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Imetelstat (GRN163L) is a potent and specific telomerase inhibitor currently in clinical development for the treatment of hematological malignancies such as myelofibrosis and myelodysplastic syndrome. It is a 13-mer N3'-P5' thio-phosphoramidate oligonucleotide covalently functionalized at the 5'-end with a palmitoyl lipid moiety through an aminoglycerol linker. As a competitive inhibitor of human telomerase, imetelstat directly binds to the telomerase RNA component sequence (hTR) in the catalytic site of the enzyme and acts as a direct competitor of human telomere binding. Administration of imetelstat causes progressive shortening of the telomeres, thereby inhibiting malignant cells' proliferation. We report here the ability of imetelstat to form stable, parallel, intermolecular G-quadruplex structures in vitro. The impact of the ionic environment on the formation and stability of imetelstat higher-order structure was investigated through circular dichroism spectroscopy, thermal denaturation analysis, and size-exclusion chromatography. We demonstrated that different structural elements, such as the 5'-palmitoyl linker and the thio-phosphoramidate backbone, critically contribute to G-quadruplex stability. Experiments further showed that G-quadruplex formation does not hamper binding to the hTR oligonucleotide sequence in vitro.

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Imetelstat is a specific and competitive inhibitor of telomerase enzymatic activity. We demonstrated that imetelstat could interfere with the DNA repair process and enhance the effect of DNA damaging agents using hematological tumor cell lines. Short-term administration of imetelstat enhanced growth suppression by anticancer agents and radiation. It also upregulated γH2AX expression induced by irradiation. Immunofluorescence staining showed that both human telomerase reverse transcriptase (hTERT) and γH2AX were upregulated and co-localized in the nucleus of peripheral blood mononuclear cells after irradiation, suggesting that hTERT was involved in the DNA-DSB repair process. Imetelstat enhanced growth inhibitory effect of cytotoxic agents in short-term culture without shortening of telomeres, indicating that this effect was attributed by telomere length independent mechanism. Our results suggest that the combination of short-term treatment with imetelstat and cytotoxic agent is a promising strategy to treat a wide variety of hematopoietic malignancies.

Increased cell proliferation is a hallmark of acute lymphoblastic leukemia (ALL), and genetic alterations driving clonal proliferation have been identified as prognostic factors. To evaluate replicative history and its potential prognostic value, we determined telomere length (TL) in lymphoblasts, B-, and T-lymphocytes, and measured telomerase activity (TA) in leukocytes of patients with ALL. In addition, we evaluated the potential to suppress the in vitro growth of B-ALL cells by the telomerase inhibitor imetelstat. We found a significantly lower TL in lymphoblasts (4.3 kb in pediatric and 2.3 kb in adult patients with ALL) compared to B- and T-lymphocytes (8.0 kb and 8.2 kb in pediatric, and 6.4 kb and 5.5 kb in adult patients with ALL). TA in leukocytes was 3.2 TA/C for pediatric and 0.7 TA/C for adult patients. Notably, patients with high-risk pediatric ALL had a significantly higher TA of 6.6 TA/C compared to non-high-risk patients with 2.2 TA/C. The inhibition of telomerase with imetelstat ex vivo led to significant dose-dependent apoptosis of B-ALL cells. These results suggest that TL reflects clonal expansion and indicate that elevated TA correlates with high-risk pediatric ALL. In addition, telomerase inhibition induces apoptosis of B-ALL cells cultured in vitro. TL and TA might complement established markers for the identification of patients with high-risk ALL. Moreover, TA seems to be an effective therapeutic target, hence, telomerase inhibitors, such as imetelstat, may augment standard ALL treatment.

Telomeres are protective chromosomal ends that shield the chromosomes from DNA damage, exonucleolytic degradation, recombination, and end-to-end fusion. Telomerase is a ribonucleoprotein that adds TTAGGG tandem repeats to the telomeric ends. It has been observed that 85 to 90% of human tumors express high levels of telomerase, playing a crucial role in the development of cancers. Interestingly, the telomerase activity is generally absent in normal somatic cells. This selective telomerase expression has driven scientists to develop novel anti-cancer therapeutics with high specificity and potency. Several advancements have been made in this area, which is reflected by the enormous success of the anticancer agent Imetelstat. Since the discovery of Imetelstat, several research groups have contributed to enrich the therapeutic arsenal against cancer. Such contributions include the application of new classes of small molecules, peptides, and hTERT-based immunotherapeutic agents (p540, GV1001, GRNVAC1 or combinations of these such as Vx-001). Many of these therapeutic tools are under different stages of clinical trials and have shown promising outcomes. In this review, we highlight the current status of telomerase-based cancer therapeutics and the outcome of these investigations.

Background: In cancer evolution, development of telomere maintenance mechanisms represents a critical feature to achieve replicative immortality and prevent cell death after shortening below a critical threshold. Several studies have indicated that excessive telomere shortening or aberrant telomeric repeats are present in cancer genomes, however, their precise role in myeloid malignancies remains largely unexplored. We stipulated that immature leukemia-initiating cells (LIC) would display higher telomerase activity and thus longer telomeres while in mature LIC lower telomerase activity would be associated with shorter telomeres due to excessive division rate. Additional lesions may also lead to disturbed differentiation programs resulting in altered expression of telomerase. Taking advantage of a vast cohort (N=1648) of patients with myeloid neoplasia analyzed by whole genome sequencing (WGS, mean. coverage 100x) we determined telomere length (TL). Aim: We set out to hypothesize that WGS data can be used to reliably measure the TL and that if systematically applied to well defined patient cohorts, we could test whether preservation of TL or its excessive shortening are prognostically or biologically relevant to clinical outcome. Methods: Our cohort included patients with AML (N=732), MDS (N=701) and MPN (N=102 incl. CML, ET, PMF, PV). Healthy subjects (N=11) and patients with the clonal non-malignant HSC disorder PNH (N=102) served as controls. To retrieve TL and telomere repeat heterogeneity via singleton analysis from WGS data we used two independent bioinformatic tools TelomereHunter and Telseq (1A). Results: First, using bidirectional cross-confirmation with independent bioinformatics pipelines, reliability of WGS-based TL measurements (1B) was confirmed and, as anticipated, age-dependency of TL in healthy controls was reproduced (1C). Next we investigated age as a possible confounder of TL shortening in patients. While in PNH age had an expected impact, patients with AML or MDS exhibited TL decoupled from its inherent age-dependence (r=.00034, P=.72). In general, we noted TL shortening as an overarching feature of myeloid proliferation irrespective of entity (1D). Furthermore, mutations in the promotor region of TERT were not correlated with TL. In AML or MDS, neither morphological nor genetic subclassification where associated with differences in TL (1E), with the exception of a small subset of AML and MDS patients (n=103, n=97), harboring abnormally long telomeres compared to healthy subjects. These were significantly associated with mutations in TP53 (AML P=1.91x10-6, mean TL 558.4; MDS P=.55x10-7, mean TL 414.5) but not with any other common mutation. This is surprising, since TP53 mutations are thought to protect cells from telomeric catastrophe following excessive shortening. Moreover, comparing the VAF of TP53mut AML samples with the corresponding measured TL, revealed a positive correlation (r=.2281, P=.021), irrespective of the blast count by cytomorphology (1F). Singleton analysis from all 1648 patients revealed that in AML and MDS patients, alternative telomere repeat- variants (chiefly TTTGGG) were increased pointing towards a possible implication of alternative telomere extension (1G). Lastly, outcome analysis revealed that in AML, the presence of longer telomeres (compared to controls) was associated with worse prognosis (P=.0035) (1H). Multivariate analysis, which included age, karyotype, TP53 status and gender, revealed that this was due to the strong association with TP53 mutations (n=630 vs n=103). The implications of our study extend beyond the realm of telomere biology and leukemia pathophysiology. Therapies targeting the telomere machinery, e.g. imetelstat, may offer an opportunity for application beyond MPN, its current indication. Ongoing studies are currently investigating its use in MDS. Based on our findings we argue that treatment may be rationally evaluated in TP53mut AML. Conclusion: Our findings provide a rationale for (1) the use of WGS to investigate TL, (2) the fundamental discovery that TL shortening is a common principle in myeloid neoplasms, (3) TP53 mutations being accompanied with increased TL and (4) the exploitation of TL to identify subgroups eligible for treatment with imetelstat. Moreover, WGS-based TL analysis in leukemias can contribute to our understanding of TL maintenance mechanisms in leukemic genome. Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.

Imetelstat (GRN163L) is a potent and selective inhibitor of telomerase. We have previously reported that GRN163L could shorten telomeres and limit the lifespan of CD18/HPAF and CAPAN1 pancreatic cancer cells. Here, we examined the effects of GRN163L on two other pancreatic cancer cell lines: AsPC1 and L3.6pl. In both lines, chronic exposure to GRN163L led to an initial shortening of telomeres followed by a stabilization of extremely short telomeres. In AsPC1 cells, telomere attrition eventually led to the induction of crisis and the loss of the treated population. In L3.6pl cells, crisis was transient and followed by the emergence of GRN163L-resistant cells, which could grow at increasing concentrations of GRN163L. The Shelterin complex is a telomere-associated complex that limits the access of telomerase to telomeres. The telomerase inhibitory function of this complex can be enhanced by drugs that block the poly(ADP-ribosyl)ation of its TRF1 and/or TRF2 subunits. Combined treatment of the GRN163L-resistant L3.6pl cells with GRN163L and 3-aminobenzamide (3AB), a general inhibitor of poly(ADP-ribose) polymerases, led to additional telomere shortening and limited the lifespan of the resistant cells. Results from this work suggest that inhibitors of telomerase and poly(ADP-ribose) polymerases can cooperate to limit the lifespan of pancreatic cancer cells.

Engaging a telomere maintenance mechanism during DNA replication is essential for almost all advanced cancers. The conversion from normal and premalignant somatic cells to advanced malignant cells often results (85%-90%) from the reactivation of the functional ribonucleoprotein holoenzyme complex, referred to as telomerase. Modulation of the human telomerase reverse transcriptase (hTERT) appears to be rate limiting to produce functional telomerase and engage a telomere maintenance mechanism. The remaining 10% to 15% of cancers overcome progressively shortened telomeres by activating an alternative lengthening of telomeres (ALT) maintenance mechanism, through a DNA recombination pathway. Exploration into the specific mechanisms of telomere maintenance in cancer have led to the development of drugs such as Imetelstat (GRN163L), BIBR1532, 6-thio-dG, VE-822, and NVP-BEZ235 being investigated as therapeutic approaches for treating telomerase and ALT tumors. The successful use of 6-thio-dG (a nucleoside preferentially recognized by telomerase) that targets and uncaps telomeres in telomerase positive but not normal telomerase silent cells has recently shown impressive effects on multiple types of cancer. For example, 6-thio-dG overcomes therapy-resistant cancers in a fast-acting mechanism potentially providing an alternative or additional route of treatment for patients with cancer. In this perspective, we provide a synopsis of the current landscape of telomeres and telomerase processing in cancer development and how this new knowledge may improve outcomes for patients with cancer.

BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p < 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p < 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p < 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p < 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p < 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p < 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.

Telomere maintenance mechanisms (i.e., telomerase activity (TA) and the alternative lengthening of telomere (ALT) mechanism) contribute to tumorigenesis by providing unlimited proliferative capacity to cancer cells. Although the role of either telomere maintenance mechanisms seems to be equivalent in providing a limitless proliferative ability to tumor cells, the contribution of TA and ALT to the clinical outcome of patients may differ prominently. In addition, several strategies have been developed to interfere with TA in cancer, including Imetelstat that has been the first telomerase inhibitor tested in clinical trials. Conversely, the limited information available on the molecular underpinnings of ALT has hindered thus far the development of genuine ALT-targeting agents. Moreover, whether anti-telomerase therapies may be hampered or not by possible adaptive responses is still debatable. Nonetheless, it is plausible hypothesizing that treatment with telomerase inhibitors may exert selective pressure for the emergence of cancer cells that become resistant to treatment by activating the ALT mechanism. This notion, together with the evidence that both telomere maintenance mechanisms may coexist within the same tumor and may distinctly impinge on patients’ outcomes, suggests that ALT may exert an unexpected role in tumor biology that still needs to be fully elucidated.

Telomeres and telomerase have become attractive targets for the development of anticancer therapeutics due to their involvement in cancer cell immortality. Currently, several therapeutics have been developed that directly target telomerase and telomeres, such as telomerase inhibitors and G-quadruplex stabilizing ligands. Telomere-specific oligonucleotides that reduce telomerase activity and disrupt telomere architecture are also in development as novel anticancer therapeutics. Specifically, GRN163L and T-oligos have demonstrated promising anticancer activity in multiple cancers types via induction of potent DNA damage responses. Currently, several miRNAs have been implicated in the regulation of telomerase activity and may prove to be valuable targets in the development of novel therapies by reducing expression of telomerase subunits. Targeting miRNAs that are known to increase expression of telomerase subunits may be another strategy to reduce carcinogenesis. This review aims to provide a comprehensive understanding of current oligonucleotide-based anticancer therapies that target telomeres and telomerase. These studies may help design novel therapeutic approaches to overcome the challenges of oligonucleotide therapy in a clinical setting.

Live-cell single-molecule imaging demonstrates that long-lasting telomerase–telomere interactions correspond to telomerase ribonucleoproteins that are engaged with the chromosome end. Furthermore, processive telomere elongation by telomerase is shown to be a key contributing factor to telomere length maintenance in vivo., The reverse transcriptase telomerase adds telomeric repeats to chromosome ends to counteract telomere shortening and thereby assures genomic stability in dividing human cells. Key parameters in telomere homeostasis are the frequency with which telomerase engages the chromosome end and the number of telomeric repeats it adds during each association event. To study telomere elongation in vivo, we have established a live-cell imaging assay to track individual telomerase ribonucleoproteins in CRISPR-edited HeLa cells. Using this assay and the drug imetelstat, which is a competitive inhibitor of telomeric DNA binding, we demonstrate that stable association of telomerase with the single-stranded overhang of the chromosome end requires telomerase-DNA base pairing. Furthermore, we show that telomerase processivity contributes to telomere elongation in vivo. Together, these findings provide new insight into the dynamics of telomerase recruitment and the importance of processivity in maintaining telomere length in human cancer cells.

Telomerase is the ribonucleoprotein reverse transcriptase that catalyzes the synthesis of telomeres at the ends of linear chromosomes and contributes to proper telomere-loop (T-loop) formation. Formation of the T-loop, an obligate step before cell division can proceed, requires the generation of a 3′-overhang on the G-rich strand of telomeric DNA via telomerase or C-strand specific nucleases. Here, it is discovered that telomerase activity is critical for efficient cell-cycle progression using transient chemical inhibition by the telomerase inhibitor, imetelstat. Telomerase inhibition changed cell cycle kinetics and increased the proportion of cells in G2-phase, suggesting delayed clearance through this checkpoint. Investigating the possible contribution of unstructured telomere ends to these cell-cycle distribution changes, it was observed that imetelstat treatment induced γH2AX DNA damage foci in a subset of telomerase-positive cells but not telomerase-negative primary human fibroblasts. Chromatin-immunoprecipitation with γH2AX antibodies demonstrated imetelstat treatment-dependent enrichment of this DNA damage marker at telomeres. Notably, the effects of telomerase inhibition on cell cycle profile alterations were abrogated by pharmacological inhibition of the DNA-damage-repair transducer, ATM. Also, imetelstat potentiation of etoposide, a DNA-damaging drug that acts preferentially during S–G2 phases of the cell cycle, depends on functional ATM signaling. Thus, telomerase inhibition delays the removal of ATM-dependent DNA damage signals from telomeres in telomerase-positive cancer cells and interferes with cell cycle progression through G2. Implications: This study demonstrates that telomerase activity directly facilitates the progression of the cell cycle through modulation of transient telomere dysfunction signals. Mol Cancer Res; 16(8); 1215–25. ©2018 AACR.

The reverse transcriptase telomerase adds telomeric repeats to chromosome ends to counteract telomere shortening and thereby assures genomic stability in dividing human cells. Key variables in telomere homeostasis are the frequency with which telomerase engages the chromosome end and the number of telomeric repeats it adds during each association event. To study telomere elongation in vivo we have established a live-cell imaging assay to track individual telomerase RNPs in HeLa cells. Using this assay and the drug imetelstat, which is a competitive inhibitor of telomeric DNA binding, we demonstrate that stable association of telomerase with the single-stranded overhang of the chromosome end requires telomerase-DNA base-pairing. Furthermore, we show that telomerase processivity contributes to telomere elongation in vivo. Together, these findings provide new insight into the dynamics of telomerase recruitment and the importance of processivity in maintaining telomere length in human cancer cells.

Imetelstat, a novel telomerase inhibitor, failed to improve significantly median PFS and OS as maintenance therapy (±bevacizumab) in advanced NSCLC. Telomere length (TL) biomarker results were consistent with the hypothesis that telomerase inhibition is of greater benefit to patients with tumors possessing shorter telomeres; the patients with shorter TL had a trend toward longer median PFS and OS. Background Continuation or ‘switch’ maintenance therapy is commonly used in patients with advancd non-small-cell lung cancer (NSCLC). Here, we evaluated the efficacy of the telomerase inhibitor, imetelstat, as switch maintenance therapy in patients with advanced NSCLC. Patients and methods The primary end point of this open-label, randomized phase II study was progression-free survival (PFS). Patients with non-progressive, advanced NSCLC after platinum-based doublet (first-line) chemotherapy (with or without bevacizumab), any histology, with Eastern Cooperative Oncology Group performance status 0–1 were eligible. Randomization was 2 : 1 in favor of imetelstat, administered at 9.4 mg/kg on days 1 and 8 of a 21-day cycle, or observation. Telomere length (TL) biomarker exploratory analysis was carried out in tumor tissue by quantitative PCR (qPCR) and telomerase fluorescence in situ hybridization. Results Of 116 patients enrolled, 114 were evaluable. Grade 3/4 neutropenia and thrombocytopenia were more frequent with imetelstat. Median PFS was 2.8 and 2.6 months for imetelstat-treated versus control [hazard ratio (HR) = 0.844; 95% CI 0.54–1.31; P = 0.446]. Median survival time favored imetelstat (14.3 versus 11.5 months), although not significantly (HR = 0.68; 95% CI 0.41–1.12; P = 0.129). Exploratory analysis demonstrated a trend toward longer median PFS (HR = 0.43; 95% CI 0.14–1.3; P = 0.124) and overall survival (OS; HR = 0.41; 95% CI 0.11–1.46; P = 0.155) in imetelstat-treated patients with short TL, but no improvement in median PFS and OS in patients with long TL (HR = 0.86; 95% CI 0.39–1.88; and HR = 0.51; 95% CI 0.2–1.28; P = 0.145). Conclusions Maintenance imetelstat failed to improve PFS in advanced NSCLC patients responding to first-line therapy. There was a trend toward a improvement in median PFS and OS in patients with short TL. Short TL as a predictive biomarker will require further investigation for the clinical development of imetelstat.

Cancer stem cells (CSCs) are thought to be responsible for tumor progression, metastasis, and recurrence. HER2 overexpression is associated with increased CSCs, which may explain the aggressive phenotype and increased likelihood of recurrence for HER2(+) breast cancers. Telomerase is reactivated in tumor cells, including CSCs, but has limited activity in normal tissues, providing potential for telomerase inhibition in anti-cancer therapy. The purpose of this study was to investigate the effects of a telomerase antagonistic oligonucleotide, imetelstat (GRN163L), on CSC and non-CSC populations of HER2(+) breast cancer cell lines. The effects of imetelstat on CSC populations of HER2(+) breast cancer cells were measured by ALDH activity and CD44/24 expression by flow cytometry as well as mammosphere assays for functionality. Combination studies in vitro and in vivo were utilized to test for synergism between imetelstat and trastuzumab. Imetelstat inhibited telomerase activity in both subpopulations. Moreover, imetelstat alone and in combination with trastuzumab reduced the CSC fraction and inhibited CSC functional ability, as shown by decreased mammosphere counts and invasive potential. Tumor growth rate was slower in combination-treated mice compared to either drug alone. Additionally, there was a trend toward decreased CSC marker expression in imetelstat-treated xenograft cells compared to vehicle control. Furthermore, the observed decrease in CSC marker expression occurred prior to and after telomere shortening, suggesting that imetelstat acts on the CSC subpopulation in telomere length-dependent and -independent mechanisms. Our study suggests addition of imetelstat to trastuzumab may enhance the effects of HER2 inhibition therapy, especially in the CSC population.

Telomerase is the ribonucleoprotein reverse transcriptase that catalyzes the synthesis of telomeres at the ends of linear human chromosomes and contributes to proper telomere-loop (T-loop) formation. Formation of the T-loop, an obligate step before cell division can proceed, requires the generation of a 3’-overhang on the G-rich strand of telomeric DNA via telomerase or C-strand specific nucleases. Here, we discover telomerase activity is critical for efficient cell cycle progression using transient chemical inhibition by the telomerase inhibitor imetelstat. Telomerase inhibition caused changes in cell cycle kinetics and increased the proportion of cells in G2 phase, suggesting delayed clearance through this checkpoint. Investigating the possible contribution of unstructured telomere ends to these cell cycle distribution changes, we observed that imetelstat treatment induced γH2AX DNA damage foci in a subset of telomerase-positive cells but not telomerase-negative primary human fibroblasts. Chromatin-immunoprecipitation with γH2AX antibodies demonstrated imetelstat treatment-dependent enrichment of this DNA damage marker at telomeres. Notably, the effects of telomerase inhibition on cell cycle profile alterations were abrogated by pharmacological inhibition of the DNA-damage-repair transducer ATM.Additionally, imetelstat potentiation of etoposide, a DNA-damaging drug that acts preferentially during S/G2 phases of the cell cycle, also depended on functional ATM signaling. Our results suggest that telomerase inhibition delays the removal of ATM-dependent DNA damage signals from telomeres in telomerase-positive cancer cells. This demonstrates for the first time that telomerase activity directly facilitates the progression of the cell cycle through modulation of transient telomere dysfunction signals.

Overview Finding specific targeted agents for cancer therapy remains a challenge. One of the hallmarks of cancer is the limitless proliferation (immortalization) of cancer cells, which correlates with the activation of the ribonucleoprotein enzyme complex called telomerase. As 85–90% of primary human cancers express telomerase activity, while most normal cells do not, telomerase is a unique and almost universal therapeutic target for cancer. Although there have been several approaches developed for telomerase-targeted therapies, there is still no agent that has been approved. This chapter focuses on the pros and cons of the different approaches to target telomerase and summarizes preclinical and clinical results. We will also review novel telomerase-mediated telomere uncapping approaches that target telomerase-expressing cancer cells, but not normal cells. Keywords: telomere; cancer therapy; G-quadruplex; immunotherapy; vaccine; targeted gene therapy; small-molecule inhibitor; Imetelstat; GRN163L; 6-thio-2′-deoxyguanosine

Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1327-6) contains supplementary material, which is available to authorized users.

Telomerase is a ribonucleoprotein that catalyses the addition of telomeric repeat sequences (having the sequence 5'-TTAGGG-3' in humans) to the ends of chromosomes. Telomerase activity is detected in most types of human tumours, but it is almost undetectable in normal somatic cells. Therefore, telomerase is a promising therapeutic target. To date, the known inhibitors of telomerase include nucleoside analogues, oligonucleotides and G-quadruplex stabilizers. This review highlights recent advances in our understanding of telomerase inhibitors, the relationships between telomerase inhibitors, cancer, and fields such as inflammation.This review summarizes new patents published on telomerase inhibitors from 2010 to 2015.The review provides a brief account of the background, development, and on-going issues involving telomerase inhibitors. In particular, this review emphasizes imetelstat (GRN163L) and some typical G-quadruplex stabilizers that participate in telomerase inhibition. Overall, the research scope of antineoplastic is becoming broader and telomerase inhibitors have been shown to be a promising therapeutic target. Therefore, novel antineoplastic agents with greater activity and higher specificity must be developed.

Purpose: Telomerase activity is one of the hallmarks of cancer and is a highly relevant therapeutic target. The effects of a novel human telomerase antagonist, imetelstat, on primary human glioblastoma (GBM) tumor-initiating cells were investigated in vitro and in vivo. Experimental Design: Tumor-initiating cells were isolated from primary GBM tumors and expanded as neurospheres in vitro. The GBM tumor-initiating cells were treated with imetelstat and examined for the effects on telomerase activity levels, telomere length, proliferation, clonogenicity, and differentiation. Subsequently, mouse orthotopic and subcutaneous xenografts were used to assess the in vivo efficacy of imetelstat. Results: Imetelstat treatment produced a dose-dependent inhibition of telomerase (IC50 0.45 μmol/L). Long-term imetelstat treatment led to progressive telomere shortening, reduced rates of proliferation, and eventually cell death in GBM tumor-initiating cells. Imetelstat in combination with radiation and temozolomide had a dramatic effect on cell survival and activated the DNA damage response pathway. Imetelstat is able to cross the blood-brain barrier in orthotopic GBM xenograft tumors. Fluorescently labeled GBM tumor cells isolated from orthotopic tumors, following systemic administration of imetelstat (30 mg/kg every day for three days), showed ∼70% inhibition of telomerase activity. Chronic systemic treatment produced a marked decrease in the rate of xenograft subcutaneous tumor growth. Conclusion: This preclinical study supports the feasibility of testing imetelstat in the treatment of GBM patients, alone or in combination with standard therapies. Clin Cancer Res; 16(1); 154–63

Telomerase is an attractive cancer target as it appears to be required in essentially all tumours for immortalization of a subset of cells, including cancer stem cells. Moreover, differences in telomerase expression, telomere length and cell kinetics between normal and tumour tissues suggest that targeting telomerase would be relatively safe. Clinical trials are ongoing with a potent and specific telomerase inhibitor, GRN163L, and with several versions of telomerase therapeutic vaccines. The prospect of adding telomerase-based therapies to the growing list of new anticancer products is promising, but what are the advantages and limitations of different approaches, and which patients are the most likely to respond?

The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of γH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p = 0.045, alvespimycin, p = 0.034; combined treatment, p = 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy.

Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy.

The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3'-->P5' thio-phosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first- (GRN163) and second-generation (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipid-conjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Malignant gliomas, including anaplastic astrocytoma and glioblastoma, are the most common malignant primary brain tumors in adults. Patients with these tumors typically undergo surgery followed by radiation therapy and chemotherapy (Levin et al., 1990; Recht et al., 1990). Despite aggressive therapy, patients with glioblastoma survive only about one year after diagnosis (Burger et al., 1990; Miller et al., 1990; Shibamoto et al., 1990), and patients with anaplastic astrocytoma usually survive only a few years (Burger et al., 1990). Clearly, there is a great need for effective therapies that will preferentially target these tumors and spare normal tissues. Telomerase is a specialized ribonucleoprotein polymerase that contains the integral RNA component hTR with a short template segment that directs the synthesis of telomeric repeats at the ends of chromosomes (Greider and Blackburn, 1985; Yu et al., 1990). Human chromosomes terminate with several kilobases of the simple telomere hexanucleotide repeat d(TTAGGG)n. Telomerase elongates telomeric DNA by these repeats and plays an important role in cellular immortalization. The vast majority of normal somatic cells either do not express telomerase activity or express it at very low levels. Therefore, gradual telomeric shortening occurs at each cell division, and this eventually leads to death of the cell. In contrast, telomerase is expressed and functionally active in a wide variety of human tumors, including bladder, breast, prostate, and head and neck tumors (Hiyama et al., 1995; Kim et al., 1994; Lin et al., 1996; Mao et al., 1996; Sommerfeld et al., 1996; Sugino et al., 1996). Thus, cancer cell DNA is continuously extended or maintained by telomerase to compensate for the loss of telomeric repeats (Counter et al., 1992), and as a result the cells become immortalized. Telomerase is also expressed in the vast majority of primary brain tumors but not in normal brain tissues (Chong et al., 1998; Langford et al., 1995; Le et al., 1998; Mori et al., 1997; Sano et al., 1998). For example, Le et al. (1998) found telomerase activity in 89% of glioblastomas and 45% of anaplastic astrocytomas, but it was absent in normal brain tissues. Therefore, inhibition of telomerase provides a therapeutic strategy for selectively targeting malignant gliomas and sparing normal brain tissue. It is important to note that a group of gliomas with exceptionally long telomeres, >17 kb, have been identified (Hakin-Smith et al., 2003). These tumors are thought to maintain their telomeres by a mechanism termed alternative lengthening of telomeres, and these tumors can be either telomerase positive or telomerase negative. Thus telomere length, by itself, is not a definitive indication of telomerase activity. The 13-mer oligonucleotide N3′→ P5′ thio-phosphoramidate GRN163 (Geron Corporation, Menlo Park, Calif.), which binds to the telomerase RNA subunit hTR at the template region (Gryaznov et al., 2001; Herbert et al., 2002), has shown potent inhibitory activity against human telomerase in several biochemical assays. In vitro, telomerase inhibition by GRN163 induces cellular senescence and apoptosis in several human tumor cell lines, and these effects are accompanied by telomere shortening (Herbert et al., 2002). We report experimental evidence showing that GRN163 inhibited the growth of s.c. human U-251 MG glioblastoma xenografts in mice. The compound also exhibited favorable i.c. uptake and biodistribution properties, prevented the growth of U-251 MG cells in rats, and increased the survival times of rats bearing i.c. tumors.

Novel treatment approaches are desperately needed for malignant rhabdoid tumor (MRT). Telomerase is an attractive therapeutic target because it is specific to cancer and critical for cancer cell immortality. We evaluated the effect of the telomerase inhibitor imetelstat in preclinical models of MRT. Three MRT cell lines, BT-12, G401, and RT-peri, were treated with the telomerase inhibitor imetelstat. The effects of imetelstat on telomere length, DNA damage response, and cell proliferation were assessed. The efficacy of imetelstat in vivo was evaluated in subcutaneous xenografts derived from each of the cell lines. Treatment with imetelstat resulted in inhibition of telomerase activity, marked telomere shortening, and activation of the DNA damage response pathway, as measured by formation of γ-H2AX nuclear foci, phosphorylation of ATM, and phosphorylation of TP53. Imetelstat-treated G401 cells underwent complete growth arrest after 16 passages. The other two cell lines exhibited growth inhibition. Imetelstat resulted in 40–50% growth inhibition compared to placebo-treated controls in all three xenograft models. The activity of imetelstat as a single agent suggests that further studies of telomerase inhibitors in combination with other agents may be warranted.

Background/Aims: Myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF), are clonal disorders characterized by unlimited proliferation of hematopoietic cells. Besides the dominant clone, in the majority of cases defined by a driver mutation (JAK2V617F, CALR or MPL mutation), sub-clones carrying additional mutations (e. g. in the genes ASXL1, CBL, DNMT3A, EZH2, IDH1, U2AF1, SF3B1, TET2, TP53) and/or normal polyclonal hematopoiesis coexist. Due to the loss of telomere sequences with each cell division, the length of telomeres can be used to describe clonal dynamics of hematopoietic cells. In neoplastic cells with a high mitotic rate shorter telomeres are typically found compared to the telomere length (TL) in normal counterparts. In the ET phase 2 study with the telomerase inhibitor imetelstat we demonstrated rapid and durable hematologic and molecular responses (Baerlocher et al. N Engl J Med 2015) and suppression of clones with non-driver mutations (Oppliger Leibundgut et al, ASH 2015). Here, our aims were 1) to evaluate the TL in subsets of leukocytes from patients (pts) with MPN compared to healthy individuals and 2) to study the TL dynamics in ET pts treated with imetelstat and to correlate those with hematologic and molecular responses. Patients and Methods: Blood samples from 63 standard of care (SOC)-treated or untreated pts with MPN (15 ET, 36 PV, 12 MF) diagnosed according to WHO 2008 criteria as well as from 14 ET pts treated with imetelstat after failure or intolerance to ≥1 prior therapy were analyzed for TL. TL percentiles from over 400 healthy individuals served as reference. Leukocytes were extracted from the peripheral blood and TL was measured in subsets of leukocytes by automated multicolor flow-FISH (Baerlocher et al, Nat Protoc. 2006). Results: 81% of the 62 MPN pts (11/15 ET, 32/35 PV, 7/12 MF) carried a JAK2 mutation, 6 (1 ET, 5 MF) a CALR mutation, 1 ET pt had a MPL mutation, 2 ET pts were triple negative and 3 PV pts were JAK2 wild type. 23 pts were treated with phlebotomy, 32 with hydroxyurea, 3 with anagrelide and 3 with IFN-alpha. The median age at study entry was 62 yrs (23-89, ET/PV/MF 64/58/66 yrs) and the median time since diagnosis was 2.7 yrs (0.3-14.2) in the ET SOC cohort. In the imetelstat cohort, 9/14 ET pts carried a JAK2V617F mutation and 5 a CALR mutation. The median age at study entry was 60.5 yrs (21-80) and the median time since diagnosis was 6.7 yrs (0.3-21). The telomere length values (TLV) in granulocytes from the 63 MPN pts on SOC were around the 10th percentile for pts with ET and PV and below the 1st percentile for most pts with MF. In the imetelstat cohort, 8 of 14 ET pts with a median of 2 prior therapies demonstrated TLV in granulocytes Conclusions: The lower TLV found in granulocytes of pts with MPN and especially with MF compared to healthy individuals reflect the higher mitotic history of malignant clones. In the ET imetelstat cohort, the very low TLV found at baseline might result from longer disease duration and resistance and/or intolerance to prior therapies. The higher TLV observed after 9 months of treatment with imetelstat and correlation with the reduction in the driver mutational burden suggests that imetelstat treatment in ET pts may suppress neoplastic clones and, in the absence of a high additional mutational burden, favor recovery of normal hematopoiesis. Disclosures Oppliger Leibundgut: Geron: Research Funding; Novartis: Research Funding. Burington:Geron Corporation: Employment. Ottmann:Fusion Pharma: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Spitzer:Trovagene Inc: Consultancy; Moldx Palmetto GBA: Consultancy; Incyte: Consultancy. Odenike:CTI/Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Algeta Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Suneisis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Research Funding. McDevitt:Salamandra Inc: Consultancy; Alexion: Membership on an entity's Board of Directors or advisory committees. Röth:Alexion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Research Funding. Snyder:Ariad: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Baerlocher:Geron: Research Funding; Janssen: Research Funding; Novartis: Research Funding.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

Telomerase is a cellular ribonucleoprotein reverse transcriptase that plays a crucial role in telomere maintenance. This enzyme is expressed in approximately 90% of human tumors, but not in the majority of normal somatic cells. Imetelstat sodium (GRN163L), is a 13-mer oligonucleotide N3'-> P5' thio-phosphoramidate lipid conjugate, which represents the latest generation of telomerase inhibitors targeting the template region of the human functional telomerase RNA (hTR) subunit. In preclinical trials, this compound has been found to inhibit telomerase activity in multiple cancer cell lines, as well as in vivo xenograft mouse models. Currently, GRN163L is being investigated in several clinical trials, including a phase II human non-small cell lung cancer clinical. trial, in a maintenance setting following standard doublet chemotherapy. In addition to the inhibition of telomerase activity in cancer cell lines, GRN163L causes morphological cell rounding changes, independent of hTR expression or telomere length. This leads to the loss of cell adhesion properties; however, the mechanism underlying this effect is not yet fully understood. In the present study, we observed that GRN163L treatment leads to the loss of adhesion in A549 lung cancer cells, due to decreased E-cadherin expression, leading to the disruption of the cytoskeleton through the alteration of actin, tubulin and intermediate filament organization. Consequently, the less adherent cancer cells initially cease to proliferate and are arrested in the G1 phase of the cell cycle, accompanied by decreased matrix metalloproteinase-2 (MMP-2) expression. These effects of GRN163L are independent of its telomerase catalytic activity and may increase the therapeutic efficacy of GRN163L by decreasing the adhesion, proliferation and metastatic potential of cancer cells in vivo.

Telomerase is a ribonucleoprotein that synthesizes telomere repeats onto chromosome ends and is involved in maintaining telomere length in germline tissues and in immortal and cancer cells. In the present study, the temporal regulation of expression of telomerase activity was examined in human germline and somatic tissues and cells during development. Telomerase activity was detected in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes. Blastocysts expressed high levels of telomerase activity as did most human somatic tissues at 16-20 weeks of development with the exception of human brain tissue. This activity could no longer be detected in the somatic tissues examined from the neonatal period onward. Neither placenta nor cultured fetal amniocytes contained detectable telomerase activity. Fetal tissues explanted into primary cell culture showed a dramatic decline in telomerase activity which became undetectable after the first passage in vitro. Elucidation of the regulatory pathways involved in the repression of telomerase activity during development may lead to the ability to manipulate telomerase levels and explore the consequences both for cellular aging and for the survival of cancer cells.

Telomeres protect DNA sequences that form the ends of chromosomes. Telomeric maintenance is a prerequisite for the indefinite proliferation of cells, cell immortalization and neoplastic growth. Telomere dysfunction also promotes chromosomal fusions that facilitate the development of human cancers. Telomerase is an active enzyme which appears in embryonic development and down-regulated in all human somatic cells prior to birth. Telomerase is transiently active in normal somatic cells throughout life and is elevated in more than 85 % of human cancers but not benign tissues. Also, expression of human telomerase reverse transcriptase (hTERT) is up-regulated in almost all human cancers but not in normal tissues. The level of telomerase activity and hTERT expression are useful diagnostic and prognostic indicator for human cancers. Progressive telomere shortening predominantly occurs during early carcinogenesis before telomerase activation. The telomere dysfunction promotes chromosomal fusions that facilitate the development of carcinomas in the p53 deficiency cells in human cancers. The activated telomerase maintains telomere length stability in almost all cancer cells. The gene encoding the endogenous telomerase inhibitor PinX1 (PIN2/TRF1), is located at human chromosome 8p23, a region frequently exhibiting heterozygosity in many common human cancers. When PinX1 binds to hTERT, it will inhibit the telomerase activity. Proteins TRF-1 and TRF-2 are negative regulators, but Tankyrase is a positive regulator of human cancers. This suggests that PinX1 could be considered as a putative tumor suppressor genes and telomerase inhibitors and considered to be used in treating cancers with highly expressed telomerase. Also, GRN163L(Imetelstat), Cisplatin, Vincristine and AZT drugs have shown potentials to decrease the telomerase activity in leukemia, lymphomas, carcinomas and sarcomas. The telomerase reactivation is vital for initiation of all human malignancies since it removes the short telomere barriers inhibitory for tumor progression. Thus, telomerase is useful biomarker for detection of cancer and aging progression. This chapter will describe the molecular characterization and regulation of telomerase activity in cancer and aging as well as its therapeutic effects.

Telomerase is mainly active in human tumor cells, which provides an opportunity for a therapeutic window on telomerase targeting. We sought to evaluate the potential of the thio-phosphoramidate oligonucleotide inhibitor of telomerase, imetelstat, as a drug candidate for treatment of esophageal cancer. Our results showed that imetelstat inhibited telomerase activity in a dose-dependent manner in esophageal cancer cells. After only 1week of imetelstat treatment, a reduction of colony formation ability of esophageal cancer cells was observed. Furthermore, long-term treatment with imetelstat decreased cell growth of esophageal cancer cells with different kinetics regarding telomere lengths. Short-term imetelstat treatment also increased γ-H2AX and 53BP1 foci staining in the esophageal cancer cell lines indicating a possible induction of DNA double strand breaks (DSBs). We also found that pre-treatment with imetelstat led to increased number and size of 53BP1 foci after ionizing radiation. The increase of 53BP1 foci number was especially pronounced during the first 1h of repair whereas the increase of foci size was prominent later on. This study supports the potential of imetelstat as a therapeutic agent for the treatment of esophageal cancer.

Specific information about how telomerase acts in vivo is necessary for understanding telomere dynamics in human tumor cells. Our results imply that, under homeostatic telomere length-maintenance conditions, only one molecule of telomerase acts at each telomere during every cell division and processively adds ∼60 nt to each end. In contrast, multiple molecules of telomerase act at each telomere when telomeres are elongating (nonequilibrium conditions). Telomerase extension is less processive during the first few weeks following the reversal of long-term treatment with the telomerase inhibitor Imetelstat (GRN163L), a time when Cajal bodies fail to deliver telomerase RNA to telomeres. This result implies that processing of telomerase by Cajal bodies may affect its processivity. Overexpressed telomerase is also less processive than the endogenously expressed telomerase. These findings reveal two major distinct extension modes adopted by telomerase in vivo.

Purpose: Cancer recurrence is one of the major setbacks in oncology. Maintaining telomeres is essential for sustaining the limitless replicative potential of such cancers. Because telomerase is thought to be active in all tumor cells and normal stem cells, telomerase inhibition may be nonspecific and have detrimental effects on tissue maintenance and development by affecting normal stem cell self-renewal. Methods: We examined telomerase activity, telomere maintenance, and stem cell maturation in tumor subpopulations from freshly resected gliomas, long-term, primary, neural tumor-initiating cells (TIC) and corresponding normal stem cell lines. We then tested the efficacy of the telomerase inhibitor Imetelstat on propagation and self-renewal capacity of TIC and normal stem cells in vitro and in vivo. Results: Telomerase was undetectable in the majority of tumor cells and specific to the TIC subpopulation that possessed critically short telomeres. In contrast, normal tissue stem cells had longer telomeres and undetectable telomerase activity and were insensitive to telomerase inhibition, which results in proliferation arrest, cell maturation, and DNA damage in neural TIC. Significant survival benefit and late tumor growth arrest of neuroblastoma TIC were observed in a xenograft model (P = 0.02). Furthermore, neural TIC exhibited irreversible loss of self-renewal and stem cell capabilities even after cessation of treatment in vitro and in vivo. Conclusions: TIC exhaustion with telomerase inhibition and lack of telomerase dependency in normal stem cells add new dimensions to the telomere hypothesis and suggest that targeting TIC with telomerase inhibitors may represent a specific and safe therapeutic approach for tumors of neural origin. Clin Cancer Res; 17(1); 111–21. ©2011 AACR.

Prostate adenocarcinoma is the most common type of cancer found in men, being second to lung cancer in terms of morbidity. While there are several therapeutic options (surgery, radiation) for the patients with localized disease, the patients with metastatic disease have only a few therapeutic options, all with limited efficacy. Therefore it is important to developed and implement new chemotherapeutic drugs for the treatment of prostate cancer. One of these novel therapeutic agents is imetelstat sodium (GRN163L), an oligonucleotide antagonist which targets the telomerase. Re-activation of telomerase, the enzyme responsible for maintenance of telomeres, is one of the hallmarks of cancer and more than 90% of prostate carcinomas possess this phenotype. In the absence of telomerase, cancer cells will progressively shorten their telomeres to a critical length and die. The majority of conventional chemotherapies are believed to leave intact small population of cells, known as tumor-initiating cells (or cancer stem cells). These cells are believed to be responsible for the subsequent relapse and metastasis of the disease and specific therapies which target these cells may achieve a more durable response. If tumorinitiating cells have telomerase activity, the imetelstat drug would target these cells in addition to the bulk tumor cells. In this study we are assessing the anti-metastatic effects of imetelstat by demonstrating that this drug targets the rare populations of tumorinitiating cells by inducing telomerase inhibition and telomere shortening.

Telomeres and telomerase play essential roles in the regulation of the lifespan of human cells. While normal human somatic cells do not or only transiently express telomerase and therefore shorten their telomeres with each cell division, most human cancer cells typically express high levels of telomerase and show unlimited cell proliferation. High telomerase expression allows cells to proliferate and expand long-term and therefore supports tumor growth. Owing to the high expression and its role, telomerase has become an attractive diagnostic and therapeutic cancer target. Imetelstat (GRN163L) is a potent and specific telomerase inhibitor and so far the only drug of its class in clinical trials. Here, we report on the structure and the mechanism of action of imetelstat as well as about the preclinical and clinical data and future prospects using imetelstat in cancer therapy.

Prostate cancer is the most common malignancy in men, and patients with metastatic disease have poor outcome even with the most advanced therapeutic approaches. Most cancer therapies target the bulk tumor cells, but may leave intact a small population of tumor-initiating cells (TICs), which are believed to be responsible for the subsequent relapse and metastasis. Using specific surface markers (CD44, integrin alpha(2)beta(1) and CD133), Hoechst 33342 dye exclusion, and holoclone formation, we isolated TICs from a panel of prostate cancer cell lines (DU145, C4-2 and LNCaP). We have found that prostate TICs have significant telomerase activity which is inhibited by imetelstat sodium (GRN163L), a new telomerase antagonist that is currently in Phase I/II clinical trials for several hematological and solid tumor malignancies. Prostate TICs telomeres were of similar average length to the telomeres of the main population of cells and significant telomere shortening was detected in prostate TICs as a result of imetelstat treatment. These findings suggest that telomerase inhibition therapy may be able to efficiently target the prostate TICs in addition to the bulk tumor cells, providing new opportunities for combination therapies.

Human telomerase, the reverse transcriptase which extends the life span of a cell by adding telomeric repeats to chromosome ends, is expressed in most cancer cells but not in the majority of normal somatic cells. Inhibition of telomerase therefore holds great promise as anticancer therapy. We have synthesized a novel telomerase inhibitor GRN163L, a lipid—attached phosphoramidate oligonucleotide complementary to template region of the RNA subunit of telomerase. Here, we report that GRN163L is efficiently taken up by human myeloma cells without any need of transfection and is resistant to nucleolytic degradation. The exposure of myeloma cells to GRN163L led to an effective inhibition of telomerase activity, reduction of telomere length and apoptotic cell death after a lag period of 2–3 weeks. Mismatch control oligonucleotides had no effect on growth of myeloma cells. The in vivo efficacy of GRN163L was confirmed in two murine models of human multiple myeloma. In three independent experiments, significant reduction in tumor cell growth and better survival than control mice was observed. Furthermore, GRN163L-induced myeloma cell death could be significantly enhanced by Hsp90 inhibitor 17AAG. These data provide the preclinical rationale for clinical evaluation of GRN163L in myeloma and in combination with 17AAG.

We determined previously that a novel human telomerase RNA (hTR) antagonist, GRN163L, inhibited the tumorigenic potential of A549-luciferase (A549-luc) lung cancer cells in vitro and in vivo. Further studies revealed that A549-luc cells were also morphologically altered by GRN163L. A549-luc cells treated before cell attachment with a single dose of GRN163L only weakly attached to the substrate and remained rounded, whereas control mismatch-treated cells exhibited typical epitheloid appearance and adhesion properties. These morphologic changes were independent of hTR expression and telomerase inhibition and were unrelated to telomere length. This effect is dependent on the molecular properties of the lipid moiety, the phosphorothioate backbone, and the presence of triplet-G sequences within the GRN163L structure. Altered adhesion was manifested by a 50% reduction in rapid cellular attachment and a 3-fold decrease in total cell spreading surface area. Administration of a single dose of GRN163L (15 mg/kg) at the time of cell inoculation, using an in vivo model of lung cancer metastasis, resulted in significant reductions in tumor burden at days 13, 20, and 27 of tumor progression. Thus, the potent antimetastatic effects of GRN163L may be related, in part, to the antiadhesive effects of this novel cancer therapeutic conferred via specific structural determinants and that these effects are independent of telomerase inhibition or telomere shortening. [Cancer Res 2007;67(3):1121–9]

Study question How does regulation of telomerase activity (TA) in human endometrial epithelial cells (EEC) by ovarian hormones impact on telomere lengths (TL) and cell proliferation? Summary answer Healthy endometrial epithelial cell proliferation is characterized by high TA and endometrial TL changes according to the ovarian hormone cycle, with shortest TL observed in the progesterone dominant mid-secretory phase, when TA is lowest, implicating progesterone in the negative regulation of TA and TL. What is known already Critical shortening of telomeres may result in permanent cell cycle arrest while the enzyme telomerase maintains telomere length (TL) and replicative capacity of cells. Telomerase expression and activity change in the human endometrium with the ovarian hormone cycle, however the effect of this on endometrial TL and cell growth is not known. Study design, size, duration A prospective observational study, which included endometrial and blood samples collected from 196 women. Participants/materials, setting, methods We studied endometrial samples from five different groups of women. Endometrial and matched blood TL and circulating steroid hormones were studied in samples collected from 85 women (Group 1). Fresh epithelial and stromal cell isolation and culture in vitro for TL and TA was done on endometrial biopsies collected from a further 74 healthy women not on hormonal therapy (Group 2) and from 5 women on medroxyprogesterone acetate (MPA) for contraception (Group 3). The epithelial TL and telomerase protein expression was examined in active, peritoneal, ectopic endometriotic and matched uterine (eutopic) endometrial samples collected from 10 women with endometriosis (Group 4); the in vivo effect of mifepristone on telomerase protein expression by immunohistochemistry (IHC) was examined in endometrium from 22 healthy women in mid-secretory phase before (n = 8), and after administering 200 mg mifepristone (n = 14) (Group 5). TA was measured by telomere repeat amplification protocol (TRAP) assay; TL by qPCR, and Q-FISH; cell proliferation was assessed by immunoblotting of histone H3 and 3D-culture to assess the ability of EECs to form spheroids; telomerase reverse transcriptase protein levels and Ki-67 (proliferative index) were assessed with IHC. Main results and the role of chance Endometrial TLs correlated negatively with serum progesterone levels (n = 58, r = -0.54) and were significantly longer than corresponding blood TLs (4893 ± 929 bp versus 3955 ± 557 bp, P = 0.002) suggesting a tissue-specific regulation. High TA and short TLs were observed in proliferating EECs in vivo and in vitro. During the progesterone dominant mid-secretory phase endometrial TL were significantly shorter compared with the proliferative phase (P = 0.0002). Progestagen treatment suppressed EEC TA in vivo and reduced endometrial TA in explant (P = 0.01) and in vitro cultures (P = 0.02) compared with untreated cells. Mifepristone (progesterone receptor antagonist) increased telomerase protein levels in vivo (P Limitations, reasons for caution The in vitro telomerase inhibition data were tested in a mono-cellular system for a short-term. Further confirmation of the results in an in vivo model is necessary. The women in group 2 included a high proportion of women although with a regular menstrual cycle, with an increased BMI (>25) therefore this may affect extrapolation of data to other groups. Wider implications of the findings The observed effects of telomerase inhibition in vitro on epithelial cell proliferation, suggest that telomerase might be an attractive target in developing new therapies for proliferative disorders of the endometrium, such as endometriosis.

Summary Telomerase activity is undetectable in most normal tissues but the vast majorities of cancers express active telomerase. Therefore, telomerase serves as an attractive target for the treatment of cancers. GRN163L is a lipidmodified oligonucleotide N3 ¢fi P5¢ thio-phosphoramidate complementary to the RNA template region of human telomerase. The anti-telomerase activity of GRN163L was evaluated using MDA-MB-231 and MDA-MB-435 human breast adenocarcinoma cell lines. Twice weekly administration of GRN163L resulted in the inhibition of telomerase activity and progressive telomere shortening. Cells treated with GRN163L did not demonstrate decreased cell proliferation for up to 2 weeks. However, after additional treatment, cell proliferation gradually decreased in GRN163L-treated cells compared to untreated or mismatch control oligoncleotide treated cells. Furthermore, anti-tumorigenic effects were seen in cells treated with GRN163L, as cells lose their ability to form colonies in soft agar and were unable to form colonies in the clonal efficiency assay upon incubation with GRN163L. Moreover, breast cancer cells that were treated with GRN163L for only 1 week prior to plating in invasion chambers, and when bulk telomere are still long, exhibit significantly diminished invasive potential. These results reveal critical information regarding the effectiveness of GRN163L as a potential therapeutic agent for the treatment of human breast cancer.

The effects of telomerase inhibition with an oligonucleotide N3′ → P5′ thiophosphoramidate (GRN163) complementary to the telomerase template region were examined on human multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines, primary MM cells, and tumor xenografts. GRN163 treatment reduced telomerase levels in all cells and induced more rapid telomeric shortening. Continuous GRN163 treatment for 7 to 14 days resulted in proliferative arrest, morphologic changes, and apoptosis characteristic of cell crisis in tumor cell lines with short (1.7-5.4 kb) but not long (9-11 kb) telomeres. Intratumoral administration of GRN163 also inhibited the growth of MM and NHL xenografts established from cell lines with short telomeres (Hs602 lymphoma, 2.7 kb; CAG myeloma, 2.7 kb) and increased tumor apoptosis. However, GRN163 therapy of NHL xenografts established from cells with long telomeres (11.0 kb) had equivocal effects on tumor growth and did not induce apoptosis during this time frame. Systemic daily intraperitoneal administration of GRN163 in myeloma xenografts with short telomere lengths also decreased tumor telomerase levels and reduced tumor volumes. These data demonstrate that telomerase is important for the replication of mature B-cell neoplasia by stabilizing short telomeres, and they suggest that telomerase inhibition represents a novel therapeutic approach to MM and NHL.