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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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

One of the hallmarks of cancer is limitless proliferative capacity, which is tightly associated with the ability to maintain telomeres. Over the last decade, the telomere biology of pediatric cancers has begun to be elucidated. Most pediatric leukemias and embryonal solid tumors activate the enzyme telomerase, a specialized reverse transcriptase that adds nucleotide repeats to telomeres. In general, high levels of tumor telomerase expression are associated with unfavorable outcome, although results vary according to tumor type. Some pediatric tumors, including osteosarcoma and glioblastoma multiforme, lack telomerase activity and maintain telomeres via a recombination-based mechanism called ALT (alternative lengthening of telomeres). Telomerase is a highly attractive therapeutic target for pediatric cancer because the enzyme plays a key role in conferring cellular immortality, is present in most tumors, and is relatively specific for cancer cells. Telomerase inhibitors have been evaluated in preclinical models of adult cancers, but few studies have been conducted on pediatric cancers. Further research is required to define how telomere biology can be used to clinical advantage in malignancies of childhood.

Purpose: A primary objective of the fifth National Wilms Tumor Study (NWTS-5) was to identify prognostic indicators for patients with favorable-histology Wilms' tumor. The prognostic significance of telomerase expression level in primary tumor samples was assessed., Patients and Methods: A case-cohort study was conducted involving 291 NWTS-5 registrants. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP). Expression levels of TERT mRNA (encoding the telomerase catalytic component) and TERC/hTR (the telomerase RNA template) were measured using quantitative real-time polymerase chain reaction., Results: After excluding samples because of lack of viable tumor, RNA degradation, or insufficient clinical information, 244 patients remained for the final analysis (96 with relapse and 148 without relapse). Univariate analysis revealed a positive correlation between relative risk (RR) of relapse and levels of TERT mRNA and TERC expression. For each doubling in TERT mRNA and TERC level, the RR increased by a factor of 1.16 (95% CI, 1.04 to 1.29; P = .01) and 1.35 (95% CI, 1.11 to 1.64; P = .003), respectively. The one third of patients whose tumors had the highest TERC expression level had an RR of 2.06 (95% CI, 1.14 to 3.70; P = .02) compared with patients with the lowest level. TERC expression level remained a significant prognostic indicator in a multivariate analysis adjusting for TERT mRNA, tumor stage, and patient age. TRAP level did not correlate with RR of relapse. Telomerase expression levels were not predictive of overall survival., Conclusion: Telomerase RNA expression level may provide a clinically useful adjunct to the current risk classification schema for favorable-histology Wilms' tumor.

Purpose: Osteosarcoma is distinct from most cancers in that the majority of osteosarcomas lack telomerase expression and use the alternative lengthening of telomeres (ALT) mechanism to maintain telomeres. Laboratory studies suggest that compared with ALT, telomerase expression is associated with increased tumor aggressiveness. We evaluated the clinical significance of telomerase expression in human osteosarcoma., Patients and Methods: Fifty-six osteosarcomas from 51 patients treated at St Jude Children's Research Hospital between 1982 and 2003 were evaluated for telomerase enzyme activity, mRNA expression of the catalytic component of telomerase (TERT), and presence of the ALT pathway., Results: Outcome analysis was based on TERT mRNA expression in the primary tumor samples from 44 patients. Fourteen primary tumors expressed TERT mRNA (32%; eight TERT only, six TERT and ALT) and 30 did not express TERT mRNA (68%; 29 ALT, one no ALT). Progression-free survival (PFS) was inferior in the TERT-positive group compared with the TERT-negative group (3-year estimates, 21.4% +/- 9.5% v 63.7% +/- 11.1%; P =.014). Likewise, overall survival was inferior in the TERT-positive group compared with the TERT-negative group (3-year estimates, 42.9% +/- 12.2% v 70.0% +/- 9.9%; P =.031). Among 31 patients with nonmetastatic disease at diagnosis, PFS was lower in the TERT-positive group compared with the TERT-negative group (3-year estimates, 33.3% +/- 13.6% v 72.0% +/- 11.5%; P =.092)., Conclusion: Telomerase expression in primary tumor samples is associated with decreased PFS and OS in patients with osteosarcoma. Additional studies are warranted to better define the clinical utility of this molecular marker.

To create immortal mesenchymal cell lines, we transduced primary human bone marrow mesenchymal cells with telomerase reverse transcriptase (TERT). TERT+ mesenchymal cells continued to grow for > 2 years; parallel TERT- cultures underwent senescence after 15 weeks. TERT+ mesenchymal cells did not form foci in soft agar, had a normal karyotype and could differentiate into osteoblasts and chondrocytes. Their capacity to support leukaemic lymphoblasts and normal CD34+ haematopoietic cells was equal to or greater than that of primary cells; 42 TERT+ mesenchymal cell clones varied in their supporting capacity. Immortalized mesenchymal cells offer a promising tool for identifying molecules that regulate human haematopoiesis.

Purpose: Recent studies associate telomerase activity with prognostic factors and survival. We compared quantitative telomerase activity in primary tumors with traditional prognostic factors and outcome in a group of invasive but nonmetastatic breast cancers., Patients and Methods: Telomerase activity was measured in 203 invasive breast cancers by the quantitative telomeric repeat amplification protocol method. Telomerase expression was compared with 28S rRNA level, tumor content, and clinical variables, including outcome. For clinical correlations, telomerase activity was standardized by two methods: (1) a correction for cellularity using 28S rRNA levels, and (2) a correction for the histologically determined invasive proportion of the specimen., Results: Telomerase activity was found in 82% of breast cancers with measurable 28S rRNA levels. Telomerase activity was associated with the proliferative index (P <.01) of the tumor but not with any other prognostic variable. Neither uncorrected nor corrected telomerase activity was associated with relapse-free or overall survival in this study., Conclusion: Telomerase activity level was associated with the proliferative index of invasive breast cancers, but its measurement in samples from this group of nonmetastatic breast cancer patients did not predict survival.

Telomerase is a reverse transcriptase that maintains chromosome ends, compensating for the progressive loss of DNA that occurs during replication. High telomerase enzyme activity is an unfavorable prognostic feature for several types of cancers. We investigated whether telomerase level predicts outcome for patients with the pediatric renal malignancy Wilms' tumor. In a case-cohort study of 78 patients with favorable histology Wilms' tumor, we compared tumor telomerase levels in patients with and without eventual recurrence. Three measures of telomerase were used: (a) telomerase enzyme activity; (b) expression of hTR, the RNA component of telomerase; and (c) mRNA expression of hTERT, the gene that encodes the catalytic component of the enzyme. Of the evaluable samples, 81% had detectable telomerase activity, 97% had detectable hTERT transcript, and 100% had detectable hTR. Weak correlations were observed between telomerase activity and hTR level (r = 0.34, P = 0.02) and between telomerase activity and hTERT mRNA level (r = 0.32, P = 0.04). Of the variables assessed, only hTERT mRNA expression correlated with outcome. The median hTERT mRNA level in tumors with recurrence was higher than that in tumors without recurrence (1.42 versus 0.97 units, P = 0.023, Wilcoxon). Univariate analysis of hTERT mRNA level as a continuous variable suggested that each unit increase in hTERT mRNA level increased the risk of recurrence (RR) by a factor of 1.66 [95% confidence interval (CI), 1.2-2.3; P < 0.005]. Compared with tumors with hTERT mRNA levels of 0-1 units, tumors with hTERT mRNA levels of 1-2 units had a RR of 2.72 (95% CI, 0.91-8.13; P = 0.074), and tumors with hTERT mRNA levels >2 units had a RR of 6.40 (95% CI, 1.49-27.67, P = 0.013). Multivariate analysis of hTERT mRNA level as a predictor of recurrence, adjusted for tumor stage and age at diagnosis, revealed a RR of 1.48 (95% CI, 0.9-2.6; P = 0.16). Measurement of hTERT mRNA level may, therefore, enable clinicians to identify a population of patients at high risk for recurrence and to adjust their therapy accordingly. A larger study will be necessary to determine whether hTERT expression is an independent prognostic indicator. Further biological investigation is warranted to discern whether the link between high hTERT expression and unfavorable prognosis is causative or correlative.

The past decade has been marked by an explosion of knowledge regarding the dysregulation of cancer at the molecular level. It has become apparent that oncogenes, tumor suppressor genes, and other ancillary molecules interact in complex pathways that govern cellular homeostasis. We review three molecular events that have been implicated in tumorigenesis and define pathways ripe for the development of new therapeutic approaches: 1) activation of telomerase, 2) dysregulation of the patched/sonic hedgehog pathway, and 3) mutation of the INK4 alpha-ARF locus.

Studies of invasive breast cancers consistently identify a subset of tumors without telomerase activity, compromising its utility as a tumor marker. Telomerase-negative tumors may represent a biologically different subset, or the result could be attributed to assay imperfections. To resolve this issue, we tested 105 invasive breast cancers for telomerase activity and found that 23 (22%) tumors were telomerase negative. Careful histological confirmation of an adjacent cryosection and/or microdissection of pure tumor cells reduced this number to 5 (5%). Thus, truly telomerase-negative invasive breast cancers are rare, making this enzyme a potentially very useful tumor marker in breast cancer.

The 1-methyl-1-nitrosourea-induced rat mammary tumor model system is well studied, reproducible, and widely used. We have investigated whether these tumors possess higher telomerase activity than normal mammary tissue. Using the telomeric repeat amplification protocol assay, we found significantly higher telomerase activity in 36 mammary carcinomas than in 72 mammary glands of virgin rats. The level of telomerase activity in virgin rats was unaffected by strain, age, stage of the estrous cycle, or ovariectomy. However, mammary glands obtained from pregnant rats exhibited telomerase activity comparable to that found in the tumors, possibly reflecting the high epithelial content of these tissues. Indeed, isolated epithelial cells from virgin and pregnant mammary glands and from carcinomas had similar telomerase activities. Thus, telomerase activity is constitutive in the rat mammary epithelium and is not a unique characteristic of malignant transformation in this tissue. These results underscore the importance of attributing biochemical properties to specific cell types in a tissue, a situation not paralleled in the interpretation of data from in vitro models.