<p>Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3A), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3A AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3A cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3) and the nucleophosmin gene (Npm1) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3A mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3A cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3A mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.</p>