Telomerase Inhibition Impairs Self-Renewal of ß-Catenin Activated Myeloproliferative Neoplasm Progenitors

Introduction

Chronic phase myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML) and myelofibrosis (MF), arise from hematopoietic stem cells. However, they harbor varying propensities to undergo blast crisis (acute leukemic) transformation based on their capacity to give rise to tyrosine kinase inhibitor (TKI) resistant myeloid progenitors that activate self-renewal pathways, such as Wnt/b-catenin signaling. Because b-catenin has been reported to transcriptionally regulate human telomerase reverse transcriptase (hTERT), we investigated the capacity of a telomerase complex inhibitor, imetelstat, to prevent malignant progenitor self-renewal. The telomerase complex consists of hTERT, an RNA template subunit (TERC), and a protective shelterin scaffold. Imetelstat is a novel, first in class covalently lipidated 13-mer oligonucleotide telomerase inhibitor with clinical activity in myeloid malignancies. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms such as MF. However, the role of imetelstat in selectively eradicating self-renewing MPN progenitors has not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized MPN progenitor primagraft studies to investigate the ability of imetelstat to selectively inhibit malignant progenitor self-renewal at doses that spare normal progenitors as well as to determine the mechanism of action.

Methods and Results

When FACS-purified progenitors from human blast crisis (BC; n=9) were compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples, RNA-seq revealed upregulation of key genes in the TERT interactome suggesting a role for TERT activation in MPN progenitor blast crisis transformation. Human MPN progenitor supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 µM significantly inhibited (p <0.001, ANOVA) in vitro replating of BC CML (n=5) compared with age-matched normal bone marrow progenitors. Also, imetelstat (5 µM) significantly reduced replating of MF (n=9) compared with normal progenitor (n=4) samples. Treatment of humanized mouse models, established with four different BC CML samples, with 30 mg/kg of imetelstat three times a week for four weeks resulted in a significant reduction in both bone marrow and spleen malignant progenitor burden compared with mismatch controls (p=0.003, and p=0.009, t test), and a significant reduction in human CD45⁺ cells in the splenic niche compared with mismatch controls (p=0.03, t test). In a MF model established in NSG-S mice, imetelstat also reduced human CD45⁺ cell engraftment (p=0.03, t test). Notably, imetelstat treatment spares normal human cells in humanized normal stem cell mouse models. Furthermore, qRT-PCR showed decreased β-catenin transcript levels in imetelstat treated MF cells (p=0.18) and CML cells (p=0.4) compared with mismatch control. Finally, FACS analysis revealed a significant reduction in activated b-catenin protein levels in engrafted human progenitors after imetelstat treatment (p=0.02) compared with vehicle control.

Conclusions

Niche responsive interactions between the telomerase complex and the Wnt/b-catenin self-renewal pathway sensitize b-catenin activated MPN progenitors to imetelstat in both in vitro and in vivo humanized pre-clinical MPN mouse models thereby providing a strong rationale for studies assessing eradication of malignant progenitors using imetelstat.

Disclosures: This work was funded by the Moores Family Foundation, the Koman Family Foundation, the California Institute for Regenerative Medicine (DR1-01430), Janssen Research & Development, LLC, and by NIH NCI R21CA189705.