Effect of combined deferasirox and 5-azacytidine treatment on human leukemia cells in vitro.

Abstract

Dear Editor, Deferasirox (DFX) is an oral iron chelator that is used to reduce iron overload in patients with bone marrow failure, such as myelodysplastic syndrome (MDS) [1]. DFX is occasionally administered to patients with iron overload who have been treated with 5-azacytidine (AZA), which is a DNA demethylating agent approved for the treatment of patients with MDS [2]. Four clinical trials are currently underway regarding treatment with a combination of AZA and DFX in higher risk MDS or acute myeloid leukemia patients (ClinicalTrials.gov identifier: NCT02038816, NCT02341495, NCT02159040, and NCT01718366). Iron is indispensable not only for the activities of DNA demethylases [3] but also for the deoxidization of ribonucleotide triphosphates [4], which is essential for the incorporation of AZA to DNA [5]. However, the pharmacological interaction between DFX and AZA has not been fully elucidated. In this study, we focused on the effect of DFX in AZA-treated human leukemia cell lines (U937 and HL-60 cells). To determine the optimal concentration of DFX, cells were incubated with 3 to 9 μM DFX. The half maximal inhibitory concentration (IC50) of DFX was 7 μM for U937 and 3.6 μM for HL-60. We therefore used 3 μM of DFX, which is less than the IC50, for the subsequent experiments. Regarding the effect of the combination of AZA and DFX on cell growth inhibition, we did not find any difference between AZA treatment and AZA and DFX treatment in U937 cells. Of note is that the growth inhibitory effect on HL-60 cells was weakened by the combined treatment with AZA and DFX (Fig. 1a). We then investigated global DNA methylation levels in DFX-treated U937 and HL-60 cells. Relative quantification of global DNA methylation by measuring 5-methylcytosine (5-mC) levels was performed using MethylFlash Methylated DNA 5-mC Quantification Kit (Colorimetric) (Epigentek, Farmingdale, NY, USA) according to manufacturer’s instructions. The level of 5-mC was significantly increased in DFX-treated HL-60 cells (Fig. 1b), whereas it was not clearly increased in DFX-treated U937 cells. We did not find an increase in the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and DNMT3A in the DFX-treated cells (data not shown). To date, the molecular mechanism by which DFX induces hematological improvement in patients with bone marrow failure syndrome has not been investigated in detail [6–8], although we have previously shown that DFX inhibits mammalian target of rapamycin (mTOR) signaling in myeloid leukemia cells [9]. More detailed analyses are required regarding the specific genomic regions of 5-mC accumulation, to understand the mechanism underlying the epigenetic modifications caused by DFX under AZA treatment. We conclude that DFX may weaken the growthinhibitory effect of AZA together with an increase in the level of 5-mC, and AZA and DFX do not exert synergistic cytotoxic effects in vitro. As hematological improvement in patients with MDS does not depend solely on the cytotoxic effects of AZA, the complex * Satoshi Imanishi s-ima@tokyo-med.ac.jp