Epigenetic Reprogramming of Cis-Regulatory Sites By R882-Mutated DNMT3A Potentiates Aberrant Stem Cell Gene Program and Acute Leukemia Development

DNA Methyltransferase 3A (DNMT3A) is frequently mutated in various hematopoietic malignancies; however, the underlying oncogenic mechanisms remain elusive. Here, we establish ex vivo and in vivo leukemogenic models recapitulating synergy between a DNMT3A 'hotspot' mutation (i.e., DNMT3A^R882H) and kinase activation, which include a leukemia-initiating stem cell (LSC) model. We show that DNMT3A^R882H cooperates with constitutively activated RAS (NRAS G12D) in transforming murine hematopoietic stem/progenitor cells (HSPCs) ex vivo and inducing acute leukemias in vivo. Genome-wide transcriptome profiling analysis of these models reveals that DNMT3A^R882H potentiates aberrant transactivation of 'stemness' gene expression programs, notably a set of transcription factors Meis1, Hox-A, Mn1 and Mycn. Further examination by ChIP-Seq and enhanced Reduced Representation Bisulfite Sequencing (eRRBS) demonstrate that R882-mutated DNMT3A enzymes directly binds to cis-regulatory elements of these genes and induces focal CpG hypomethylation reminiscent of what was seen in human leukemias bearing DNMT3A R882 mutation. Furthermore, DNMT3A^R882H-induced DNA hypomethylation facilitates gene enhancer/promoter activation and recruitment of Dot1l-associated transcription elongation machineries. Lastly, with these established leukemogenic model systems, we also demonstrate profound differences between R882-mutated and wild-type DNMT3A in mediating oncogenic transformation, epigenetic dysregulation and gene mis-regulation. Collectively, these findings not only mechanistically explain clonal and malignant hematopoiesis found associated with DNMT3A mutation but also establish targeting transcription elongation machineries as novel therapeutic avenues for DNMT3A-mutated hematological malignancies.