Transient Telomerase Inhibition with Imetelstat Impacts DNA Damage Signals and Cell-Cycle Kinetics.

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 G2Implications: 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 accumulation and not the specific activity of telomerase ribonucleoprotein determines telomere maintenance deficiency in X-linked dyskeratosis congenita.

X-linked dyskeratosis congenita (X-DC) is caused by mutations in the housekeeping nucleolar protein dyskerin. Amino acid changes associated with X-DC are remarkably heterogeneous. Peripheral mononuclear blood cells and fibroblasts isolated from X-DC patients harbor lower steady-state telomerase RNA (TER) levels and shorter telomeres than healthy age-matched controls. Previously, we showed that retroviral expression of recombinant TER, together with expression of recombinant telomerase reverse transcriptase, restored telomere maintenance and proliferative capacity in X-DC patient cells. Using rare X-DC isoforms (ΔL37 and A386T dyskerin), we showed that telomere maintenance defects observed in X-DC are solely due to decreased steady-state levels of TER. Disease-associated reductions in steady-state TER levels cause deficiencies in telomere maintenance. Here, we confirm these findings in other primary X-DC patient cell lines coding for the most common (A353V dyskerin) and more clinically severe (K314R and A353V dyskerin) X-DC isoforms. Using cell lines derived from these patients, we also examined the steady-state levels of other hinge-ACA motif RNAs and did not find differences in their in vivo accumulations. We show, for the first time, that purified telomerase holoenzyme complexes from different X-DC cells have normal catalytic activity. Our data confirm that dyskerin promotes TER stability in vivo, endorsing the development of TER supplementation strategies for the treatment of X-DC.

Telomerase inhibition potentiates the effects of genotoxic agents in breast and colorectal cancer cells in a cell cycle-specific manner.

Previous studies have shown that telomerase facilitates DNA-damage repair and cell survival following stress. It is not clear how telomerase promotes DNA repair, or whether short-term telomerase inhibition, combined with genotoxic stress, can be exploited for cancer therapy. Here, we show that transient inhibition of telomerase activity by the specific inhibitor, GRN163L, increases the cytotoxicity of some, but not all, DNA-damaging agents. Such synergistic inhibition of growth requires the use of DNA-damaging agents that are toxic in the S/G(2) phase of the cell cycle. Notably, inhibition of Ataxia Telangiectasia Mutated (ATM) kinase, together with telomerase inhibition, synergistically increases the cytotoxicity induced by the G(2)-specific topoisomerase II inhibitor etoposide. By varying the timing of telomerase inhibition, relative to the timing of DNA damage, it is apparent that the prosurvival functions of telomerase occur at early stages of DNA damage recognition and repair. Our results suggest that the protective role of telomerase in cell cycle-restricted DNA damage repair could be exploited for combined anticancer chemotherapy.

Adenoviral oncoprotein E1B55K mediates colocalization of SSBP2 and PML in response to stress.

Transient expression of adenoviral oncoprotein E1B55K in normal cells induces aggresome formation and sequestration of critical host proteins in aggresomes. Our previous studies reported that Sequence Specific Binding Protein 2 (SSBP2), a candidate tumor suppressor is recruited to aggresomes in adenovirally transformed human embryonal kidney 293 (HEK293) cells. To understand the extent and significance of the E1B55K-SSBP2 interactions in these cells, we have examined SSBP2 localization under conditions of stress in HEK293 cells. SSBP2 localizes to PML- Nuclear Bodies (PML-NBs) in response to inhibition of nuclear export, treatment with etoposide, hydroxyurea or gamma irradiation only in HEK293 cells. Furthermore, the PML-NBs grow in size and number in response to radiation over a 24 hour period in HEK293 cells analogous to previous findings for other cell types. Nonetheless, we conclude that E1B55K subverts SSBP2 function in HEK293 cells. These findings demonstrate the limitations in using HEK293 cells to study DNA damage response and other cellular processes since SSBP2 and similar regulatory proteins are aberrantly localized due to constitutive E1B55K expression.

Telomerase as a clinical target: current strategies and potential applications.

Chromosome ends are capped by telomeres, protective DNA-protein complexes that distinguish natural ends from random DNA breaks. Telomeres erode with each successive cell division, and such divisions cease once telomeres become critically short. This proliferation limit is important as a tumor suppressive mechanism, but also contributes to the degenerative conditions associated with cellular aging. In cell types that require continuous renewal, transient expression of telomerase delays proliferation arrest by the de novo synthesis of telomere repeats. Data from our work and others' has shown that deficient telomerase activity has a negative impact on normal human physiology. In the bone marrow failure syndrome dyskeratosis congenita, telomerase enzyme deficiency leads to the premature shortening of telomeres. Premature telomere shortening most grievously affects tissues that have a rapid turnover, such as the hematopoietic and epithelial compartments. In the most severe cases, compromised renewal of hematopoietic stem cells leads to bone marrow failure and premature death. Telomerase activation/replacement shows potential as a therapy for telomere maintenance deficiency syndromes, and in tissue engineering for the degenerative conditions that are associated with normal aging. Conversely, clinical researchers are developing telomerase inhibition therapies to treat tumors, which overcome the short-telomere barrier to unrestricted proliferation by over-expressing telomerase.