/mTR null mice displaying strand invasion junction are ideal targets for DNA- reduced tumor formation and the p53/mTR null mice interactive agents. This is because the T-loop has a showing increased tumor formation (Chin et al., 1999; duplex telomeric repeat, providing multiple targets of Greenberg et al., 1999). These results suggest that loss of the same sequence, and also speci®c secondary DNA telomerase activity may have di€erent e€ects on tumor structures associated with the invasion complex. This development and progression based on the genetic presents us with several unique opportunities to background of a given cell. The fact that loss of achieve unusual speci®city with DNA-interactive telomerase activity can promote tumor formation in agents. the presence of mutant p53 is cause for concern of the use Telomeric duplex DNA has a 6-base-pair repeating of telomerase inhibitors in the clinic, especially in sequence (5'-TTAGGG)n, and the binding of TRF1 prolonged treatment schedules. However, how and if dimers may result in the curvature necessary to form these mouse models translate to is still in the T-loop. The c-Myb-like domain of TRF1 bound to question, especially in light of the fact that telomere duplex DNA structure shows major groove binding of maintenance in laboratory mouse cells is already known the protein, leaving the minor groove accessible for to di€er from that of human cells, especially in the area drug modi®cation (Konig et al., 1998). Thus drugs that of maintenance of telomere length (Blasco et al., 1997). interact in the minor groove of DNA and have a With many questions still existing about the validity sequence speci®city that targets them toward telomeric and feasibility of the use of telomerase inhibitors as sequences and distorts DNA structure may well disrupt anticancer agents, it is worth noting that no matter the T-loop structure.

Oncogene Targeting telomere maintenance DJ Bearrs et al 6637 G-quadruplex targets At the very ends of telomeres telomeres, the primary targets of G-quadruplex-inter- are 150- to 200-base single-stranded DNA regions of active agents at this stage are still telomerase and the 3' (G-rich) strand. Because of the unique properties telomeres, primarily due to the lack of data concerning of G-rich sequences of DNA, they can form an other cellular roles (Hurley et al., 2000; Sharma et al., unusual variety of secondary structures known as G- 1997). quadruplexes (Han and Hurley, 2000). Our research group, along with several others, has During the cell cycle, most likely at the end of the S adopted a structure-based design and synthesis ap- phase, it seems reasonable that this single-stranded end proach to the development of lead compounds that must become accessible to telomerase or to other interact with G-quadruplexes, which has been very factors prior to re-sequestration within the T-loop successful. To date, several groups of compounds have junction at the so called D-loop site by TRF2 (see been identi®ed (Figure 3), and their interaction with G- Figure 1). We have proposed that within this window, quadruplexes has been extensively studied. the presence of small molecular weight `driver Anthraquinone analogs were originally developed as molecules' may facilitate the formation of secondary DNA-interactive agents with cytotoxicity to a range of DNA structures such as G-quadruplexes (Figure 2), tumor cell lines (Agbandje et al., 1992). The 2,6- making these ends unavailable for either telomerase or diamido anthraquinones were shown to act as selective sequestration into the D-loop structure (Han and triplex-interactive compounds with reduced anity for Hurley, 2000). duplex DNA (Haq et al., 1999). Molecular modeling A number of groups have been seeking to identify studies predicted that they might bind to G-quadruplex small organic molecules that bind to G-quadruplex structures by a threading intercalation model (Tanious structures with the aim to inhibit telomerase and thus et al., 1992). We ®rst showed by 1H NMR that one of disrupt telomeres (Hurley et al., 2000; Sharma et al., the analogs of the 2,6-diamido anthraquinone BSU- 1997). Although the biological e€ects of G-quadru- 1051 (Figure 3) binds to and stabilizes G-quadruplex plexes might not be restricted to telomerase and structures. We also demonstrated by using the direct telomerase assay that this compound inhibits telomer- ase through its interaction with the intramolecular G- quadruplex formed from the telomeric primer (Sun et al., 1997). A number of anthraquinone analogs have

Figure 2 G-tetrad and G-quadruplexes. (a) Four Figure 3 G-quadruplex-interactive compounds. BSU-1051: 2,6- residues forming a planar structure, G-tetrad through Hoogsteen diamido anthraquinone; TMPyP4: tetra-(N-methyl-4-pyridyl)por- hydrogen bonding. (b) A parallel G-quadruplex model. (c)An phine; TMPyP2: tetra-(N-methyl-2-pyridyl)porphine; PIPER: intermolecular antiparallel G-quadruplex model. (d) An intramo- N,N' - bis[2 - (1 - piperidino)- ethyl]-3,4,9,10-perylenetetracarboxylic lecular foldover G-quadruplex model. Each parallelogram in b, c diimide; DODC: 3,3'-diethyloxyadicarbocyanine; EtBr: ethidium and d represents a G-tetrad bromide

Oncogene Targeting telomere maintenance DJ Bearrs et al 6638 been recently identi®ed to interact with G-quadruplexes and inhibit telomerase, including some of the most potent small-molecule inhibitors of telomerase reported to date (Perry et al., 1999). Porphyrins have long been of particular interest in photodynamic cancer therapy, largely because of their ability to accumulate to a greater extent in tumor tissues rather than in normal tissues (Schuitmaker et al., 1996). The planar arrangement of the aromatic rings in porphyrin analogs led us to propose that such compounds might be able to bind to G-quadruplexes through interactive stacking with the G-tetrads (Wheel- house et al., 1998a,b). By using , circular dichroism, and NMR, it has been shown that the porphyrin analog TMPyP4 (Figure 3) binds to and stabilizes both parallel and antiparallel G-quadruplex structures (Anantha et al., 1998; Wheelhouse et al., 1998a,b). We have further shown that TMPyP4 can inhibit telomerase through G-quadruplex interaction with telomeric DNA, whereas its positional isomer TMPyP2 does not (Figure 3) (Han et al., 1999a; Wheelhouse, 1998a,b). A photocleavage assay revealed that the di€erence in G-quadruplex interaction between Figure 4 NMR-derived structure of the 1 : 1 d(TAGGGTTA)4 ± these two isomers results from their di€erent binding PIPER complex (Briem and Kuntz, 1996). Thymine is shown in sites in G-quadruplexes (Han et al., 1999a). Two cyan, adenine in purple, guanine in yellow, and PIPER in green di€erent G-quadruplex-binding models have been proposed for porphyrins. The ®rst model, based upon photocleavage data, predicts that porphyrin molecules However, one of the most striking properties of this externally stack to G-tetrads located at the ends of G- compound is its driver-like role in the assembly of G- quadruplex, while the other proposes, on the basis of quadruplex structures. PIPER not only converts the molecular modeling and stoichiometry measurements, dimerization reaction of the dimeric G-quadruplex that porphyrin molecules intercalate between two G- formation from second order to ®rst order, but it also tetrads (Han et al., 1999a; Haq et al., 1999). Although increases the initial formation rate about 100-fold at a the spectroscopy data and molecular modeling studies 10 mM concentration (Han et al., 1999b). A similar e€ect support both models, photocleavage data provides the has been observed in the facilitation of G-quadruplex best experimental evidence for the external stacking formation by short G-rich oligonucleotides and by the model (Han et al., 1999a). b-subunit of the Oxytricha telomere binding protein, Comparative studies using tumor cell lines found which is reportedly a G-quadruplex chaperone (Fang that TMPyP4 has a higher slowing e€ect on cell and Cech, 1993; Marco-Haviv et al., 1999). This ®nding growth than the non- quadruplex-interactive com- suggests that in addition to passive binding and pound TMPyP2, which is in accord with the telomerase stabilization, this type of compound may be able to inhibition data (Izbicka et al., 1999a,b). TMPyP4 also play an inductive role in the formation of G-quadruplex induces anaphase chromosomal bridges in sea urchins, structures within cells. In very recent studies we have while TMPyP2 does not (Izbicka, 1999b). This result also shown that PIPER, by binding to G-quadruplexes, indicates that G-quadruplex-interactive compounds inhibits their unwinding by Sgs1, a G-quadruplex- might target the telomeres directly inside cells. speci®c from (Han et al., 2000). Perylene as a potential G-quadruplex-interactive It has been proposed, not unreasonably, that the agent was ®rst identi®ed by using the structure-based invasion of the single-stranded telomeric end into the design program DOCK, developed by Irwin Kuntz at duplex telomeric region may involve a D-loop. It is UCSF (Briem and Kuntz, 1996). On the basis of also conceivable that an alternative secondary structure molecular modeling data, Fedoro€ and co-workers such as a G-quadruplex may be involved. Successful designed and synthesized the N,N'-bis[2-(1-piperidino)- drug targeting of this structure will be dependent upon ethyl] - 3, 4, 9, 10 - perylenetetracarboxylic diimide (PI- identi®cation of this DNA secondary structure; thus, as PER), which contains two positive charges at the ends a prerequisite to drug design, this alternative secondary (Figure 3). This compound turned out to be a very structure will need to be solved. speci®c G-quadruplex-interactive agent that has little interaction with single- or double-stranded DNA. The Posttranslational modification of telomere binding NMR structure of a PIPER ± G-quadruplex complex proteins (Figure 4), the ®rst de®nitive structure of a ligand ± G- quadruplex complex, showed that the binding mode of An attractive feature of the T-loop model is that it PIPER to G-quadruplexes is similar to that proposed provides an explanation for how telomeres escape by us for the porphyrins, i.e. external stacking to G- detection by DNA damage checkpoint proteins. tetrads (Fedoro€ et al., 1998). However, it also seems logical that this structure must As with other G-quadruplex-interactive compounds, be disassembled and reassembled to allow telomere PIPER showed very good telomerase and DNA maintenance mechanisms to function. Figure 5 shows a polymerase inhibitory activities (Fedoro€ et al., 1998). scheme proposed by the Jacobsons for the involvement

Oncogene Targeting telomere maintenance DJ Bearrs et al 6639 DNA. Telomere maintenance mechanisms where tan- kyrase and PARG may be involved include telomere replication, telomere repair, telomere length stabiliza- tion by telomerase, stable integration of telomerase into the telomere, and telomere recombination. The unlimited proliferation potential of cancer cells is closely linked with their ability to maintain stable telomere structures. Thus, the disruption of telomere stability by targeting tankyrase suggests a new approach for cancer treatment. Most inhibitors devel- oped to inhibit PARP-1 act at or near the nicotinamide region of the NAD-binding site of the enzyme, which is likely to have a similar structure in all PARPs. This is supported by the observations that 3-aminobenzamide inhibits the activity of each of the four known PARPs (Ame et al., 1999; de Murcia and Menissier de Murcia, 1994; Kickhoefer et al., 1999). The discovery of Figure 5 A speculative scheme for the involvement of tankyrase multiple PARPs now dictates the design of inhibitors and poly(ADP-ribose) glycohydrolase (PARG) in the disassembly speci®c for di€erent PARPs for both elucidation of and reassembly of a telomere loop structure (Jacobson and Jacobson, 1999). The black lines represent non-telomere DNA function and therapeutic targeting of ADPR polymer and the yellow and red lines represent C-rich and G-rich strands cycles. Finally, PARG may be an important therapeutic of telomere DNA, respectively. The telomere-speci®c proteins target. The dynamic nature of polymer cycles indicates TRF1 and TRF2 form a loop structure to stabilize the G-rich that PARPs and PARG function coordinately, thus overhang. Tankyrase-catalyzed addition of negatively charged ADP-ribose (ADPR) polymers to TRF1 results in dissociation of inhibition (or activation) of PARG is expected to a€ect TRF1 from telomere DNA and disassembly of the loop. In turn, functions modulated by polymer cycles. The low PARG-catalyzed degradation of TRF1-associated polymers homology of PARG to other known proteins and its allows reassembly of the loop. Nam refers to nicotinamide structurally unique substrate suggest that highly selective PARG inhibitors could be developed. Further- more, since each of the known PARPs undergoes of tankyrase and poly(ADP-ribose) glycohydrolase automodi®cation, modulation of PARG might be an (PARG) in the disassembly and reassembly of a e€ective way to modulate these enzymes. telomere loop structure (Jacobson and Jacobson, 1999). The discovery of a component of telomeres contain- Conclusion ing poly(ADP-ribose) polymerase (PARP) activity has provided evidence for a direct involvement of ADP- It is clear that telomere maintenance is important to all ribose polymer cycles in telomere maintenance mechan- dividing cells, including cancer cells. The integrity of isms (Smith et al., 1998). This PARP, termed telomere maintenance mechanisms is essential for tankyrase, is a 142-kDa protein with a catalytic genomic stability and without proper telomere main- domain homologous to PARP-1, but otherwise it has tenance cells are subject to the activation of pathways a very di€erent domain structure. Most notably, leading to cell cycle arrest or cell death. This makes the tankyrase contains 24 tandem repeats of the ankyrin telomere maintenance mechanisms prime targets for motif, a 33 amino acid sequence motif that often links the development of anticancer agents. membrane proteins to the cell cytoskeleton. In contrast Although telomerase activation is not the sole to the better-known PARP-1 and PARP-2, tankyrase mechanism responsible for maintaining telomeric does not appear to require DNA for activity, and it sequences, the enzyme appears to be crucial in the interacts with and catalyzes polymer modi®cation of maintenance of telomeres for most cancer cells. In pre- the telomere-speci®c protein TRF1 in vitro. The clinical studies, telomerase inhibitors have shown domain structure for tankyrase and TRF1 and their promise as e€ective agents for a wide variety of interactions are shown in Figure 5. malignancies, but their usefulness in the clinic have Thus, the telomere loop model suggests a role for yet to be proven. In addition to telomerase, new tankyrase and PARG in disassembly and reassembly of discoveries have shed light on other proteins involved the telomere loop structure as speculated in Figure 5. in the telomere maintenance mechanisms. Identifying The structure of ADP-ribose polymers results in a very and understating how these proteins are involved in high density of negative charges on the polymers, telomere maintenance has added and will continue to making them e€ective polyanions that can compete add to the list of possible targets for therapeutic with DNA for DNA binding proteins. The tankyrase- intervention. Other possible targets for the disruption catalyzed addition of negatively charged polymers to of telomere maintenance are speci®c DNA structures, TRF1 is postulated to result in disassociation of TRF1 such as G-quadruplexes, that can form from telomeric from telomere DNA and disassembly of the loop. In sequences. Results from our group and others indicate turn, PARG-catalyzed degradation of TRF1-associated that G-quadruplex-interactive compounds might target polymers would result in the removal of the competing telomeres and disrupt telomere maintenance in cells, polyanion and thus facilitate reassembly of the loop. making these compounds attractive potential anti- The model shown in Figure 5 is not meant to imply cancer agents. that ADP-ribose polymer cycles necessarily result in While telomerase and telomere maintenance mechan- complete disassociation of the T-loop structure, only isms may not be the universal targets for cancer that that they are capable of increasing access to telomere they once were thought to be, we certainly believe that

Oncogene Targeting telomere maintenance DJ Bearrs et al 6640 they will be important targets in future research aimed (CA67760). Particular thanks are due Drs Mike and Elaine toward a successful strategy for curing cancer. Jacobson for insight into tankyrase and PARG as drug targets. We are grateful to other members of the NCDDG Acknowledgments group for valuable discussions and to Ruben Munoz and Our research involving the targeting of telomerase was Dr David M Bishop for preparing, proofreading, and supported by a National Cooperative Drug Discovery editing the manuscript and ®gures. Group Grant from the National Cancer Institute

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