Eukaryotic DNA methylation, an integral component of the epigenetic machinery, prevents genomic instability by regulating the expression of oncogenes and tumor suppressor genes. The importance of dysregulated DNA methylation in diverse blood cancers including Acute Myeloid Leukemia (AML) pathogenesis is highlighted by the strong correlation between mutations in the de novo DNA methyltransferase gene, DNMT3A, and adult patients with poor prognoses. We show that clinically observed DNMT3A mutations result in dramatic changes in enzyme activity, including mutations that lead to 6‐fold hypermethylation and 3‐fold hypomethylation on the p15 human promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN‐AML) constitutes 40–50% of all AML cases, is the most epigenetically diverse AML subtype and has pronounced changes in DNA methylation in non‐CpG regions. We identified a subset of mutations in DNMT3A that lead to a 2–8 fold enhancement in the enzyme's ability to perform non‐CpG methylation. Many of the mutations studied map to regions on the protein that are well known to interact with partner proteins, which themselves contribute to AML, such as the DNA base excision repair (BER) enzyme Thymine DNA glycosylase (TDG). Here we show that DNMT3A mutations cause significant and diverse changes in the ability of these regulatory partner proteins to affect DNMT3A function. Our results present a link between DNMT3A mutations and the disruption of the epigenetic landscape in AML. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A myriad of protein partners modulate the activity of the human DNA methyltransferase 3A (DNMT3A), whose interactions with these other proteins are frequently altered during oncogenesis. We show here that the tumor suppressor p53 decreases DNMT3A activity by forming a heterotetramer complex with DNMT3A. Mutational and modeling experiments suggested that p53 interacts with the same region in DNMT3A as does the structurally characterized DNMT3L. We observed that the p53-mediated repression of DNMT3A activity is blocked by amino acid substitutions within this interface, but surprisingly, also by a distal DNMT3A residue, R882H. DNMT3A R882H occurs frequently in various cancers, including acute myeloid leukemia, and our results suggest that the effects of R882H and other DNMT3A mutations may go beyond changes in DNMT3A methylation activity. To further understand the dynamics of how protein-protein interactions modulate DNMT3A activity, we determined that p53 has a greater affinity for DNMT3A than for DNMT3L and that p53 readily displaces DNMT3L from the DNMT3A:DNMT3L heterotetramer. Interestingly, this occurred even when the preformed DNMT3A:DNMT3L complex was actively methylating DNA. The frequently identified p53 substitutions (R248W and R273H), whereas able to regulate DNMT3A function when forming the DNMT3A:p53 heterotetramer, no longer displaced DNMT3L from the DNMT3A:DNMT3L heterotetramer. The results of our work highlight the complex interplay between DNMT3A, p53, and DNMT3L and how these interactions are further modulated by clinically derived mutations in each of the interacting partners. This work was supported by Center for Hierarchical Manufacturing NationalScience Foundation award 1808775. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S3.

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DNA methylation and histone tail modifications are interrelated mechanisms involved in a wide range of biological processes, and disruption of this crosstalk is linked to diseases like acute myeloid leukemia (AML).  In addition, DNMT3A activity is modulated by several regulatory proteins, including p53 and TDG.  However, the relative role of histone tails and regulatory proteins in the simultaneous coordination of DNMT3A activity remains obscure.  We observed that DNMT3A binds H3 tails and p53 or TDG at distinct allosteric sites to form DNMT3A-H3 tail-p53 or -TDG multiprotein complexes.  Functional characterization of DNMT3A-H3 tail-p53 or -TDG complexes on human-derived synthetic histone H3 tails, mono- or polynucleosomes shows p53 and TDG play dominant roles in the modulation of DNMT3A activity.  Intriguingly, this dominance occurs even when DNMT3A is actively methylating nucleosome substrates.  The activity of histone-modifiers is influenced by their ability to sense modifications on histone tails within the same nucleosome or histone tails on neighboring nucleosomes.  In contrast, we show here that DNMT3A acts on DNA within a single nucleosome, on nucleosomal DNA within adjacent nucleosomes, and DNA not associated with the DNMT3A-nucleosome complex.  Our findings have direct bearing on how the histone code drives changes in DNA methylation and highlight the complex interplay between histone tails, epigenetic enzymes and modulators of enzymatic activity.

We previously identified two structurally related pyrazolone (compound 1) and pyridazine (compound 2) allosteric inhibitors of DNMT3A through screening of a small chemical library. Here, we show that these compounds bind and disrupt protein–protein interactions (PPIs) at the DNMT3A tetramer interface. This disruption is observed with distinct partner proteins and occurs even when the complexes are acting on DNA, which better reflects the cellular context. Compound 2 induces differentiation of distinct myeloid leukemia cell lines including cells with mutated DNMT3A R882. To date, small molecules targeting DNMT3A are limited to competitive inhibitors of AdoMet or DNA and display extreme toxicity. Our work is the first to identify small molecules with a mechanism of inhibition involving the disruption of PPIs with DNMT3A. Ongoing optimization of compounds 1 and 2 provides a promising basis to induce myeloid differentiation and treatment of diseases that display aberrant PPIs with DNMT3A, such as acute myeloid leukemia.