Abstract 1386 Suppress the dominant-negative effect of DNMT3A mutants in leukemogenesis

DNA methyltransferase DNMT3A-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) give rise to a dominant-negative effect in DNA hypomethylation in cells, which in turn contributes to pathogenesis of acute myeloid leukemia (AML) and other human diseases. To decipher the mechanism underlying the dominant effect of DNMT3A R882 mutations, we perform structural, biochemical, and cellular characterizations of wild-type and R882-mutated DNMT3A homo-oligomers. Our study uncover large enthalpy-entropy compensation associated with the formation of one oligomeric interface of DNMT3A (a.k.a. RD interface), which confers a high sensitivity of DNMT3A assembly to amino acid variation within this interface, and the distinct assembly behavior between DNMT3A and closely related DNMT3B. Mechanistically, the DNMT3A R882 mutations introduce subtle structural and dynamic perturbations to the RD interface, resulting in a shift of DNMT3A assembly toward macro-oligomeric forms. Formation of the DNMT3A macro-oligomers lead to reduced DNA binding and chromatin association, which underpins the dominant effects of DNMT3A R882 mutants in DNA hypomethylation. Taking advantage of the subtle interface variation between DNMT3A and DNMT3B, we introduce DNMT3B-converting mutations to the RD interface, which effectively inhibits the oligomerization-promoting behavior of the DNMT3A R882 hotspot mutants. In cells, fine-tuning of DNMT3A oligomerization by the rescue mutant leads to increased chromatin association of DNMT3A and genomic DNA methylation, and offsets the growth advantage of TF-1 cells conferred by the disease mutants. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered DNA hypomethylation in AML and identifies a strategy to suppress the dominant negative effect of DNMT3A hotspot mutants in leukemogenesis. This work was supported by NIH grants (R35GM119721 to J.S, R01 CA215284 and R01 CA211336 to G.G.W, and R35 ES031707 to Y.W.) and University of California Cancer Research Coordinating Committee (UC CRCC) grant (CRR-20-634140) to J.S. G.G.W. is American Cancer Society (ACS) Research Scholar and a Leukemia & Lymphoma Society (LLS) Scholar.