Combination Treatment with Imetelstat, a Telomerase Inhibitor, and Ruxolitinib Depletes Myelofibrosis Hematopoietic Stem Cells and Progenitor Cells
Authors: Hu Cing Siang , Huang Fei , Hoffman Ronald , Wang Xiaoli 
Published: November 2019

Therapy with imetelstat (Ime), a telomerase inhibitor, has been shown to have disease-modifying effect in patients with myelofibrosis (MF) (Tefferi N Engl J Med 2015; Mascarenhas Blood 2018). This has been attributed to the ability of Ime to selectively deplete MF hematopoietic stem cells (HSC)/progenitor cells (HPC) (Wang Blood Adv 2018). As reported, the primary toxicities of Ime are cytopenias, however, they appear to be reversible, and most importantly, manageable without meaningful clinical consequences (Mascarenhas Blood 2018, Fenaux EHA 2019). Ruxolitinib (Rux) is the first and only JAK 1/2 inhibitor approved for use in patients with intermediate- or high-risk MF. Based on different mechanisms of action for Ime and Rux, we hypothesize that Ime in combination with Rux might create a regimen that would be more efficacious than single agent alone in depleting MF HSCs/HPCs. Using in vitro HPC assays and in vivo HSC assays, we evaluated the therapeutic potential of Rux and Ime.

MF splenic (n=7) or cord blood (CB) (n=3) CD34+ cells (2.5×104/mL) were incubated in serum free expansion medium containing cytokines in the presence of Rux (50nM) alone, Ime alone (1.8uM), Rux+Ime (simultaneous treatment) or Rux→Ime (sequential treatment). Rux was dosed for 3 days and Ime for 14 days under treatment conditions containing either or both drugs. In addition, parallel cultures with mismatched oligonucleotides (MM) or vehicle alone were performed. Compared to either drug alone or to MM control, Rux followed by Ime sequential treatment resulted in significant reductions in the numbers of MF Lin-CD34+ cells (Rux→Ime vs. Rux alone, p=0.001; vs. Ime alone, p=0.059; vs. Rux→MM, p=0.052) and assayable HPCs [(CFUGM+BFU-E+CFU-GEMM), Rux→Ime vs. Rux alone, p=0.02; vs. Ime alone, p=0.05; vs. Rux→MM, p=0.04], while the simultaneous treatment with Rux+Ime did not significantly reduce MF Lin-CD34+ cells and assayable HPCs. By contrast, none of these treatment conditions affected the behavior of normal HSCs/HPCs. Genotyping of individual colonies showed that sequential drug treatment of CD34+ cells from 2 JAK2V617+ MF patients resulted in greater reductions in both the percentage and absolute numbers of JAK2V617F+ myeloid progenitors than that achieved with Ime alone or simultaneous combination treatment. These findings suggest that an additive inhibitory activity against MF HSCs and malignant HPCs can be achieved with sequential treatment of Rux followed by Ime. We next assessed if treatment with Ime (10mg/kg, 3 times/week for 4 weeks, I.P. injection) and Rux (45mg/kg, daily for 7 days, oral gavage) had additive effects on MF or normal HSCs by directly treating NSG mice transplanted with MF or CB CD34+ cells with these two drugs either alone, in combination simultaneously or sequentially. Mice were sacrificed 4 months after the transplantation. Both simultaneous and sequential drug treatment of mice transplanted with MF splenic CD34+ cells resulted in greater reductions in the degree of human CD45+ marrow cell chimerism (relative to vehicle alone: Rux→Ime: 54.0%; Rux+Ime: 72.4%; Rux alone: 76.6%, Ime alone: 80.0%) and human CD45+ splenic cell chimerism (relative to vehicle alone: Rux→Ime: 13.8%; Rux+Ime: 31.9%; Rux alone: 163.1%; Ime alone: 42.4%) as compared with either drug alone treatment. Similar to our findings in the above in vitro studies, a greater reduction in the absolute number of human CD34+ cells was observed with sequential but not simultaneous combination treatment as compared with either drug alone treatment in the marrow of the mice transplanted with MF CD34+ cells (relative to vehicle alone: Rux→Ime: 36.0%; Rux+Ime: 61.0%; Rux alone: 47.5%, Ime alone: 57.3%). Furthermore, similar results were also observed in depletion of MF long-term HSCs in mice receiving splenic CD34+ cells from an additional patient. However, this same sequential drug schema did not affect normal HSC function. Collectively, these data indicate that alterations of scheduling of the administration of Rux and Ime affect the efficacy of this drug combination in depleting MF HPC/HSCs. We propose that cycles of Rux followed by Ime represents a potentially effective therapeutic strategy that is capable of eliminating MF HSCs/HPCs with an acceptable toxicity profile.

Disclosures

Hoffman: Merus: Research Funding.