Epigenetic Perturbations by Arg882-Mutated DNMT3A Potentiate Aberrant Stem Cell Gene-Expression Program and Acute Leukemia Development

Abstract

SUMMARY DNA methyltransferase 3A (DNMT3A) is frequently mutated in hematological cancers; however, the underlying oncogenic mechanism remains elusive. Here, we report that DNMT3A mutational hotspot at Arg882 (DNMT3AR882H) cooperates with NRAS mutation to transform hematopoietic stem/progenitor cells and induce acute leukemia development. Mechanistically, DNMT3AR882H directly binds to and potentiates transactivation of stemness genes critical for leukemogenicity including Meis1, Mn1 and Hoxa gene cluster. DNMT3AR882H induces focal epigenetic alterations, including CpG hypomethylation and concurrent gain of active histone modifications, at cis-regulatory elements such as enhancers to facilitate gene transcription. CRISPR/Cas9-mediated ablation of a putative Meis1 enhancer carrying DNMT3AR882H-induced DNA hypomethylation impairs Meis1 expression. Importantly, DNMT3AR882H-induced gene expression programs can be repressed through Dot1l inhibition, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.

Structural basis for DNMT3A-mediated de novo DNA methylation

“…To achieve co-expression of the wildtype (WT) and mutant DNMT3A at equal levels in cells, we engineered a T2A-based fusion construct consisting of the mutant cDNA, which was added with an N-terminal 3×Flag-(GGGGS) 3 -Myc tag to differentiate its protein size from non-tagged WT DNMT3A, followed by a T2A peptide sequence at its C-terminus and the cDNA of non-tagged WT DNMT3A. Myc-tagged full-length human DNMT3A isoform 1 were cloned into MSCV Pac retroviral vector as previously described 24 . All plasmid sequences were verified by sequencing.…”

“…Genomic DNA of each sample was added with 0.5% of unmethylated lambda DNA (Promega) as spike-in control and subjected to eRRBS using MethylMidi-seq (Zymo Research) as described before 24 . In brief, approximately 300 ng of DNA were digested with three restriction enzymes (80 units of MspI, 40 units of BfaI and 40 units of MseI) to improve genomic DNA fragmentation and coverage.…”

“…Sanger bisulfite sequencing was carried out as previously described 24 . Briefly, genomic DNA was prepared using the DNeasy Blood & Tissue Kit (Qiagen) and 1 μg genomic DNA subject to bisulfite conversion using the EZ DNA methylation gold kit according to manufacturer’s instructions (Zymo Research).…”

Epigenetic plasticity and the hallmarks of cancer

“…DNA methyltransferase mutations can lead to hypo-methylation and aberrant activation of enhancers that drive leukemogenic gene patterns (77, 78). Fidelity may also be perturbed by altered cellular contexts, including the increased demands of a rapidly replicating cancer genome.…”

BCR/ABL can promote CD19+ cell growth but not render them long-term stemness

“…The fusion gene encodes a constitutively active tyrosine kinase, which alters signaling pathway, subverts the controls of normal proliferation, blocks differentiation and promotes resistance to apoptosis (5,6). The fusion protein was found in all of the human chronic myelogenous leukemia (CML) (6) and in part of B-ALL.…”