Leukemic T-PLL Cells Exhibit Short and Narrowly Distributed Telomere Lengths and High Telomerase Activities, Associated with Rapid and Dose-Dependent Cell Death Following Telomerase Inhibition by Imetelstat Sodium.

Reflecting their proliferative history, malignant cells generally have shorter telomeres compared to their normal counterparts. We have reported that leukemic cells from patients with T-prolymphocytic leukaemia (T-PLL) have exceptionally short telomeres (telomere lengths around 1 kb) compared to other malignancies, and high levels of telomerase activity (Roeth et al., Leukemia, 2007). We have also previously presented (Roeth et al., ASCO 2008) cytotoxic effects of the telomerase inhibitory agent imetelstat sodium (GRN163L), which is currently in clinical trials. The objective of this study was to further characterize the relationship between mean telomere length (TL), the single chromosome telomeric size distributions (single TL), telomerase levels and imetelstat-induced telomerase inhibition and cytotoxicity in T-PLL, B-CLL, and normal cells.

Cells were obtained from peripheral blood of normal donors and patients with T-PLL and B-CLL and cryopreserved. TL was measured by flow-FISH (which yields a multicellular average measurement across all chromosomes) and the distribution of individual telomere lengths by single telomere length analysis (STELA, XpYp specific). Telomerase activity (TA) was assessed by TRAP. Effects of imetelstat upon cell survival, apoptosis and TA were studied in short-term cell cultures. Apoptosis was measured by Annexin V-PI double staining.

Average (±SD) TL of the T-PLL cells was 1.8 ± 0.73 kb (n=13) which was shorter than for B-CLL cells (ZAP-70⁺/CD38⁺ CLL: 2.46 ± 1.08 kb (n=30); ZAP-70^neg/CD38^neg CLL: 5.06 ± 1.76 kb (n=29), Roeth et al, Br. J. Haem., 2008). T-PLL cells were found to have a narrow range of single chromosome telomere lengths, while both subtypes of B-CLL cells demonstrated a broader distribution of telomere lengths. No attrition of mean telomere length was observed over time in two patients with T-PLL who were followed over time, despite continued clinical progression. In contrast, we have observed that most patients with B-CLL do have decreases in telomere length over the course of their disease.

Leukemic T-PLL cells exhibited high levels of telomerase activity, whereas there was little or no detectable telomerase activity in normal, unstimulated T-cells and most B-CLL cells. Imetelstat (GRN163L, Geron Corporation), a potent inhibitor of telomerase, induced significant and dose-dependent inhibition of telomerase activity in T-PLL cells after 2 hours of culture at 1μM and 10μM. Imetelstat significantly reduced cell viability of T-PLL cells in vitro after 7-10 days of culture (viability as % of control (mean ± SD) with 1, 3, 10 μM imetelstat: 69.9 ± 27.3, 51.7 ± 30.2, 37.1 ± 27.6). No significant cytotoxicity of imetelstat was observed in CLL samples, or in unstimulated and stimulated T-cells from 5 normal donors. Leukemic cells from one T-PLL patient early in the course of the disease did not exhibit loss of viability when exposed to imetelstat at levels up to 10 μM; however, when cells from this patient were tested later during the progression of the disease significant cytotoxicity was observed.

In summary, leukemic T-PLL cells exhibit extremely short telomere lengths, narrower ranges of single chromosome telomere size distributions, and higher telomerase activity compared to B-CLL cells and normal T cells. These observations may explain why inhibition of telomerase activity in T-PLL cells by imetelstat is associated with rapid and dose-dependent cell death.

Baerlocher:Geron Corporation: Research Funding. Bassett:Geron Corporation: Employment, Equity Ownership. Elias:Geron Corporation: Employment, Equity Ownership. Go:Geron Corporation: Employment, Equity Ownership. Roeth:Geron Corporation: Research Funding.