Imetelstat (GRN163L), a Telomerase Inhibitor Selectively Affects Malignant Megakaryopoiesis in Myeloproliferative Neoplasms (MPN)

Abstract

Imetelstat is a telomerase inhibitor which has been shown to have therapeutic activity in patients with myelofibrosis (MF) (Tefferi A. et al., 2013 ASH Annual Meeting, Abstract 662) and essential thrombocythemia (ET) (Baerlocher G. et al., 2012 ASH Annual Meeting, Abstract 179). In clinical trials involving patients with a variety of other malignancies, the primary dose-limiting toxicity of GRN163L has been thrombocytopenia. We utilized GRN163L in order to explore the possible role of telomerase on human megakaryocytes (MK) and the mechanisms underlying the drug’s inhibitory effects on platelet production. MK were generated from normal primary CD34+ cells using an ex vivo culture system previously described by our laboratory (Iancu-Rubin C. et al, Blood 2011, 117:17; 4580-4589). We first showed that both telomerase activity and the expression of its catalytic unit hTERT were elevated in proliferating normal CD34+ cells, declined transiently during the initial stages of MK differentiation but were then partially restored during the final stages of terminal maturation. Treatment of normal CD34+ cells with GRN163L did not affect the numbers of CFU-MK assayed. Furthermore, exposure to GRN163L during the first 7 days in a liquid culture system did not interfere with the ability of CD34+ cells to commit to MK. When the same cultures were allowed to mature for 7 additional days, the proportion of CD34+/CD41+ MK precursors in drug-treated cultures was twice that observed in control and the drug-treated cultures contained 70% fewer mature CD41+/CD42b+MK than control cultures. The inhibitory effects of GRN163L on MK maturation were supported by observations showing that the cultures treated with the drug contained 60% fewer polyploid MK than control cultures. These results suggest that that GRN163L-mediated inhibition of telomerase does not affect normal MK progenitors but blocks the maturation of MK precursor cells.Previous studies have shown that GRN163L inhibits neoplastic CFU-MK colony formation by CD34+ cells from patients with ET (Brunold C. et al., 2011 ASH Annual Meeting, Abstract 3843). We, therefore, examined the effects of GRN163L on malignant MK from patients with MF and ET. Unlike normal CD34+ cells, treatment of MPN PB-MNCs with GRN163L decreased the numbers of assayable CFU-MK from 6 out of 11 patients. CFU-MK colony formation by PB-MNCs from these 6 patients was reduced by 33% (ranging from 13% to 50% reduction in CFU-MK formation) as compared to PB-MNCs treated with an inactive form of the drug (p value= 0.00473). Of note, in two out of 5 patients in which the total number of CFU-MK was not affected by GRN163L treatment, the drug decreased the size of the CFU-MK colonies formed. The ability of MPN PB-MNCs to differentiate into MK was next assessed. Although the total number of cells in PB-MNC liquid cultures exposed to GRN163L was decreased as compared to those treated with the inactive drug (n=6; p value=0.03204), the proportion of CD34+/CD41+MK precursors generated was not significantly affected. We then evaluated the effects of GRN163L on the genotype of MPN MK generated in the presence and absence of GRN163L by assessing the JAK2V617F allele burden. Treatment with GRN163L but not the inactive form of the drug reduced the JAK2V617F allele burden in MK derived from two patients: in one patient, JAK2V617F allele burden in MK generated in the presence of the inactive drug was 91.86% while MK generated in the presence of GRN163L was 19.75%; MK generated from a second patient had a JAK2V617F allele burden of 10.84% in the presence of the inactive drug which was reduced to 2.14% in the presence of GRN163L.We conclude that GRN163L-mediated inhibition of telomerase affects normal megakaryopoiesis by blocking the terminal maturation of CD34+/CD41+ MK precursors, providing a possible explanation for GRN163L’s propensity to induce thrombocytopenia in patients with normal bone marrow. By contrast, GRN163L treatment inhibited the ability of MPN CD34+ cells but not normal CD34+ cells to form CFU-MK colonies and drug treatment reduced the numbers of malignant MK generated. We propose that the amelioration of fibrosis observed in a clinical trial of MF patients treated with GRN163L might be due to, at least in part, two potential modes of action: 1) inhibiting malignant MK progenitors cells and 2) preventing terminal MK maturation thus depleting the pool of mature MKs which are the major source of fibrogenic cytokines in MF. DisclosuresIancu-Rubin:Geron Corporation: Research Funding. Parker:Geron Corporation: Employment. Eng:Geron Corporation: Employment. Hoffman:Geron Corporation: Consultancy.