Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine

May Levin,Bluma Berman,Yehuda G Assaraf,Michal Stark

doi:10.1038/s41419-019-1626-x

May Levin, Bluma Berman + Show 2 more

Open Access

https://doi.org/10.1038/s41419-019-1626-x

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Abstract

Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines’ sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.

Highlights

Acute myeloid leukemia (AML) is a hematopoietic malignancy of the myeloid lineage displaying rapid proliferation and accumulation of undifferentiated myeloid cells in the bone marrow, thereby interfering with the production and maturation of normal blood cells
Cytarabine uptake via equilibrative nucleoside transporters (ENTs)[5] and concentrative nucleoside transporter 3 (CNT3)[6], is followed by three consecutive phosphorylation steps resulting in the cytotoxic metabolite AraCTP1
We found that Cytarabine-resistant sublines displayed hypersensitivity to a combination of HU and azidothymidine (AZT), compared to parental cells; this combination exhibited a marked synergistic activity on hematopoietic cells including primary cells from AML patient specimens, which was potentiated upon acquisition of Cytarabine-resistance

Summary

Introduction

Acute myeloid leukemia (AML) is a hematopoietic malignancy of the myeloid lineage displaying rapid proliferation and accumulation of undifferentiated myeloid cells in the bone marrow, thereby interfering with the production and maturation of normal blood cells. Since 1973, AML treatment relies on the anchor pro-drug Cytarabine (cytosine arabinoside, Ara-C), a cytidine analog[1,2,3]. Whereas 70–80% of AML patients of them relapse for which no salvage regimen currently exists[2,3,4]. The rate-limiting enzyme in Cytarabine phosphorylation is deoxycytidine kinase (dCK), a central enzyme in the nucleoside salvage pathway (NSP)[7,8,9], which phosphorylates the naturally occurring deoxycytidine, deoxyadenosine and deoxyguanosine to their monophosphate form[8]. Loss of dCK activity is a bona fide

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