DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells

Abstract

The gene encoding DNA methyltransferase 3A (DNMT3A) is mutated in ∼20% of acute myeloid leukemia cases, with Arg882 (R882) as the hotspot. Here, we addressed the transformation ability of the DNMT3A-Arg882His (R882H) mutant by using a retroviral transduction and bone marrow transplantation (BMT) approach and found that the mutant gene can induce aberrant proliferation of hematopoietic stem/progenitor cells. At 12 mo post-BMT, all mice developed chronic myelomonocytic leukemia with thrombocytosis. RNA microarray analysis revealed abnormal expressions of some hematopoiesis-related genes, and the DNA methylation assay identified corresponding changes in methylation patterns in gene body regions. Moreover, DNMT3A-R882H increased the CDK1 protein level and enhanced cell-cycle activity, thereby contributing to leukemogenesis.genomic variation | epigenetic abnormality | leukemogenic effect D NA methylation represents one of the major epigenetic modifications and plays a key role in a number of regulatory mechanisms of life processes (1-3). In mammals, the executors of genome methylation are members of the DNA methyltransferase (DNMT) family, including DNMT1, DNMT3A and DNMT3B. It has been well established that DNMT3A forms complex with DNMT3L to catalyze the de novo DNA methylation (4, 5). Both DNMT3A and DNMT3B show high expression levels at the early embryogenesis, and their expressions are down-regulated along with the embryonic development and cell differentiation (6, 7).It is well known that all blood-cell lineages originate from the multipotent hematopoietic stem cells (HSCs). A number of regulations are involved in directing HSCs activities, and the epigenetic modifications are of great importance (8,9). It has been shown that loss of Dnmt3a in a Dnmt3a-conditional knockout mouse results in progressive impairment of HSCs differentiation and expansion (10). Notably, DNMT3A recently has been reported to be mutated in up to 20% of cases of acute myeloid leukemia (AML), mostly in cases with monocytic lineage (AML-M5 or -M4) and clinical features including old age, normal karyotype, leukocytosis and thrombocytosis, and poor prognosis (11,12). Although a variety of DNMT3A mutations have been identified, the majority (∼50%) affect Arg882 (R882) located at the catalytic domain, and the most common substitution is Arg882His (R882H) (11,13,14). It also has been suggested that R882 mutation may interfere with oligomerization of DNMT3A and thereby exert an aberrant effect on its enzymatic function (15). Evidence has been obtained supporting DNMT3A mutations as the fundamental genetic event at the initiation stage of AML pathogenesis (16,17). However, the in vivo transformation power of DNMT3A mutations needs to be addressed, and the relevant molecular and cellular mechanisms of these mutations in AML pathogenesis remain obscure.In the present work, using retroviral transduction and bone marrow transplantation (BMT) technology, we were able to investigate the in vivo effect of the DNMT3A-R882H mutation on the transforming po...

Conditional knockin of Dnmt3a R878H initiates acute myeloid leukemia with mTOR pathway involvement

“…The importance of DNMT3A in hematopoiesis and leukemogenesis has been demonstrated by studies using Dnmt3a knockout or DNMT3A R882-overexpressing mice (7,12,22). In this study, the conditional knockin mouse model allowed the inducible allelic switch from Dnmt3a WT/WT to Dnmt3a R878H/WT in the hematopoietic system of adult animals, which indeed possessed some major features of human AML associated with the DNMT3A mutation.…”

“…This kind of DNMT3A mutation confers reduced methyltransferase activity and promotes the possibility of dominant-negative consequences compared with the wild-type (WT) allele (2,9,10). Moreover, the DNMT3A mutation causes aberrant DNA hypomethylation and up-regulates a series of target genes involved in AML pathogenesis (11)(12)(13).…”

“…Although these results indicate a major role of Dnmt3a deletion in facilitating the development of leukemia, the in vivo roles of DNMT3A mutants in leukemogenesis still need to be addressed. In our previous work, bone marrow transplantation (BMT) of bone marrow (BM) cells retrovirally transduced with DNMT3A R882H induced chronic myelomonocytic leukemia (CMML) with mild invasive behaviors through disturbing DNA methylation and gene expression in recipient mice (12). In contrast to the aggressive manifestations of DNMT3A mutation-related AML in clinical settings, this phenotype can be ascribed to an inappropriate expression of the mutant DNMT3A gene as driven by a retroviral promoter instead of the endogenous promoter/ enhancer.…”

Epigenetic dysregulation of hematopoietic stem cells and preleukemic state

“…In vivo analysis using a retroviral transduction of DNMT3A R882H mutant construct and bone marrow transplantation (BMT) assay showed development of chronic myelomonocytic leukemia (CMML)-like disease in mutant recipients, possibly through increased CDK1 protein level and enhanced cell-cycle activity [30]. Similarly, mice transplanted with Dnmt3a-null whole bone marrow (BM) or HSCs developed a spectrum of hematologic malignancies, including MDS, AML, myeloproliferative neoplasms (MPN), and T-and B-cell acute lymphocytic leukemia (ALL) [31,32].…”

Epigenetics in the hematologic malignancies

“…Intriguingly, heterozygous mutations are most common with recent data demonstrating that R882 DNMT3A mutations have a dominant negative effect through inhibition of DNMT3A oligomerization. [35][36][37] DNMT3A mutations have also been identified in patients with myelodysplastic syndromes (MDS) 38 and myeloproliferative neoplasms (MPN), 39 and are associated with increased likelihood of progression to AML. Indeed, in some studies, the same DNMT3A mutation as the antecedent hematologic disorder is identified in secondary AML, suggesting that these mutations may be an early event in malignant clonal evolution.…”