Treatment of myelofibrosis (MF) patients with imetelstat (Imet), a telomerase inhibitor, has been reported to lead to clinical, morphologic and molecular remissions in a subset of patients (Tefferi A, et al. N Engl J Med. 2015; 373:908), suggesting that Imet has disease-modifying activity. The precise mechanism by which Imet induces such responses has however not been reported. In this study, we investigated the effects of Imet on MF hematopoietic stem/progenitor cells (HSC)/(HPC) to address this question.
Cord blood (CB) CD34+ cells were cultured with cytokines alone or cytokines plus increasing doses of Imet (1.8uM, 3.75uM, 7.5μM) or mismatched oligonucleotide control (Mis), for 1-2 weeks. Similar numbers of Lin-CD34+, Lin-CD34+CD38- cells, CD34+ cells that expressed aldehyde dehydrogenase (ALDH) and assayable HPCs (CFU-GM+BFU-E+CFU-GEMM) were generated under each of these culture conditions (P all>0.05). By contrast, culture of MF CD34+ cells (n=14; 9 spleens and 5 peripheral blood) with 7.5uM Imet led to reduction in the numbers of Lin-CD34+ (P<0.05), Lin-CD34+CD38- (P=0.07), CD34+ALDH+ cells (P=0.05), and HPCs (CFU-GM+BFU-E+CFU-GEMM) (P<0.05) as compared with cells treated with Mis. Furthermore, Imet treatment (7.5uM) for 1 week reduced the absolute numbers of JAK2V617F+ CFU-GM by 45.8±22.5% (P=0.09, n=4) and the absolute number of JAK2V617F homozygous CFU-GM by 53.6±19.3% (P=0.03, n=4) as compared to treatment with Mis. These findings suggest Imet can preferentially deplete malignant MF HPCs, resulting in the reduction of malignant cell burden.
We further investigated the effects of Imet on normal and MF HSCs in vivo by treating NOD/SCID/IL2R null (NSG) mice transplanted with CB or MF splenic CD34+ cells directly with vehicle alone, Mis or Imet. The mice receiving CB CD34+ cells (5-10 ×105/mouse, n=3) were treated with 5mg/kg, 15mg/kg or 30mg/kg Imet using IP injection 3 times a week for 4 weeks. Four months after the transplantation (3 months after the treatment), the number of hCD45+ cells and the number of hCD34+ HSCs and HPCs in the marrows of mice receiving 5 mg/kg Imet were not reduced as compared to mice receiving vehicle alone or equal doses of Mis. By contrast mice receiving 15mg/kg Imet had modest reduction in the numbers of hCD45+ cells in the marrow (reduced by 20.9% as compared to vehicle alone and by 33.9% as compared to equal dose of Mis). In addition, the numbers of hCD45+ cells and hCD34+ cells were reduced by 25.4% and 38.0%, respectively in the marrow of the mice treated with 30mg/kg Imet as compared to the mice treated with vehicle alone. These findings indicate that the ability of Imet treatment to deplete normal HSCs is dose-dependent. In order to limit the effects of Imet on the normal HSC, 15mg/kg Imet treatment for 4 weeks was selected to treat mice transplanted with MF CD34+ cells. Four month after the transplantation, the number of hCD45+ cells in the marrow was dramatically depleted by Imet treatment in mice receiving CD34+ cells from 2 MF patients (% of hCD45+ cells relative to vehicle alone treatment: Pt 1: Mis: 54.2%, Imet: 15.7%; Pt 2: Mis: 4.1%, Imet: 0.3%). The various lineage cells belonging to both myeloid and lymphoid lineages generated by the grafts of these 2 patients were also correspondingly depleted. Moreover, depletion of hCD34+ cells in the same mice was also achieved by Imet treatment (% of hCD34+ cells relative to vehicle alone treatment: Pt 1: Mis: 44.5%, Imet: 21.9%; Pt 2: Mis: 7.0%, Imet: 4.1%). These data suggest that Imet is capable of selectively eliminating MF HSCs.
To explore the mechanisms by which Imet might affect MF CD34+ cells, the effect of the drug on apoptosis was studied. Two days after the treatment of MF CD34+ cells (n=4) with 7.5uM Imet, the percentage of MF CD34+ Annexin V+ PI− cells was significantly greater than the percentage of cells treated with Mis (P=0.002) or cytokines alone (P=0.003). The absolute number of CD34+ Annexin V+ PI− cells present in Imet containing cultures was 3.4±1.0- and 5±1.4-fold greater than in cultures treated with Mis or cytokines alone, respectively. Furthermore, Imet treatment did not induce apoptosis of CB CD34+ cells (n=3). These findings suggest that Imet is capable of selectively promoting apoptosis and inhibiting the proliferation of phenotypically and functionally defined MF but not normal HSCs/HPCs. Imet, therefore, represents a potentially promising drug for the treatment of MF which appears to affect primitive MF HSCs.
Huang: Janssen Research & Development, LLC: Employment. Wang: Janssen Research & Development, LLC: Research Funding.
Asterisk with author names denotes non-ASH members.