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Key Points Clinically relevant imetelstat concentrations significantly inhibit CFU-MEG formation from MNCs of ET patients and reduce hTERT expression. However, similar concentrations of imetelstat do not inhibit cytokine-induced CFU-MEG from MNCs of healthy donors.

3843 Background/Aims: Essential thrombocythemia (ET), a clonal myeloproliferative neoplasia, is characterized by elevation of platelets in peripheral blood (PB) and excessive proliferation of megakaryocytes (megs) in bone marrow. Most ET patients exhibit spontaneous growth of megakaryocytic colony-forming units (CFU-Meg) in vitro. One of the most important pathways involved in clonal expansion is reactivation of telomerase, an enzyme complex which is able to compensate for the loss of telomere repeats due to cell divisions. Over 90% of malignancies demonstrate up-regulation of telomerase, which is an essential component for their immortalization. Imetelstat sodium (GRN163L) is a potent and specific telomerase inhibitor that has demonstrated in vitro and in vivo activity against various tumor types and cancer stem cells. Our aims were to study the growth of CFU-Meg and their levels of telomerase activity (TA) from cord blood (CB), mononuclear cells (MNC) from healthy individuals and ET patients, as well as to test whether TA inhibition by imetelstat could suppress CFU-Meg formation. Agents which inhibit clonal expansion in vitro may have promising clinical activity. Methods: CB cells were enriched for CD34 expressing cells using a negative cell separation system. Cells were incubated with imetelstat (1–15μM) in serum free liquid medium, StemSpan® SFEM containing a cytokine formulation designed for the development of meg progenitor cells. CB cells were cultured for a total of 17 days; at various time point cells were enumerated, assessed by flow cytometry for differentiation markers (CD41) and for TA. Mononuclear cells (MNC) from healthy individuals and from 11 ET patients (WHO 2009 criteria) were isolated from PB and suspended in IMDM or plated into collagen ± cytokines (TPO, IL3, IL6, SCF, EPO) and treated with 0, 0.1, 1 and 10 μM imetelstat or a mismatch control, and incubated for several hours (cell suspensions) or 10–12 days (collagen plus 5% CO2) at 37° C. Megs were stained and the number of CFU-Meg was scored. In addition, TA was measured in CD34+ cells, megs and MNC by TRAP assay. Results: TA is low in CD34+ cells from CB of healthy individuals, but increases up to 9 times during early megakaryocytic differentiation (CD41a+CD34-) and peaks at day 3, followed by a decrease as the cells differentiate. Imetelstat (1–15 μM) significantly inhibited TA in a concentration-dependent manner in these progenitor cells (65% to 99%). Despite considerable TA inhibition, no significant inhibition in cellular growth and differentiation of megs was found in these CD34+ cell cultures from healthy CB donors. TA is low in MNC isolated from the PB of healthy individuals and of patients with ET. Imetelstat did not inhibit CFU-Megs in healthy individuals. In contrast, imetelstat significantly (p < 0.001) and concentration-dependently inhibited the spontaneous growth of CFU-Meg in each patient with ET (n=11, 7 JAKV617F-positive and 4 JAKV617F-negative). The growth of CFU-Meg (% of control) with various imetelstat concentrations in μM was: 100% (0 μM), 107% ± 8.6% (0.1 μM), 79% ± 11.8% (1 μM) and 33% ± 9.4% (10 μM). No correlation was found with the JAKV617F mutational status, other laboratory and clinical parameters, or with cytoreductive therapies. Conclusions: In summary, we can demonstrate telomerase activation with megakaryocytic proliferation and differentiation which precedes the decrease in cellular proliferation. Furthermore, we demonstrated concentration-dependent inhibition of TA by imetelstat in cells from healthy donors, but without inhibiting megakaryocytic proliferation. In contrast, TA inhibition by imetelstat results in a dose-dependent inhibition of spontaneous growth of CFU-Meg in ET patients. Inhibition was demonstrated at clinically relevant concentrations. This in vitro CFU-Meg inhibition is independent of the JAKV617F mutational status and of cytoreductive therapy. It appears that the transient inhibition of TA in normal megakaryocytic cells by imetelstat does not inhibit cellular proliferation, whereas TA inhibition by imetelstat in malignant cells significantly inhibited CFU-Meg proliferation. These findings suggest a specificity of imetelstat for malignant megakaryocytic cells. The impact of imetelstat's clinical activity is being explored in an ongoing phase 2 study in ET patients who have failed at least one prior therapy or who refuse standard of care. Disclosures: Go: Geron Corporation: Employment. Ninomoto:Geron Corporation: Employment. Kashani:Geron Corporation: Employment. Stuart:Geron Corporation: Employment. Oppliger Leibundgut:Geron Corporation: Service Contract; Novartis: Membership on an entity's Board of Directors or advisory committees, Service Contract; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Baerlocher:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Geron Corporation: Research Funding, Service Contract; Pfizer: Membership on an entity's Board of Directors or advisory committees.