Telomerase Inhibition, Telomere Shortening and Apoptotic Cell Death in Multiple Myeloma Cells Following Exposure to a Novel and Potent Telomerase Inhibitor (GRN163L), Targeting RNA component of Telomerase.

Telomeres, the specialized nucleoprotein structures at the ends of chromo-somes, shorten at each DNA replication, and if unopposed leads to chromosomal erosion and cell death. Telomere shortening below a critical length is prevented by telomerase. We have previously observed elevated telomerase activity and shortened telomeres in multiple myeloma (MM), making the telomere maintenance mechanism an important target for therapy. Based on success with other non-specific telomerase inhibitors, in this study, we evaluated the effects of a thio-phosphoramidate oligonucleotide specifically targeting the RNA component of telomerase (GRN163L), with modifications to facilitate its delivery into human cells. Nuclear uptake of GRN163L without need for transfection enhancer at 24h was confirmed in >99% MM cells using fluorescein isothiocyanate-tagged GRN163L and confocal microscopy. Next we evaluated the effects of different concentrations and length of exposure of GRN163L on telomerase activity in diff MM cell lines (ARP and INA6). Whereas control oligonucleotide did not significantly affect telomerase activity, ≥ 80% loss of telomerase activity was observed in MM cell lines at day 3 at submicromolar concentrations of GRN163L. This inhibition of telomerase activity was associated with inhibition of myeloma cell growth and survival. Treatment of INA6 cells with GRN163L for three weeks induced 96±4% and 100% cell death at 0.5 and 1 μM concentrations, respectively, while ARP cells with higher telomerase activity and longer telomeres showed 67 ± 4% cell death at 5 weeks with 0.5 μM inhibitor and 82 ± 3% and 100% cell death at 4 and 5 weeks respectively with 2 μM concentration. The cell death was predominantly apoptotic, as determined by 51% annexin V-positive INA6 cells at two weeks and >80 % annexin V positive ARP cells at four weeks. The apoptotic cell death was associated with reduction in telomere length as analyzed using Telomere-FISH. While the control oligo treated ARP cells showed mean Telomere Fluorescence Intensity (TFI) on interphase chromosomes of 17.2 ± 1.5 (range 1.3–146.4), GRN163L treated cells showed reduction of mean TFI to 13.6 ± 0.46 (range 0.74 – 65.7). Although values above 100 were observed on 2 chromosomes in control oligo treated cells, GRN163L-treated cells had no chromosome with a TFI of more than 70.0. A similar reduction in telomere length was observed for INA6 cells. Subsequently, we were also able to increase cytotoxicity of DNA damaging agents in MM cells treated with GRN163L and shortened telomeres; providing a rationale for evaluation of combination therapies. These data demonstrate GRN163L as a potent and specific telomerase inhibitor able to disrupt telomere integrity and inducing apoptotic death of multiple myeloma cells. Evaluation of this agent in a SCIDhu model of myeloma is underway prior to its clinical evaluation.