Inhibition of Telomerase with Imetelstat Is Detrimental to Leukemia Stem Cells in Acute Myeloid Leukemia (AML)
Authors: Bruedigam Claudia , Bagger Frederik Otzen , Heidel Florian H , Kuhn Catherine , Guignes Solene , Song Axia , Austin Rebecca , Vu Therese , Lee Erwin M , Moore Andrew S. , Lock Richard B. , Bullinger Lars , Hill Geoff R , Lane Steven W 
Published: October 2019

Abstract

Telomerase is essential to maintain the long-term replicative potential in many cancers including AML. We have recently shown that genetic deletion of telomerase eradicates long-term LSC function in murine AML, mediated by p53-dependent cell cycle arrest and LSC apoptosis.
Novel inhibitors of telomerase have recently entered clinical trials for a variety of malignancies including imetelstat (GRN163L), a competitive inhibitor that binds to the RNA template (TERC) of the telomerase holoenzyme. Here we investigated the therapeutic potential of pharmacologic telomerase inhibition in vivo using primary AML patient sample xenograft transplantation models.
Xenografts were established using the NSGS model as recipient and donor patient samples of various AML subtypes including MLL-AF9 translocations (AML-X, AML-18), normal cytogenetics with FLT3-ITD mutation (AML-16), or complex cytogenetics (AML-5).
AML engraftment was confirmed by flow cytometry prior to the commencement of GRN163L therapy and found to be similar between groups. Administration of imetelstat prevented the in vivo expansion of AML cells from all subtypes tested as measured by human-specific CD45 flow cytometry in the peripheral blood and spleen.
Prolonged treatment with imetelstat (AML-X) prevented AML expansion in vivo and remained cytostatic after imetelstat treatment, until disease progression 5 weeks after the completion of treatment. Imetelstat treatment was associated with prolonged overall survival compared to vehicle control (p < 0.0001).
In order to identify the cellular mechanism of imetelstat-mediated inhibition of AML expansion, we performed cell cycle flow cytometry of LSCs from AML xenografts at least three weeks after start of treatment. LSCs from imetelstat-treated xenografts showed marked reduction in G0 (p < 0.0002) but unchanged G1 and S/G2/M percentages demonstrating enforced cell cycle entry or impaired re-entry into quiescence. Gamma-H2AX staining demonstrated significantly increased DNA damage (p < 0.0001) in all AML subtypes analyzed.
Next, to determine the effect of imetelstat on bone marrow LSCs, we examined LSCs by immunophenotype. There was depletion of immunophenotypic LSCs and expansion of the differentiated AML population in AML-16 (normal cytogenetic, Flt3 ITD+ve) xenografts. Concordantly, the engraftment and proliferation of AML-16 cells in secondary recipients was significantly delayed, impairing the in vivo expansion of this sample. In contrast, imetelstat prevented AML expansion without depletion in immunophenotypic LSCs or delay in disease relapse after transplantation in the AML-5 xenografts (containing complex cytogenetics, predicting resistance to chemotherapy and dismal clinical outcome). Interestingly, telomerase activity was ten fold higher in this sample than AML-16.
We therefore investigated whether imetelstat could impair disease relapse after chemotherapy, a highly clinically relevant model of LSC function. AML-5 transplanted recipient mice were treated with chemotherapy, imetelstat, a combination of imetelstat and chemotherapy, or vehicle control. Chemotherapy alone caused a striking depletion in AML cells, however there was rapid relapse and expansion of the residual hCD45+ AML cells. In contrast, chemotherapy combined with imetelstat prevented hCD45+ cell expansion in recipient mice, an effect that was more pronounced than treatment with imetelstat alone.
These data demonstrate the efficacy of pharmacologic inhibition of telomerase using imetelstat in human AML and propose a novel paradigm of AML therapy that is to delay or prevent relapse by targeting AML stem cells in vivo.
Together, these data demonstrate a requirement for telomerase in human LSC function from a wide range of AML subtypes and identify telomerase as a tractable therapeutic target in human AML.

Disclosures
No relevant conflicts of interest to declare.