Effects of Imetelstat on CD34+ Cells of Patients with Myelofibrosis
Authors: Wang Xiaoli , Hu Cing Siang , Li Yan , Qiu Jiajing , Lam Morgan , Eng Kevin , Hoffman Ronald 
Published: October 2019

At present, there is no known therapeutic agent capable of altering the natural history of myelofibrosis (MF); this can only be achieved by allogeneic stem cell transplantation. Imetelstat is a potent and specific inhibitor of telomerase. An investigator initiated clinical trial in MF showed that imetelstat can achieve complete clinical remissions by IWG criteria. This includes the reversal of bone marrow fibrosis and induction of morphologic and molecular remissions in a subset of patients with MF. This suggests that imetelstat has disease-modifying activity in MF (Tefferi A, et al. 55th ASH Annual Meeting; December 7-10, 2013; New Orleans, LA). However, the mechanism by which imetelstat achieves these beneficial effects remains poorly understood. Therefore we investigated the effects of imetelstat on MF CD34+cells.

MF splenic or CB CD34+ cells (2.5×104/mL) were incubated in serum free medium (SFEM) alone or SFEM supplemented with 50 ng/ml SCF, 100 ng/ml FLT-3 ligand (FL), 100 ng/ml TPO, and 50 ng/ml IL-3 in the presence of imetelstat or a mismatched oligonucleotide (Mismatch, 1.8uM, 3.75uM, 7.5μM, Geron) or vehicle alone for 2-14 days. Cells were then enumerated and stained with CD34, a lineage cocktail, CD38, CD15, and Glycophorin A (GlyA) mAbs and analyzed by flow cytometry. After the treatment of cultured normal CB CD34+ cells for one week with imetelstat (1.8uM, 3.75uM, 7.5μM), mismatch or cytokines alone, numbers of total cells, Lin-CD34+ (HSCs/HPCs), Lin-CD34+CD38- (primary HSCs), Lin-CD34+CD38+ cells (HPCs), assayable hematopoietic colonies (HC, CFU-GM, BFU-E) as well as CD34-GlyA+ (erythrocytes) and CD34-CD15+ cells (myeloid cells) were not significantly changed (P all>0.05). Moreover, the amount of CB CD34+cells that expressed aldehyde dehydrogenase (ALDH), a functional stem cell marker, was not significantly changed with imetelstat or mismatch treatment (P all >0.05). These findings suggest that imetelstat at the doses studied has minimal effects on normal CB hematopoiesis.

By contrast, imetelstat was capable of selectively inhibiting in vitro MF hematopoiesis. After treatment of MF CD34+ cells (n=7; 6 MF spleens and 1 MF PB) for one week with 7.5 uM imetelstat, the total number of MF cells (36.4%, P=0.058), Lin- CD34+ (50.1%, P=0.02), Lin- CD34+CD38- (41.9%, P=0.07), CD34+ALDHhigh (49.1%, P=0.01), Lin-CD34+CD38+ cells (48.1%, P=0.04), CFU-GM (34.6%, P=0.02) and BFU-E (15%, P=0.2) as well as CD34-CD15+ (47.1%, P=0.09) and CD34-GlyA+cells (47.7%, P=0.02) was reduced compared to treatment with mismatch. After one week of culture, individual colonies (CFU-GM and BFU-E/CFU-E) from 3 JAK2V617F-positive MF patients (JAK2V617F allele burden: 78-90%) were plucked and analyzed for the JAK2V617Fusing a nested allele-specific PCR. Two out of three patients did not have any wild type (WT) JAK2 HCs before or following treatment with imetelstat. In one patient with a reservoir of JAK2V617F WT HCs, the percentage of JAK2V617F-positive HCs was reduced to 9% by imetelstat treatment as compared to 41% observed in cultures exposed to cytokines alone and 57% in cultures exposed to mismatch. In this single patient the JAK2V617Fhomozygous HCs were completely eliminated by imetelstat treatment, while 29% and 50% of the HCs remained homozygous after exposure to cytokines alone or mismatch, respectively. To explore the mechanisms by which imetelstat might reduce MF CD34+ cell counts, the ability of the drug to induce apoptosis was studied. After two days of treatment with 7.5uM of imetelstat (n=4), the percentage of MF CD34+ Annexin V+ PI− cells was significantly greater (32.2% ± 6.0%) than cells treated with mismatch (11.21± 1.4%; P=0.002) or cytokines alone (4.7% ± 0.2%, P =0.003). The absolute number of CD34+ Annexin V+ PI− cells present in imetelstat containing cultures was 3.4±1.0-and 5±1.4-fold greater than in cultures treated with mismatch or cytokines alone, respectively. Furthermore, imetelstat treatment did not induce apoptosis of CB CD34+ cells. These findings suggest that imetelstat is capable of selectively promoting the apoptosis and inhibiting the proliferation of phenotypically and functionally defined MF but not normal CD34+ cells. Furthermore, imetelstat in some patients can preferentially deplete malignant MF HPCs. Imetelstat, therefore, represents a potentially promising drug for the treatment of MF which appears to affect primitive MF HSCs.

Disclosures

Wang: Geron Corporation: Research Funding. Lam:Geron Corporation: Employment. Eng:Geron Corporation: Employment. Hoffman:Geron Corporation: Consultancy.