The cytosine analogues azacytidine and decitabine are currently being developed 
as drugs for epigenetic cancer therapy. Although various studies have shown that 
both drugs are effective in inhibiting DNA methylation, it has also become clear 
that their mode of action is not limited to DNA demethylation. Because 
azacytidine is a ribonucleoside, the primary target of this drug may be cellular 
RNA rather than DNA. We have now analyzed the possibility that azacytidine 
inhibits the RNA methyltransferase DNMT2. We found that DNMT2 is variably 
expressed in human cancer cell lines. RNA bisulfite sequencing showed that 
azacytidine, but not decitabine, inhibits cytosine 38 methylation of tRNA(Asp), 
a major substrate of DNMT2. Azacytidine caused a substantially stronger effect 
than decitabine on the metabolic rate of all the cancer cell lines tested, 
consistent with an effect of this drug on RNA metabolism. Of note, drug-induced 
loss of RNA methylation seemed specific for DNMT2 target sites because we did 
not observe any significant demethylation at sites known to be methylated by 
other RNA methyltransferases. Our results uncover a novel and quantifiable drug 
activity of azacytidine and raise the possibility that tRNA hypomethylation 
might contribute to patient responses.