The cytosine analogues 5-azacytosine (azacytidine) and 2′-deoxy-5-azacytidine (decitabine) display substantial therapeutic potential in patients with AML and MDS. Besides causing DNA demethylation, azanucleosides also mediate cytotoxic effects, and it appears that clinical responses are influenced by both epigenetic alterations and by apoptosis induction. However, the molecular changes induced by these drugs are still poorly understood. NK cells play an important role in tumor-immunosurveillance by confining development and progression of hematopoietic malignancies and are also important after therapeutic intervention like e.g. haploidentical stem cell transplantation. Thus it is important to define how a given therapeutic agent influences NK cell reactivity. Here we studied the effect of pharmacological concentrations of azacytidine and decitabine on NK cell effector functions. After preincubation with the azanucleosides (12h or more), NK cell cytotoxicity was found to be significantly enhanced by decitabine while, in contrast, azacytidine nearly completely abolished NK cell lysis of K562 and Raji target cells (up to 50% increase and 88% reduction, respectively; E:T ratio 10:1). Of note, modulation of NK cell cytotoxicity was also observed when the compounds were, after NK cell pretreatment, absent during the cytotoxicity assays. In contrast, neither the presence of either agent in cytotoxicity assays without preincubation nor pretreatment of target cells with either agent for 4h (corresponding to the time of the cytotoxicity assay) altered NK cell reactivity. These results indicate that azanucleosides are capable to modulate NK cell responsiveness to activating stimuli. In line, after pretreatment with the compounds NK cell IFN-γ production upon stimulation with IL-2 and IL-15 or in cocultures with target cells was found to be enhanced by decitabine but inhibited by azacytidine (up to 46% increase and 85% reduction, respectively). NK cell effector functions were not affected by deoxycytidine and cytidine, the physiological counterparts of the azanucleosides. While azacytidine treatment substantially induced NK cell apoptosis (about 30% after 24h) which may explain its inhibitory effect, no induction of apoptosis by decitabine was observed. Our data demonstrate that azacytidine and decitabine differentially affect NK cell anti-tumor reactivity and suggest that, while azacytidine causes NK cell apoptosis, decitabine enhances NK cell responsiveness via a yet unknown mechanism which is presently under study.

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