Telomerase activity is a strong indicator of cellular malignancy (17
), and in GBM tumors, high levels of telomerase expression correlate with tumor progression and poor prognosis (19
). Here we report that a novel human telomerase antagonist, imetelstat, is a potent inhibitor of telomerase activity in primary human GBM tumor-initiating cells. The ability to target these crucial subpopulations of cells that evade conventional and targeted therapies could be a significant step in developing effective strategies for GBM treatment.
For this study we used primary GBM tumor-initiating cells which produce orthotopic tumors with accurate GBM histopathology. The isolated tumor-initiating cells were also cultured as non-adherent neurospheres, conditions which maintain their stem-like properties (11
). Imetelstat produced a dose-dependent, reversible inhibition of telomerase activity over a wide dose range that persists for several days, raising the possibility that the pharmacokinetics may be well suited for the clinical setting. The negative impact of imetelstat on proliferation was not evident until after ~15-20 population doublings, when progressive telomere attrition leads to the induction of DNA damage signaling, end-to-end fusions and genetic instability, processes that can lead to apoptotic cell death.
Because tumor cells share the same telomere elongation machinery with normal proliferative stem-like cells, one major concern associated with the use of a telomerase inhibitors is the potential decline of regenerative capacity in normal stem cells. Such an adverse possibility is of particular concern for the organs with high rates of cellular turnover and especially relevant in elderly patients. Because telomere shortening occurs in most human tissues during aging and is accelerated in response to chronic diseases (33
), it is important to determine not only the telomere length in tumor-initiating stem cells but also in normal stem cells. The results of our present studies clearly show that the average telomere lengths of GBM tumor cells are approximately three times shorter compared to normal human brain cells (~10 vs. 3.5 kb). In principle, assuming equivalent rates of proliferation, this difference offers an ample therapeutic window to cause malignant tumor cells to undergo critical telomere shortening while telomeres in the normal stem cell compartment remain of adequate length. Moreover, following removal of imetelstat, telomerase activity rapidly recovers to normal levels which suggest that potential adverse telomere shortening in the normal stem cells may be reversible. Clearly, this is a significant advantage over conventional chemotherapies which often produce irreversible damage to all proliferative cells including stem cells of renewal tissues.
Recent studies have shown that combination of temozolomide (TMZ) and ionizing radiation (IR) have some therapeutic benefits. We explored whether addition of imetelstat would increase the therapeutic potential of these agents and demonstrate that GBM neurospheres cells treated with imetelstat for long periods of time are more sensitive to IR and TMZ (). While the additive effects seen with TMZ or IR and long-term imetelstat treated cultures were consistent with previous data on irradiated breast cancer cells (34
), the effects of TMZ on short-term imetelstat-treated cells were surprising because this combination led to increased toxicity in the absence of telomere shortening (). This result appears to be specific for TMZ because the cells irradiated with a dose of 5Gy do not show significant cytotoxicity compared to the un-irradiated controls, but we cannot exclude the possibility that GBM cells subjected to other IR regimens may exhibit a similar response. One explanation for this mechanism of drug synergy may be that IR and TMZ induced telomeric DNA breaks which could not be repaired in the absence of telomerase, leading to an increased DNA damage response (). Another explanation is that treatment with imetelstat may activate an elevated autophagy response in GBM cells. It is documented that TMZ triggers the GBM cell death by autophagy (35
) and some reports suggest that GBM tumor-initiating cells are resistant to the TMZ-induced autophagy due to the down-regulation of critical proteins (37
). Interestingly, the gene encoding one of these autophagy proteins (APG5) was found to be significantly up-regulated in myeloma cells treated with imetelstat (38
). Moreover, it was shown that a conditionally replicating hTERT adenovirus can induce autophagic cell death in malignant glioma cells (39
). Taken together, these data suggest that the telomere length-independent effects of imetelstat along with its primary effect on telomere elongation may be uniquely effective against GBM tumor-initiating cells.
One of the major challenges in brain tumor therapy remains the difficulty of delivering drugs across the blood-brain barrier (BBB). Building on previous biodistribution studies (40
), our hypothesis was that imetelstat could penetrate the BBB and efficiently inhibit telomerase in orthotopic GBM tumors. Despite strong evidence of tight junctions, our data clearly shows for the first time that intraperitoneal-introduced imetelstat was able to penetrate the BBB in sufficient intra-parenchymal concentration to block ~70% of telomerase activity in human GBM orthotopic xenograft cells.
The in vitro data indicated that GBM tumor-initiating cell must undergo at least 15-20 population doublings before significant telomere attrition may occur. Because in the orthotopic model 5 × 104 cells were injected into the brain, the total cell number following 20 population doublings would be 5 × 1010 cells, which would exceed the 1-2 mm3 maximum tumor mass an adult mouse cranium can accommodate without brainstem compression, herniation and death. For this reason, we used subcutaneous xenografts to investigate the in vivo effect of imetelstat on the tumor growth rate and size, because this model can accommodate much larger tumor volumes (2 cm3) without significant morbidity. Furthermore, imetelstat penetration data shows that telomerase inhibition of GBM tumors cells is very similar for the subcutaneous and intracranial tumors, therefore the subcutaneous xenograft model is more than adequate for a therapeutic proof of concept. Drug treatment was initiated once the subcutaneous tumors were clearly visible (~1 mm) and the results show significant differences between the imetelstat- and vehicle-treated mice (). Regular monitoring of tumor size by bioluminescence initially showed little difference between the treated and control cohorts, but as the tumor masses approached 25% of maximal tolerate size (500 mm3), imetelstat-treated tumors were showing a significantly slower tumor growth, presumably due to a subset of tumor cells having undergone the required number of population doublings for critical telomere shortening. Most GBM patients undergo aggressive de-bulking resection (unless contraindicated by tumor location or other co-morbidities) which will ensure that there is sufficient space to permit tumor growth and erosion of telomeres to critical levels that trigger cellular quiescence and/or cell death. We predict an even greater therapeutic efficacy and perhaps a durable response when imetelstat is combined with radiation and temozolomide, which currently provide only a partial response. Taken together, the in vitro and in vivo data is encouraging for pursuing testing of imetelstat in GBM patients
Statement of Translational Relevance
Glioblastoma is one of the most lethal human cancers and the chemotherapeutic options are still limited by the reduced capacity of drugs to penetrate the blood-brain barrier. Cancer relapse is believed to be caused by small populations of tumor-initiating cells which can escape conventional therapies. Imetelstat is a novel telomerase inhibitor that inhibits telomerase and induces telomere shortening in glioblastoma tumor-initiating cells in addition to the bulk tumor mass. This pre-clinical study demonstrates that imetelstat is a highly efficient and specific agent, both in vitro and in mouse orthotopic primary glioma xenografts. Imetelstat not only penetrates the blood brain barrier but also shows increased efficacy in combination with ionizing radiation and temozolomide, the current standard of care for glioblastoma. The experimental data supports the future implementation of imetelstat in clinical studies for glioblastoma.