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DNMT3A Mutants Provide Proliferating Advantage With Augmentation of Self-Renewal Activity in the Pathogenesis of AML in KMT2A-PTD-positive Leukemic Cells


DNMT3A Mutants Provide Proliferating Advantage With Augmentation of Self-Renewal Activity in the Pathogenesis of AML in KMT2A-PTD-positive Leukemic Cells

Rabindranath Bera et al. Oncogenesis.


Acute myeloid leukemia (AML) with partial tandem duplication of histone-lysine N-methyltransferase 2A (KMT2A-PTD) is a subtype of AML and is associated with adverse survival, yet the molecular pathogenesis of KMT2A-PTD is not fully understood. DNA methyltransferase 3A (DNMT3A) is mutated in various myeloid neoplasms including AML, especially at the Arg882. Recently, it has been found that DNMT3A mutations frequently coexisted with KMT2A-PTD and are associated with inferior outcomes. We aimed to understand the biological role of DNMT3A mutation in KMT2A-PTD-positive cells. Herein, we found that overexpression of DNMT3A mutants (MT) in KMT2A-PTD-positive EOL-1 cells augmented cell proliferation and clonogenicity. Serial colony replating assays indicated that DNMT3A-MT increased the self-renewal ability of Kmt2a-PTD-expressing mouse bone marrow cells with immature morphology. At 10 months post bone marrow transplantation, mice with the combined Kmt2a-PTD and DNMT3A-MT showed hepatosplenomegaly and leukocytosis with a shorter latency compared to control and DNMT3A-wild-type. Gene expression microarray analyses of bone marrow samples from human AML with KMT2A-PTD/DNMT3A-MT showed a stem cell signature and myeloid hematopoietic lineage with dysregulation of HOXB gene expression. In addition, human bone marrow AML cells carrying KMT2A-PTD/DNMT3A-MT showed abnormal growth and augmented self-renewal activity in primary cell culture. The present study provides information underlying the pathogenic role of DNMT3A-MT with KMT2A-PTD in proliferating advantage with augmentation of self-renewal activity in human leukemia, which may help to better understand the disease and to design better therapy for AML patients with these mutations.

Conflict of interest statement

The authors declare that they have no conflict of interest.


Fig. 1
Fig. 1. DNMT3A-MT deregulates cell proliferation and clonogenicity in EOL-1 cells.
a Immunoblot of DNMT3A overexpressed with WT and mutant-DNMT3A in KMT2A-PTD positive EOL-1 cells. b Growth curves of EOL-1 cells stably transduced with WT- and mutant-DNMT3A. Representative results from three independent replicates are shown. c, d Colony-formation ability in methylcellulose containing medium after stable overexpression of WT; and mutant-DNMT3A in EOL-1 cells (original magnification: ×100). Colonies of more than 50 cells were scored on day 10 of cultures. e Cell viability of transformed EOL-1 cells in the presence of 200 nM ATRA, 600 nM SAHA and the combination of 100 nM ATRA with 500 nM SAHA at 72 h. Error bars represent ± s.d. of the mean of duplicate cultures and each experiment repeated at least three times. *P < 0.05, **P < 0.03, ***P < 0.01, either compared with the control or as indicated in figures. Two-sided Student’s t test was used to calculate the P value.
Fig. 2
Fig. 2. Primary human KMT2A-PTD/DNMT3A mutants BM cells exhibited hyperproliferation, clonogenicity and self-renewal activity.
a Representative cell morphology with Liu’s reagents stained smears (original magnification: ×400) of primary human KMT2A-PTD AML cells with DNMT3A-WT/MT. DNMT3A-WT: AML#1, AML#2, AML#3, AML#4; DNMT3A-MT: AML#5, AML#6. b, c Colony-forming potentials and self-renewal activity of KMT2A-PTD/DNMT3A-WT/MT AML bone marrow cells in human MethoCult H4535 medium-enriched without erythropoietin (EPO), representative images of colonies in the second-round replating were shown. Colonies of more than 30 cells were scored using an inverted microscope on day 10 of cultures (b), original magnification: ×100. Columns represent the number of serially replated colonies (c). d Proportion of CFU-colonies of first-round plating. e Primary AML cell proliferation at day 3 (3d) and day 6 (6d) in a liquid culture medium containing 20% FBS and 20% conditional medium. f Cell viability of primary AML cells harboring KMT2A-PTD/DNMT3A-WT/MT in the presence of 200 nM ATRA, 600 nM SAHA incubated for 72 h. Each experiment repeated two times and error bars represent ±s.d. of duplicate cultures. g Representative flow cytometry data to determine the Annexin-V and propidium iodide (PI)-positive primary AML cells. h Primary BM cells were cultured in RPMI medium containing 20% FBS, 20% conditional medium with antibiotic for 3 days and the percentage of Annexin V and PI-positive cells were analyses by flow cytometry. Data are presented as means ± s.d. (n = 3). Two-sided Student’s t test was used to calculate the P value and compared between KMT2A/DNMT3A-WT and KMT2A/DNMT3A-MT groups, n.s. not significant.
Fig. 3
Fig. 3. DNMT3A mutations deregulate HSC activation, proliferation, and RNA modifying-associated genes.
a Heatmap representation of HSC activation, positive regulation of cell proliferation, HOXB gene expression identified as being differentially expressed in human primary AML cells harboring KMT2A-PTD with DNMT3A-WT and mutants. Red indicates upregulated genes compared to WT (green). b GO analyses of upregulated genes in DNMT3A-MT with KMT2A-PTD AML cells showing a series of genes enriched in different cellular and molecular processes including cell proliferation, angiogenesis, and regulation of apoptosis. cf GSEA determining specific gene sets or pathways that are positively or negatively regulated by DNMT3A mutants with KMT2A-PTD. Compared with KMT2A-PTD/DNMT3A-MT AML (right side), KMT2A-PTD/DNMT3A-WT (left side) negatively correlated with gene sets downregulated in platelet activation (c), positively correlated with those of mRNA processing (d), RNA splicing (e) and RNA methylation (f). The enrichment scores (ES), normalized enrichment scores (NES) and P values were shown in figures.
Fig. 4
Fig. 4. DNMT3A-MT deregulates HOXB gene expression in EOL-1 and primary AML cells.
a HOXB expression in EOL-1 cells transduced with DNMT3A-WT/MT by quantitative RT-PCR analyses showing the same patterns observed in gene expression microarray analysis of patient samples with KMT2A-PTD/DNMT3A-WT and mutants. Data are expressed as mean ± s.d. of three independent experiments. *P < 0.05, ***P < 0.005 compared to EV. Two-sided Student’s t test was used to calculate the P value. b Immunoblot data showing H3K4me3 and H4Ac protein levels increased and decreased respectively in EOL-1 cells expressing DNMT3A-MT. β-Actin was used as a control for equal loading. c Quantitation of indicated proteins in transduced EOL-1 cells. Error bars presented as mean ± s.d. of three independent experiments. *P < 0.02, **P < 0.006; n.s. not significant. Two-sided Student’s t test was used to calculate the P value. d Levels of H4Ac at the promoters of HOXB genes in DNMT3A-WT and DNMT3A-R882H-expressing EOL-1 cells as detected by ChIP-qPCR. The relative amounts of immunoprecipitated DNA were depicted as a percentage of input DNA. Error bars presented as mean ± s.d. of two independent experiments. *P < 0.05, ***P < 0.005. Two-sided Student’s t test was used to calculate the P value. e Relative expression levels of HOXB genes were examined by quantitative RT-PCR in BM cells derived from normal control (n = 3) and from primary AML cells with KMT2A-PTD/DNMT3A-WT/MT. The values were normalized by GAPDH mRNA levels and expression was shown as relative to normal BM cells (average of 3). Experiment was repeated twice and data are expressed as mean ± s.d. of two experiments.
Fig. 5
Fig. 5. DNMT3A-R882 mutant induces modifications of genomic methylation patterns in transduced EOL-1 cells.
a Distribution of genomic methylation pattern (β value) in the whole genome of EOL-1 cells transduced with EV control, DNMT3A-WT, and DNMT3A-R882C. b Hypo- and hypermethylation probes count obtained from transduced EOL-1 cells shown as bar-diagram. β value < 0.25 and >0.75 considered as hypomethylation and hypermethylation peaks, respectively. c, d The total hypermethylation and hypomethylation probes counted in each region defined by genomic structure shown in bar graph. e, f Methylation patterns in four regions defined by the distance from CpG islands, such as CpG islands, Shore, Shelf, and Open Sea region were shown.
Fig. 6
Fig. 6. Addition of DNMT3A-MT in Kmt2a-PTD mouse BM cells increased colony-forming and self-renewal activity.
a Lentivirus-mediated transduction of DNMT3A-WT/mutants in Kmt2a-PTD-positive mouse BM cells and DNMT3A mRNA level was detected by quantitative RT-PCR using first colony-forming cells. b, c Colony-forming potentials of Kmt2a-PTD+ mouse BM cells transduced with indicated plasmids in methylcellulose colony-forming media, representative images of colonies in the second-round plating were shown. Colonies at least 50 cells were scored on day 8 of cultures. Colonies were photographed and counted manually, original magnification: ×100. Columns represent the number of serially replated colonies. Error bars represent the mean ± s.d. from duplicated cultures. *P < 0.05, **P < 0.03, ***P < 0.01, compared with the control. d Proportion of CFU-colonies of second-round serial replating were shown. e Cytospin smeared preparations of cultured cells in the first plating colonies assays by Liu’s reagents staining were shown. f Statistical analysis of the numbers of immature cells in the first plating colonies cells. Error bars represent the mean ± s.d. of 4–5 different microscopic fields. **P < 0.01, ***P < 0.001, compared with the control. Two-sided Student’s t test was used to calculate the P value.
Fig. 7
Fig. 7. The collaboration of DNMT3A-MT with Kmt2a-PTD shows shorter latency in vivo.
a Kaplan−Meier curve shows the survival of mice transplanted with BM cells harboring Kmt2a and transduced with empty vector (EV, n = 5) control, DNMT3A-WT, DNMT3A-R882C/H/S (n = 5). P values were calculated using a log-rank test. b, c Quantification of hepatomegaly (b) and splenomegaly (c) in mice transduced with EV, DNMT3A-WT, DNMT3A-R882C/H/S were shown (n = 5). df Peripheral blood counts of transduced mice were shown (n = 5 for each group). *P < 0.05, **P < 0.01, ***P < 0.005, compared with the DNMT3A-WT group.

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