Epigenetic silencing of tumor suppressor genes has been implicated as a mechanism of resistance to chemotherapy but target genes are not well defined. DNA methylation is one such mediator of epigenetic silencing. Drug-induced increased methylation is well documented, and has been suggested to be a part of acquired drug resistance [1
]. In acute myelogenous leukemia (AML), DNA methylation increases at relapse post-chemotherapy-induced complete remission (CR) [3
], and methylation at remission predicts for a high rate of disease relapse [4
]. Drugs that reverse DNA methylation are now in routine use in myeloid leukemias [5
], and it has been hypothesized that hypomethylation could reverse drug resistance in AML.
Azacitidine (5-AC) is a ring analog of the naturally occurring pyrimidine nucleoside, cytidine with replacement of carbon by nitrogen in position 5 of the cytidine heterocyclic ring. The initial step in activation of 5-AC intracellularly is the conversion to 5-azacytidine monophosphate (5-ACMP) by uridine-cytidine kinase [6
]. 5-ACMP is further phosphorylated to 5-AC di- and triphosphate by CMP-UMP-dCMP kinases and nucleoside diphosphate kinases, respectively. 5-AC triphosphate can potentially be incorporated into RNA [7
]. 5-AC diphosphate on the other hand can also be reduced to 5-azadeoxycytidine diphosphate (5-AdCDP) by ribo-nucleotide reductase. 5-AdCDP in turn can be phosphorylated by nucleoside diphosphate kinase to 5-azadeoxycytidine trisphosphate (5-AdCTP) and can be incorporated into DNA [9
]. Incorporation into RNA can inhibit the processing of ribosomal RNA from higher-molecular-weight species, disassembly of polyribosomes, and markedly inhibit protein synthesis [11
]. At millimolar concentrations of 5-AC, incorporation into DNA could inhibit DNA synthesis and induce cell cycle arrest [12
]. At lower dose, 5-AC (2–10 μM) inhibits DNA methylation through stoichiometric binding with DNA-methyltransferase (DNMT) after incorporation to DNA [13
]. Randomized phase III trials have established the activity of 5-AC in myelodysplastic syndrome (MDS) with improvement in cytopenias, decrease in progression to AML [16
] and more recently improvement in survival compared with conventional care regimens [18
Cytarabine is a nucleoside analog of deoxycytidine and is the most potent and most widely used antileukemic agent against AML. Mechanisms of resistance to cytarabine include but are not limited to reduced influx of by the hENT1 transporter, reduced phosphorylation by deoxycytidine kinase (dCK), and increased degradation by cytoplasmic 5′-nucleotidase (5NT) and/or cytidine deaminase (CDD) [19
]. In vitro
, hypomethylation induction by 5-aza-2′-deoxycitidine (decitabine) sensitizes to cell killing by cytarabine, and the combination is synergistic in myeloid leukemia cell lines [24
]. However, cytarabine and decitabine have similar intracellular metabolic pathways and shared mechanisms of resistance [25
]. Study of resistance to decitabine in leukemia cells indicated decreased incorporation of decitabine in DNA as the major mechanism and results from reduced dCK activity, increased transporters and deamination by CDD, reminiscent of resistance to cytarabine. Despite the fact that decitabine causes DNA hypomethylation at low doses, resistance to decitabine does not appear to be related to the expression of DNMT.
Based on these data, we hypothesized that reversal of DNA methylation in leukemia cells can potentially modify sensitivity to cytarabine in vivo
. Because of shared mechanisms of resistance between decitabine and cytarabine, we chose to test this hypothesis by combining 5-AC with cytarabine in patients with refractory or relapsed AML or high risk MDS. This study used a novel adaptive clinical trial design [26
] that seamlessly transitions from phase I to a randomized phase II component, allowing simultaneous assessment of toxicity and (preliminary) efficacy across a range of doses.