Structure-guided functional suppression of AML-associated DNMT3A R882 mutations [methylation]

DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote macro-oligomer formation, leading to aberrant DNA methylation that in turn contributes to pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the hotspot mutation-induced functional mis-regulation of DNMT3A remains unclear. Here, we report the crystal structure of DNMT3A methyltransferase (MTase) domain, revealing a molecular basis for its DNMT3B-distinct oligomerization behavior. Introducing DNMT3B-converting mutations to DNMT3A R882 mutants also led to structure determination of R882H- and R882C-mutated DNMT3A, which show enhanced intermolecular contacts than wild-type DNMT3A. Consistently, our in vitro and genomic DNA methylation analyses reveal that the DNMT3B-converting mutations eliminate the gain-of-function effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy. TF-1 cell line was stably expressed with DNMT3A (isoform 1), including wild-type (WT) or AML-associated hotspot mutant (either R882C or R882H) or the one carrying an additional macro-oligomerization-decreasing mutation, namely, R676K (i.e., WT-K), M674T/R676K (WT-TK), R882C/R676K (CK), R882C/M674T/R676K (CTK), R882H/R676K (HK), or R882C/M674T/R676K (HTK). The total genomic DNA was extracted, subjected for bisulfite conversion, and then analyzed with the Illumina Infinium Methylation EPIC arrays. Replicated samples (n = 3-4) were used per group.