# DNMT3B Controls Enhancer-Linked Chromatin and Cell Cycle Networks in Acute Myeloid Leukemia

## TL;DR

High levels of DNMT3B in acute myeloid leukemia (AML) help cancer cells survive, and blocking DNMT3B makes these cells more vulnerable to treatment with venetoclax.

## Contribution

DNMT3B overexpression in AML is driven by enhancer-linked chromatin changes, not mutations, and inhibiting it increases sensitivity to BCL-2 inhibitors.

## Key findings

- DNMT3B overexpression in AML is linked to enhancer-associated chromatin activation, not mutations.
- Inhibiting DNMT3B disrupts DNA methylation and chromatin at cell-cycle and survival genes, inducing apoptosis.
- Combining DNMT3B inhibition with BCL-2 inhibitors like venetoclax shows cooperative cytotoxicity in AML models.

## Abstract

Acute myeloid leukemia (AML) cells with high levels of the epigenetic regulator DNMT3B show poor patient outcomes and appear to rely on DNMT3B-controlled DNA regulatory elements to maintain growth and survival. We found that DNMT3B overexpression is driven by changes in gene control regions rather than mutations, and that inhibiting DNMT3B selectively remodels DNA methylation and chromatin at key cell-cycle and survival genes without globally erasing methylation. This disruption induces replication stress and programmed cell death and, importantly, rewires the balance of pro- and anti-apoptotic signals to make leukemia cells markedly more sensitive to the BCL-2 inhibitor venetoclax. These findings identify DNMT3B as a central epigenetic stabilizer of leukemic fitness and support combined DNMT3B and BCL-2 targeting as a rational therapeutic strategy for high-risk AML.

Background: DNMT3B is frequently overexpressed in molecular subsets of acute myeloid leukemia (AML) and is associated with poor prognosis. Unlike DNMT3A, DNMT3B is rarely mutated, suggesting dysregulation through epigenetic mechanisms. The regulatory basis and downstream consequences of DNMT3B overexpression in AML remain incompletely defined. Methods: We integrated analyses of BeatAML, TCGA, and BLUEPRINT cohorts with multi-omic profiling (RNA-seq, DNA methylation, ATAC-seq, and proteomics) in DNMT3B-high AML models. Nanaomycin A (NanA) was used as a DNMT3B-directed functional probe to interrogate cis-regulatory remodeling, transcriptional circuitry, and apoptotic dependencies. Results: DNMT3B overexpression was linked to enhancer-associated chromatin activation rather than recurrent genetic mutation, particularly in CEBPA- and NPM1-mutant AML. NanA exposure produced focal epigenomic remodeling, including 6900 differentially methylated CpGs, with 268 CpGs located within regions of altered chromatin accessibility. These changes were accompanied by coordinated transcriptomic and proteomic reprogramming enriched for cell-cycle, checkpoint, and stress-response pathways. Functionally, DNMT3B perturbation induced redistribution of cell-cycle phases with increased S-phase fraction and progressive apoptosis. Transcriptional profiling demonstrated induction of BH3-only sensitizers (NOXA, PUMA), repression of BCL2, and compensatory upregulation of MCL1 and BCL-XL, collectively reshaping apoptotic dependency. Combined DNMT3B perturbation and BCL2 inhibition produced cooperative cytotoxicity in DNMT3B-high AML models. Conclusion: DNMT3B functions as a context-dependent epigenetic regulator linking enhancer-associated chromatin organization with proliferative control and apoptotic resistance in AML. DNMT3B-directed epigenetic perturbation remodels cis-regulatory circuitry and is associated with increased venetoclax responsiveness, supporting DNMT3B-governed networks as a candidate co-targeting axis in high-risk AML.