Bone Marrow Ribonucleotide Reductase mRNA Levels and Methylation Status As a Prognostic Factor in Patients with Myelodysplastic Syndrome Treated with 5-Azacytidine

Ribonucleotide Reductase (RNR) is the enzyme that converts ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of two subunits, termed subunit 1 (RRM1) and 2 (RRM2). Imbalance in the regulation of RNR activity and control of dNTPs' pool leads to genomic instability and increases mutation rate. The amount of the enzyme present in the cell and allosteric mechanisms with positive and negative effectors control its activity. 5-Azacytidine (AZA) has been found to be a potent RRM2 inhibitor in acute myeloid leukemia. High expression of RNR is associated with chemoresistance and poorer overall survival in several malignancies and antisense inhibitors that bind to the enzyme mRNA have shown promising results. Moreover, RNR overexpression is a potential mechanism for chemoresistance to nucleoside analogs competing for DNA incorporation. These important roles have made RNR an attractive therapeutic target. Aim. We investigated the potential role of RNR as a prognostic factor in patients with MDS and the correlation of bone marrow RNR mRNA levels with treatment response to AZA. Methods. Bone marrow samples were collected from patients with MDS at diagnosis. RNA extraction and reverse transcription were performed using standard protocols. A Taqman based real-time PCR was performed on a CFX96 RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). For both the housekeeping and target genes, a Taqman primer/probe mix was used according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). RRM1 and RRM2 mRNA levels were expressed as an RRM1-2/beta-actin ratio. A High-Resolution Melt (HRM) analysis for mutation detection of 8 genes of interest in MDS (IDH1, IDH2, SRSF2, SFEB1, UAF1, ASXL2, DNMT3A, BCOR) and quantification of the methylation levels of RRM1/RRM2 promoters were also carried out in a subset of 63 samples. IBM SPSS statistics, version 23.0 (IBM Corporation, North Castle, NY, USA) was used for the analysis of the results. Results The study included 123 patients diagnosed with MDS per the WHO 2008/2016 classification; 98 of them were treated with AZA. The basic characteristics of the patients are shown in Table 1. The median mRNA levels of RRM1 were 4.2 times higher in non-responders (p=0.019) in comparison to responders. The levels of RRM2 did not differ between the two groups. We found no correlation of any other of the studied factors (MDS type, IPSS, IPSS-R, WPSS, karyotype risk, number of c