There are several telomere or telomerase based anticancer therapies currently in different phases of development. These are illustrated in Figure 1.4, and include:
(A) direct telomerase inhibition, (B) telomerase interference, (C) TERT or TERC promoter driven strategies, (D) telomerase-based immunotherapy and (E) telomere- based approaches.[19, 29] The following sections will discuss each of these approaches.
8 Figure 1.4 Various telomere/telomerase based strategies for killing cancer cells:(A) Direct telomerase inhibition using an oligonucleotide (Imetelstat) to bind to the TERC template; (B) Telomerase interference using reprogrammed telomerase to add mutant telomeric DNA that evokes a DNA-damage response; (C) TERT promoter driven adenovirus genes in an oncolytic virus inducing cellular lysis in cancer cells by viral replication; (D) Telomerase vaccines inducing cytotoxic T lymphocytes; and (E) Telomere-based approach using G- quadruplex stabilisers to prevent telomerase from interacting with its 3´ overhang substrate.
Adapted from various references.[19, 20, 29]
A. Direct telomerase inhibition: This approach involves the use of small molecules such as 2-[(E)-3-naphthalen-2-yl-but-2-enoylamino]-benzoic acid (BIBR1532) (Figure 1.5 (a)) to directly inhibit the activity of telomerase by targeting one of its critical regions.[30] This compound had been shown previously via in vitro and in vivo studies to exhibit a high degree of selective inhibition of telomerase in leukemia cells, compared to normal stem cells.[30] Recently, Bryan and co-workers
AATCCC TTAGGGTTA
TERT-TERC
AAUCCC GGG
3´
G
G G G
Telomerase inhibitors
…(TTAGGG)…
A
E
MT-hTer-47A
B Telomerase interference
A TAGGGTTAGAC A
C
Telomere DNA
Antigen-presenting cell
D
Tumour cell T cell activation for
cell lysis
G
G G G G
G G G
Reprogrammed telomerase Imetelstat
G-quadruplex stabiliser
9 showed that BIBR1532 binds to the thumb domain of TERT, thereby obstructing TERT-TERC assembly and inhibiting the activity of telomerase.[31] However, despite the promising results obtained during preclinical testing, BIBR1532 has not yet entered into clinical trials.[32] Another approach involves modified oligonucleotides such as 5´-TAGGGTTAGACAA-3´ (GRN163L; Imetelstat, Figure 1.5 b), which was designed by Geron Corporation in 2003.[33] Imetelstat interferes with TERC/TERT interactions, by directly binding to TERC,[33] and was found to induce telomere shortening in vitro, resulting in DNA damage and cell death in brain, bladder, prostate, lung, liver and breast cancer cells. This has led to clinical studies for a range of cancer types.[29]
(a) (b)
Figure 1.5 Examples of direct telomerase inhibition agents:(a) BIBR1532; (b) Imetelstat.
Although Imetelstat is the most promising candidate for this kind of therapeutic approach, it has not produced notable results in phase I trials for breast cancer or phase II trials for non-small-cell lung cancer (NSCLC).[34] Full details of the mechanism by which Imetelstat operates are still not clearly understood, since there is no direct or clear correlation between the response of patients in clinical trials and the variation of telomere length in their cancer cells after treatment. The explanation for why there is a therapeutic response in some patients whereas others do not show a response is also not clear.[29] In addition, both small molecule and oligonucleotide
10 based approaches require a long treatment period in order to induce cell death, during which malignant tumours can continue to grow, thereby intensifying the risk of side effects from anticancer drugs.[18]
B. Telomerase interference: This approach to cancer therapy centres on interference with the telomerase expression process, and subsequent blocking of the biogenesis functions of telomerase. This can be achieved through the use of altered RNA template sequences for TERC, such as the Mutant-Template human Telomerase 47A (MT-hTer-47A).[35, 36] In one such study, mutant TERC templates were transfected into cancer cells using lentiviral vectors, resulting in DNA mutations located in telomeric regions.[35, 36] As telomerase could not successfully use these abnormal template sequences, telomere shortening occurred which triggered the DNA damage response, and finally induced senescence and apoptosis in vitro.[35]
Even though this approach has shown a promising ability to eliminate various cancer types in vitro, the accurate expression and introduction of mutant TERC into cancer cells remains an enormous challenge before in vivo evaluation of this approach can commence.[29]
C. TERT/TERC promoter driven strategies: In the majority of benign cells the hTERT genes are inactive, since the DNA promoters initiating transcription of TERT and TERC proteins are deactivated. However, during cancer cell development, specific hTERT promoters are mutated, leading to increased transcription of hTERT and overexpression of telomerase.[18] These observations highlight the potential of therapeutic strategies that selectively target DNA promoter regions, by using either oncolytic virus or suicide gene therapy to infect the cancer cells with engineered adenovirus vectors. Adenovirus genes in the oncolytic virus particles directly induce cellular lysis in cancer cells by viral replication. In comparison, in the suicide gene
11 therapy approach, an enzyme named nitroreductase is produced. This enzyme converts prodrugs such as CB1954 into active cytotoxic molecules that have been shown to cause cell death in different cancer cell lines.[37] The most promising candidate to emerge from oncolytic virus therapy studies is OBP-301 which has now entered a phase I clinical trial for hepatocellular carcinoma.[38] In contrast, suicide gene therapy drugs are yet to reach clinical trials.
D. Telomerase-based immunotherapy: Tumor-Associated Antigens (TAAs) are substances generated in tumor cells that stimulate an immune response in patients. They include peptides and protein fragments derived from the degradation of telomerase by proteasomes that are present on the cancer cell membrane. TAAs are recognised via the human leukocyte antigen (HLA) class I pathway, leading to the release of cytotoxic T cells which can then kill tumor cells.[18] The existence of TAAs in some malignant cells was discovered in the early 1990s, and has now led to a novel immunotherapeutic approach for cancer treatment.[39] Telomerase immunotherapy could be conducted by an in vivo approach involving immune system activation by using injectable peptides such as GV1001. Alternatively, it could be performed using an ex vivo approach in which the dendritic cells of patients are collected and transduced outside the body with mRNA encoding hTERT,[18] and subsequently introduced back into patients via intradermal injections.[29] Several promising examples of this therapeutic approach are currently being tested in clinical trials.[18] Despite this, it must be remembered that immunotherapy approaches to cancer treatment are generally hampered by the low concentration of telomerase present even in cancer cells.[29, 40]
E. Telomere-based approaches: These strategies involve the use of telomere targeting agents such as XAV939, JW55 (Figure 1.6) to interfere with telomeric
12 regions, rather than directly interrupting the activity of telomerase. Therefore, these therapies may even be successful for cancer cells which are telomerase-negative, as the latter can preserve telomere length through the ALT pathway.[29] One approach of this kind involves inhibiting the activity of tankyrase, a poly-ADP ribose polymerase (PARP) protein which plays an important role in the regulation of telomere length. When tankyrase is over expressed in the nucleus of cells, TRF1 is separated from the telomere, and then degraded by a ubiquitin-mediated pathway.[41] This suggests that inhibition of tankyrase will help to maintain the association between TRF1 and telomeres in the T-loop structure, thereby preventing the elongation of telomeric sequences.[29]
(a)
(b)
(c)
(d)
Figure 1.6 Examples of tankyrase inhibitors:(a) XAV939; (b) JW55; (c) flavone; (d) IWR1 (R1
= H) and IWR2 (R1 = CH3).
Many small molecules such as IWR1, IWR2, and flavone shown in Figure 1.6, have been shown to effectively inhibit tankyrase expression,[42] however, none of them has yet produced promising results in clinical trials. These approaches also involve transfecting malignant cells with an 11-mer oligonucleotide (T-oligo) that is analogous to the 3´-single stranded overhang present in telomeres in order to
13 simulate the telomere uncapping process, and elicit a DNA damage response followed by cell growth arrest and apoptosis.[29]
In another example of a telomere-based approach, it has been shown that telomerase functions most effectively when the DNA substrate is present in a double helical or single strand DNA (ssDNA) structure.[19] In view of this observation, and the strong correlation between telomerase activity and cancer cell growth, it is reasonable to suggest that inducing telomeric DNA sequences to form other types of secondary structures such as quadruplex DNA (qDNA or G-quadruplex DNA), may prevent telomerase from interacting with its normal substrate (Figure 1.7).[15, 43]
This would remove the pathway by which cancer cells become effectively “immortal”, thereby making them more susceptible to conventional cancer treatment methods.[43]
Figure 1.7 Inhibition of telomerase activity via stabilisation of G-quadruplex DNA structures in the enzyme’s substrate. Adapted from various references.[20, 22, 44]
5´ 3´
T T A GGG T T A A A T C C C
GGG
T T A GGG A A T CC C
. . .
T T A GGGT T A
T T A GGG T T A
TERT-TERC complex
Inhibition of elongation
X
. . . . . .
No binding + G4 ligand
A A UC C C
A A UC C C 5´ 3´
. . . G
G G G G G G G G G G G 5´
5´
3´
3´
G4 ligand
Elongation of telomeric DNA
14 The validity of the above approach has been shown by a number of investigations, and recently several G-quadruplex stabilising agents have emerged as promising candidates for telomere-based therapy. Many of these compounds show an ability to stabilise G-quadruplex structures formed from proto-oncogenes, e.g. pyridostatin (Figure 1.8 a),[45] or downregulate the expression of oncogenes which are involved in telomere maintenance, e.g. MM41 (Figure 1.8 b).[46] Some of these compounds have been shown to intensify cancer cell senescence in vitro, e.g.
RHPS4 (Figure 1.8 c),[47] or induce shrinkage of xenotransplantation tumors in vivo, e.g. quarfloxin (Figure 1.8 d).[48]
(a) (b)
(c) (d)
Figure 1.8 Examples of qDNA stabilising compounds:(a) pyridostatin; (b) MM41; (c) RHPS4;
(d) quarfloxin.
15 To date, quarfloxin is one of the most successful G-quadruplex stabilising compounds, and was the first such drug to enter phase I and II clinical trials for cancer treatment.[29, 49] It should be noted that quarfloxin was originally designed as a c-myc quadruplex stabilising agent. However, it subsequently was shown to prefer acting on putative quadruplex sequences of ribosomal DNA (rDNA), leading to a significant decrease in tumor cell volume in xenograft models of MIA-PACA-2 pancreatic cancer and MDAMB- 231 breast cancer.[48, 49]