Targeting the Limitless Replicative Potential of Cancer: The Telomerase/Telomere Pathway
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Molecular Pathways
Targeting the Limitless Replicative Potential of Cancer:
The Telomerase/Telomere Pathway
Lloyd Kelland
Abstract The maintenance of telomeric DNA underlies the ability of tumors to possess unlimited replicative
potential, one of the hallmarks of cancer. Telomere length and structure are maintained by the
reverse transcriptase telomerase and a multiprotein telomere complex termed shelterin. Telomer-
ase activity is elevated in the vast majority of tumors, and telomeres are critically shortened in
tumors versus normal tissues, thus providing a compelling rationale to target the telomerase/
telomere pathway for broad-spectrum cancer therapy. This strategy is supported by a variety of
genetic-based target validation studies. Both telomerase inhibitors and telomere interactive mol-
ecules have shown stand-alone antitumor activity at nontoxic doses against a variety of human
tumor xenografts in mice. These translational advances have resulted in the first antitelomerase
agent, the oligonucleotide-based GRN163L targeting the telomerase RNA template, entering
clinical evaluation. Additional translational approaches, such as targeting telomeres using
G-quadruplex ligands, should result in antitelomere agents, such as RHPS4, entering the clinic in
the near future. These prototype trials will be extremely informative in determining the role of the
telomerase/telomere pathway in clinical oncology and, moreover, whether drugs targeting the
unlimited replicative potential of cancer will find a place in cancer chemotherapy.
angiogenesis, migration, or invasion), and ultimately whether
Background
small-molecule or biological modulators provide a benefit to
Current cancer drug development is largely focused on cancer patients (2). Pioneering studies done over the past 2
combating the major phenotypical features of cancer [i.e., decades have shown that one of the above-described and to
uncontrolled growth (through inactivation of tumor suppres- date therapeutically untapped phenotypic hallmarks of cancer,
sors, activation of oncogenes, and evasion of apoptosis), unlimited replicative potential, is intimately related to the
unlimited replicative potential, formation of new blood vessels maintenance of telomeres, repetitive TTAGGG sequences of
(angiogenesis), and tissue invasion and metastatic spread; DNA located on the ends of human chromosomes. Whereas
ref. 1]. In recent years, this strategy has resulted in some notable mortal cells shorten their telomeres during each round of
success stories with new approved molecularly targeted drugs replication due to the ‘‘end-replication problem,’’ in tumor cells
that have made a significant effect in lengthening the survival of telomere length is stabilized mainly via reactivation of the
cancer sufferers (e.g., imatinib in chronic myelogenous reverse transcriptase termed telomerase, composed of a RNA
leukemia, trastuzumab in breast cancer, and bevacizumab in component (hTR or hTERC) and a catalytic protein (hTERT;
colorectal cancer; ref. 2). see ref. 3 for a recent review). Moreover, the introduction of
hTERT into normal human cells extends their life span (4) and
The Relevance of theTelomere Maintenance hTERT (along with two oncogenes, large T and H-ras)
expression results in the direct tumorigenic conversion of
Pathway to Cancer
normal human epithelial and fibroblast cells (5). Other studies,
Validation of a cancer target encompasses information on the however, suggest that telomerase may also play a role in
prevalence and role of the target or pathway in human cancer, maintaining telomeres in some normal human cells (6) and
whether modulation of the target or pathway in preclinical may possess additional roles to telomere maintenance, such
model systems results in a robust anticancer phenotype (e.g., as regulation of chromatin state and DNA damage responses,
growth inhibition, induction of apoptosis or prevention of as cells lacking hTERT possessed a diminished capacity for
DNA double-strand break repair and fragmented chromosomes
(7). In addition, in some tumors (particularly those of
neuroepithelial and mesenchymal origin), telomeres are main-
Author’s Affiliation: Head of Biology, Cancer ResearchTechnology Development
Laboratory, Wolfson Institute for Biomedical Research, University College London,
tained by an alternative lengthening of telomere mechanism
London, United Kingdom (8). In patients with glioblastoma multiforme tumors, alterna-
Received 2/20/07; revised 3/16/07; accepted 3/20/07. tive lengthening of telomere is associated with mutant TP53
Requests for reprints: Lloyd Kelland, Head of Biology, Cancer Research and a favorable prognosis (9); characteristics of alternative
Technology Development Laboratory, Wolfson Institute for Biomedical Research, lengthening of telomere cells are alternative lengthening of
University College London, London WC1E BBT, United Kingdom. Phone: 44-20-
76796949; E-mail: lkelland@ cancertechnology.com.
telomere – associated promyelocytic leukemia bodies and cir-
F 2007 American Association for Cancer Research. cular extrachromosomal telomeric DNA (telomeric t circles,
doi:10.1158/1078-0432.CCR-07-0422 which require Xrcc3 and Nbs1 for their production; ref. 10).
Clin Cancer Res 2007;13(17) September 1, 2007 4960 www.aacrjournals.org
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Cancer Research.Targeting theTelomerase/Telomere Pathway
Finally, a group (six to date) of telomere-associated proteins, resulted in growth inhibition but this occurred with a different
collectively termed shelterin, comprising TRF1 and TRF2 that lag time dependent on this initial telomere length (15). Such
bind to double-stranded telomeric DNA, POT1 that binds the studies supported the view that, following inhibition of
single-stranded 3¶ G-rich overhang, and three interconnecting telomerase, telomeres needed to be gradually eroded down to
proteins (TIN2, TPP1, and RAP1), acts to shape and safeguard a critical length before any phenotypic effect occurred. Hence,
telomeres (Fig. 1; refs. 11, 12). small-molecule mimics of this effect would be best suited to the
The interest in telomerase and telomeres in a cancer treatment of tumors possessing very short telomeres.
therapeutics context has emerged from two main observations: Subsequent studies, however, targeting components of
first, telomerase activity, typically using a PCR-based assay, telomerase or telomeres/shelterin have shown that much more
telomeric repeat amplification protocol, is elevated in the great rapid apoptosis or induction of senescence effects can occur.
majority (f85%) of tumor types in comparison with normal For example, antisense oligonucleotide-mediated inhibition of
tissues, and second, telomeres are generally shorter in tumors hTERT, but not hTERC, caused a rapid reduction in cell growth
than in corresponding normal tissues (see refs. 13, 14 for and induction of apoptosis without telomere shortening in
reviews). Furthermore, several studies provide genetic valida- human prostate cancer cells (16). Second, the introduction of
tion of telomerase as an anticancer target in that inactivation of mutant template telomerase RNA into human tumor cells
hTR and/or hTERT by dominant-negative mutants or antisense rapidly decreased cell viability and increased apoptosis,
strategies resulted in an inhibition of tumor cell proliferation. possibly via a telomere uncapping mechanism and triggering
For example, transfection of a dominant-negative mutant of a DNA damage response (17, 18). Third, ribozymes targeting
hTERT into cancer cell lines of differing initial telomere length murine telomerase RNA induced antitumor effects, including
Fig. 1. The telomerase/telomere pathway
and main points of possible therapeutic
intervention. Pathway modulators may
result in either mainly telomere erosion
(e.g., BIBR1532) resulting in relatively
slow phenotypic anticancer effects or
predominantly telomere uncapping (e.g.,
RHPS4) producing more rapid anticancer
effects. Agents such as GRN163L probably
mainly affect telomere erosion but also seem
to induce telomere uncapping.
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Cancer Research.Molecular Pathways
reducing melanoma tumor invasion and metastases in vivo; derivative of GRN163, a 13-mer thio-phosphoramidate oligo-
these studies also showed that telomerase activity may control nucleotide complementary to the RNA template of hTR (28).
the glycolytic pathway (19, 20). Finally, other genetic and Although cell-based studies with GRN163 generally showed a
biochemical studies suggest that targeting components of phenotypic lag of at least a few weeks between the onset of
shelterin, such as TRF2 (21) or POT1 (12, 22, 23), or exposing exposure and growth inhibition (e.g., ref. 28), the addition of a
the telomere 3¶ overhang (24) can induce apoptosis or 5¶ lipid (palmitate) moiety in GRN163L increased potency and
telomere-initiated senescence and thereby activate a DNA resulted in a more rapid loss of telomeres and inhibition of cell
damage checkpoint response (25). growth (29). Furthermore, in vivo inhibition of tumor growth
Because telomerase activity is relatively high in many tumors by GRN163L has been reported in mice in A549 human lung
and telomeres are generally shorter in tumors than normal cancer metastases treated with 15 mg/kg thrice weekly for 3
tissues, taken together with the above-described genetic-based consecutive weeks (30). Antitumor activity, observable from 2
validation studies, provide a compelling argument to suggest to 3 weeks of thrice-weekly dosing, has also been reported in
that the telomerase/telomere pathway is a well-validated target mice bearing orthotopic MDA-MB-231 human breast cancer
at the preclinical level. Therefore, appropriate modulators xenografts (31).
should be tested in patients. Over the past decade, many chemical classes of G-quadruplex
ligands have been described, the most potent of which,
Clinical Translational Advances telomestatin, inhibits telomerase in the telomeric repeat
amplification protocol assay at low nanomolar concentrations
As shown in Fig. 1, the burgeoning knowledge about the (32). Other compounds have been rationally designed based
structure of telomeres and the roles of various proteins involved on the crystal structure of parallel quadruplexes from human
in telomere maintenance provides several possible targets for telomeric DNA (33, 34). Several reduce the growth of various
modulation. Some of these, such as modulating the interaction cancer cell lines in vitro after 1 to 2 weeks of exposure to low
of TRF2 or POT1 with telomeres, may hold promise as cancer micromolar concentrations, often accompanied by cellular
drugs but, as yet, have not been described. To date, two areas senescence (34 – 37), apoptosis (32, 36), and/or chromosomal
have received major drug discovery attention: first, the targeting fusions/anaphase bridges (32, 35, 36). Several G-quadruplex
of components of telomerase (e.g., hTR RNA template or ligands have exhibited antitumor activity in vivo at nonacute
hTERT protein), and second, the targeting of telomeres (e.g., toxic doses in mice bearing various human tumor xenografts:
G-quadruplex ligands that induce the formation of guanine- the 3,6,9-trisubstituted acridine BRACO19 in combination with
rich telomeres to stably fold into four-stranded G-quadruplex paclitaxel against vulval carcinoma (34) or as monotherapy
structures, thereby adversely affecting telomere function). against DU145 prostate cancer (38) or UXF1138L uterine
Although, as described above, telomerase represents an cancer (39), the cationic porphyrin TMPyP4 against PC3
attractive cancer target, surprisingly few potent small-molecule prostate cancer (40), telomestatin against U937 acute myelo-
inhibitors have been discovered to date. It is unclear whether monocytic leukemia (41), and the pentacyclic acridine RHPS4
this is because of a general lack of interest in pursuing this against CG5 breast cancer (42). In general, antitumor effects
target within the pharmaceutical industry (perhaps because of were apparent relatively rapidly following the onset of drug
the phenotypic lag arguments described above) or because the treatment, typically after f10 days (34, 38 – 42). A consistent
enzyme is not particularly tractable to the main current drug mechanism of action schema is emerging involving displace-
discovery approach using high-throughput screening of large ment of POT1 and TRF2 from telomeres and erosion of 3¶
compound libraries resulting in few chemical hits or leads single-stranded telomeric overhangs (G-tails) with activation
emerging. One small-molecule nonnucleoside inhibitor of of a DNA damage response [e.g., reported with telomestatin
hTERT, BIBR1532, has been studied in preclinical models, (32, 43, 44) and RHPS4 (42)]. Such findings suggest that the
including in vivo (26, 27). BIBR1532 inhibited telomerase in G-quadruplex ligands can induce relatively rapid telomere
cell-free assays with an IC50 of 93 nmol/L and was shown to be uncapping and may provide an explanation as to why
a mixed-type noncompetitive inhibitor with a drug binding site reductions in telomere length are not always observed in cells
distinct from the sites for deoxyribonucleotides and the DNA exposed to this class of compound. The cell-based effects
primer. Cell-based studies with BIBR1532 revealed a significant (senescence, apoptosis, activation of a DNA damage response
phenotypic lag, typically of f100 days, between the onset of including gH2AX phosphorylation, etc.) are also consistent
telomerase inhibition and cellular growth arrest, hence with modulation of the currently known biological roles of
mimicking the above-described studies with dominant-negative POT1 (23) and TRF2 (21). Finally, measurements of POT1 or
hTERT. Antitumor effects were demonstrable in vivo in mice TRF2 displacement from telomeres or gH2AX phosphorylation
following inoculation of HT1080 fibrosarcoma cells that had may provide sensitive and appropriate biomarkers of target
been previously exposed to the compound for 100 days in vitro modulation in clinical trials. At least some of the G-quadruplex
(and leading to a reduction in telomere length to 1.6 kb). ligands, such as RHPS4, are expected to enter clinical trials in
An oligonucleotide-based molecule that acts as a telomerase the near future.
RNA template antagonist, GRN163L, represents the most
advanced antitelomerase therapeutic. Phase I clinical trials
Concluding Remarks
have been initiated in patients with either chronic lymphocytic
leukemia or solid tumors.1 GRN163L is a more potent The telomerase/telomere field has come a long way in a
relatively short time and is now at a particularly exciting
juncture, as the first telomerase inhibitor, GRN163L, undergoes
1
http://www.clinicaltrials.gov/ early clinical development. Other small-molecule modulators
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Cancer Research.Targeting theTelomerase/Telomere Pathway
of telomerase/telomere biology, notably G-quadruplex ligands a broad-spectrum of cancers, most likely in combination with
such as RHPS4, are in preclinical development and are existing chemotherapeutic drugs. Although there is relatively
anticipated to enter the clinic in the near future. These clinical little to guide rational clinical combinations of antitelomerase/
studies will be extremely informative in determining whether telomere therapeutics with other drugs at present (and
the positive anticancer findings from various genetic-based although dose-limiting toxicities remain unknown), the asso-
preclinical validation studies are borne out in cancer patients. ciation of telomerase/telomeres with DNA repair, particularly
Furthermore, based on the high prevalence of elevated double-strand break repair, pathways is suggestive of this new
telomerase and/or shortened telomeres in multiple tumor types class of agent being usefully combined with cytotoxics that
in comparison with normal tissue counterparts, targeting induce DNA damage (e.g., platins and topoisomerase inhib-
telomerase/telomeres could be applicable to the treatment of itors) or ionizing radiation.
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Cancer Research.Targeting the Limitless Replicative Potential of Cancer: The
Telomerase/Telomere Pathway
Lloyd Kelland
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