RNA-dependent inhibition of ribonucleotide reductase is a major pathway for 5-azacytidine activity in acute myeloid leukemia

Abstract

5-Azacytidine (5-azaC) is an azanucleo-side approved for myelodysplastic syndrome. Approximately 80%-90% of 5-azaC is believed to be incorporated into RNA, which disrupts nucleic acid and protein metabolism leading to apoptosis. A smaller fraction (10%-20%) of 5-azaC inhibits DNA methylation and synthesis through conversion to decitabine triphos-phate and subsequent DNA incorporation. However, its precise mechanism of action remains unclear. Ribonucleotide reductase (RR) is a highly regulated enzyme comprising 2 subunits, RRM1 and RRM2, that provides the deoxyribonucle-otides required for DNA synthesis/repair. In the present study, we found for the first time that 5-azaC is a potent inhibitor of RRM2 in leukemia cell lines, in a mouse model, and in BM mononuclear cells from acute myeloid leukemia (AML) patients. 5-azaC-induced RRM2 gene expression inhibition involves its direct RNA incorporation and an attenuated RRM2 mRNA stability. Therefore, 5-azaC causes a major perturbation of deoxyribonucleotide pools. We also demonstrate herein that the initial RR-mediated 5-azaC conversion to decitabine is terminated through its own inhibition. In conclusion, we identify RRM2 as a novel molecular target of 5-azaC in AML. Our findings provide a basis for its more widespread clinical use either alone or in combination. (Blood. 2012;119(22):5229-5238) Introduction 5-Azacytidine (5-azaC; Vidaza) is a pyrimidine analog that was first synthesized 45 years ago and later found to be a natural product from Streptoverticillium ladakanus. 1-3 Currently, 5-azaC is approved by the US Food and Drug Administration (FDA) for the treatment of myelodysplastic syndrome (MDS), but it has also been widely used for the treatment of acute myeloid leukemia (AML). 4 Despite decades of efforts made to delineate the mechanisms of the antileukemia activity for this compound in terms of its interference with RNA and DNA metabolism, 5 the precise basis of its clinical efficacy remains to be elucidated fully. 6 After cell uptake, 5-azaC is first anabolized to its nucleoside monophosphate, 5-aza-CMP, by uridine-cytidine kinase (UCK) and is eventually phosphorylated by diphosphate kinase to its triphosphate metabolite, 5-aza-CTP, which is thought to be incorporated into RNA. 7,8 Approximately 80%-90% of 5-azaC is processed through the entire sequence and incorporated into RNA. 7 This ultimately disrupts mRNA and protein metabolism, leading to apoptosis. 4,6 A small fraction (10%-20%) of the 5-azaC disphos-phate intermediate product 5-aza-CDP is instead converted to 5-aza-dCDP by ribonucleotide reductase (RR), followed by further phosphorylation to 5-azaC-dCTP (5-aza-2'-deoxycyridine triphos-phate; DAC-TP). 4,7 DAC-TP is incorporated into DNA and cova-lently binds DNA methyltransferases (DNMTs), thereby inhibiting the activity of these proteins. It has been recognized that DNMTs add methyl groups to the cytosine in the context of the CpG dinucleotides in promoter region thereby promoting gene hyper-methylation and epigenetic silencing. Thus 5-azaC has be...

Mountain high and valley deep: epigenetic controls of pluripotency and cell fate

“…To this purpose, de-methylating agents, which are wellcharacterized DNA methyltransferase (DNMT) inhibitor, were selected and used to erase DNA epigenetic restrictions. An example comes from 5-azacytidine (5-aza-CR), that, when used at low doses, substitutes for cytosine and incorporate into DNA and RNA during replication (Stresemann and Lyko, 2008;Aimiuwu et al, 2012), forming covalent adducts with DNMT1. Thanks to its powerful effects, 5-aza-CR is able to induce global DNA hypo-methylation (Christman, 2002;Stresemann and Lyko, 2008), gene reactivation (Jones, 1985), and can facilitate adult somatic cell switch from one phenotype to a different one (Taylor and Jones, 1979;Glover et al, 1986;Harris et al, 2011).…”

Demethylation Therapy as a Targeted Treatment for Human Papillomavirus–Associated Head and Neck Cancer

“…On the other hand, demethylation of CpG loci within gene bodies can paradoxically decrease gene transcription (23). Furthermore, 5-aza is incorporated into RNA (24,25), where it can alter mRNA stability (26) and therefore change expression independently of its effect on transcription. Finally, 5-aza and decitabine cause DNA damage and activate the DNA damage response in some cells; however, the nature of this DNA damage has not been fully characterized and is likely cell type-dependent (27).…”

Alteration of Runt-related Transcription Factor 3 Gene Expression and Biologic Behavior of Esophageal Carcinoma TE-1 Cells after 5-Azacytidine Intervention

“…5-AzaC, a pyrimidine ring analogue, had antiproliferation and anti-neoplasia activity in acute acute myeloid leukemia (Aimiuwu et al, 2012) and hepatocellular carcinoma (Acun et al, 2011). But the overall clinical utility of 5-AzaC was disappointing because of its activities of induce-neoplasia in mammalian cells.…”