Comparison of DNMT1 inhibitors by methylome profiling identifies unique signature of 5-aza-2'deoxycytidine (expression)

Dacogen (DAC/ 5-aza-2’deoxycitidine/Aza) is currently used to treat myeloid dysplastic syndrome (MDS) and is in trials for Acute Myeloid Leukaemia (AML) and some solid cancers. As a hypomethylating agent it is thought to act by inhibiting the enzymes which add methyl groups to DNA, chief among them DNMT1. Improved targeting has been hindered by a lack of understanding of the off-target effects following treatment. Both Dacogen and DNMT1 siRNA caused overall hypomethylation in the treated cells, with the latter proving more efficient at demethylation at gene bodies in particular. Amongst the targets experiencing demethylation, some hypomethylated promoters were unique to Dacogen treatment and therefore off-target with respect to the reduction in DNMT1. An unexpected phenomenon almost exclusively caused by DAC treatment was gain in methylation. Our results suggest Dacogen is also having an important effect on methylation unrelated to the inhibition of DNMT1, suggesting further avenues for therapeutic improvements. We performed a comparative treatment of the same normosomic, non-transformed fibroblast cell line hTERT1604 over three days with either the pharmacological 5-Aza-2’deoxycitidine (Aza) or with SMARTpool siRNA directly targeting DNMT1. DNA was collected for analysis of methylation levels using Illumina Human Methylation 450k BeadChip methylation arrays. Data was analysed in R using the tailored RnBeads pipeline and in-house scripts.