Mutation of DNA methyltransferase 3A at arginine 882 DNMT3AR882mut relied on a motif involved in heterodimer-(DNMT3AR882mut) is prevalent in hematologic cancers and ization, whereas its various chromatin-binding domains were disorders. Recently, DNMT3AR882mut has been shown to have dispensable. Mutation of the heterodimerization motif that hypomorphic, dominant-negative, and/or gain-of-function interferes with DNMT3AR882mut binding to endogenous wild-effects on DNA methylation under different biological con-type DNMT proteins partially reversed the CpG hypomethyla-texts. However, the causal role for such a multifaceted effect of tion phenotype caused by DNMT3AR882mut, thus supporting a DNMT3AR882mut in leukemogenesis remains undetermined. dominant-negative mechanism in cells. In mice, bromodo-Here, we report TF-1 leukemia cells as a robust system useful main inhibition repressed gene-activation events downstream for modeling the DNMT3AR882mut-dependent transformation of DNMT3AR882mut-induced CpG hypomethylation, thereby and for dissecting the cause–effect relationship between mul-suppressing leukemogenesis mediated by DNMT3AR882mut. tifaceted activities of DNMT3AR882mut and leukemic transfor-Collectively, this study reports a model system useful for mation. Ectopic expression of DNMT3AR882mut and not wild-studying DNMT3AR882mut, shows a requirement of the dom-type DNMT3A promoted TF-1 cell transformation character-inant-negative effect by DNMT3AR882mut for leukemogenesis, ized by cytokine-independent growth, and induces CpG hypo- and describes an attractive strategy for the treatment of leu-methylation predominantly at enhancers. This effect was dose kemias carrying DNMT3AR882mut. dependent, acted synergistically with the isocitrate dehydro-genase 1 (IDH1) mutation, and resembled what was seen in Significance: These findings highlight a model system human leukemia patients carrying DNMT3AR882mut. The to study the functional impact of a hotspot mutation of transformation- and hypomethylation-inducing capacities of DNMT3A at R882 in leukemia.