Functional Analysis of DNMT3A DNA Methyltransferase Mutations Reported in Patients with Acute Myeloid Leukemia

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3–15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis.

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Variants ofDNMT3Acause transcript-specific DNA methylation patterns and affect hematopoiesis

Life Science Alliance ◽

10.26508/lsa.201800153 ◽

2018 ◽

Vol 1 (6) ◽

pp. e201800153 ◽

Cited By ~ 7

Author(s):

Tanja Božić ◽

Joana Frobel ◽

Annamarija Raic ◽

Fabio Ticconi ◽

Chao-Chung Kuo ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Dna Methyltransferase ◽

De Novo ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Acute Myeloid

De novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing ofDNMT3Ahas characteristic epigenetic and functional sequels. SpecificDNMT3Atranscripts were either down-regulated or overexpressed in human hematopoietic stem and progenitor cells, and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that, particularly, transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating thatDNMT3Avariants also affect malignancies. Our results demonstrate that specificDNMT3Avariants have a distinct epigenetic and functional impact. Particularly, DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in acute myeloid leukemia.

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Expression of myeloperoxidase in acute myeloid leukemia blasts mirrors the distinct DNA methylation pattern involving the downregulation of DNA methyltransferase DNMT3B

Leukemia ◽

10.1038/leu.2014.15 ◽

2014 ◽

Vol 28 (7) ◽

pp. 1459-1466 ◽

Cited By ~ 9

Author(s):

H Itonaga ◽

D Imanishi ◽

Y-F Wong ◽

S Sato ◽

K Ando ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Dna Methyltransferase ◽

Methylation Pattern ◽

Dna Methylation Pattern ◽

Acute Myeloid

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DNMT3A mutations mediate the epigenetic reactivation of the leukemogenic factor MEIS1 in acute myeloid leukemia

Oncogene ◽

10.1038/onc.2015.359 ◽

2015 ◽

Vol 35 (23) ◽

pp. 3079-3082 ◽

Cited By ~ 30

Author(s):

H J Ferreira ◽

H Heyn ◽

M Vizoso ◽

C Moutinho ◽

E Vidal ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Dna Methyltransferase ◽

De Novo ◽

Alternative Pathway ◽

Missense Mutations ◽

Cytogenetic Aberrations ◽

Genome Bisulfite Sequencing ◽

Acute Myeloid

Abstract Close to half of de novo acute myeloid leukemia (AML) cases do not exhibit any cytogenetic aberrations. In this regard, distortion of the DNA methylation setting and the presence of mutations in epigenetic modifier genes can also be molecular drivers of the disease. In recent years, somatic missense mutations of the DNA methyltransferase 3A (DNMT3A) have been reported in ~20% of AML patients; however, no obvious critical downstream gene has been identified that could explain the role of DNMT3A in the natural history of AML. Herein, using whole-genome bisulfite sequencing and DNA methylation microarrays, we have identified a key gene undergoing promoter hypomethylation-associated transcriptional reactivation in DNMT3 mutant patients, the leukemogenic HOX cofactor MEIS1. Our results indicate that, in the absence of mixed lineage leukemia fusions, an alternative pathway for engaging an oncogenic MEIS1-dependent transcriptional program can be mediated by DNMT3A mutations.

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Faculty Opinions recommendation of DNA methylation signatures identify biologically distinct subtypes in acute myeloid leukemia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1528964.1017064 ◽

2010 ◽

Author(s):

Lee Grimes

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Acute Myeloid

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Identification and validation of obesity-related gene LEP methylation as a prognostic indicator in patients with acute myeloid leukemia

Clinical Epigenetics ◽

10.1186/s13148-021-01013-9 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Ting-juan Zhang ◽

Zi-jun Xu ◽

Yu Gu ◽

Ji-chun Ma ◽

Xiang-mei Wen ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Related Gene ◽

Targeted Bisulfite Sequencing ◽

Specific Pcr ◽

Frequent Event ◽

Acute Myeloid

Abstract Background Obesity confers enhanced risk for multiple diseases including cancer. The DNA methylation alterations in obesity-related genes have been implicated in several human solid tumors. However, the underlying role and clinical implication of DNA methylation of obesity-related genes in acute myeloid leukemia (AML) has yet to be elucidated. Results In the discovery stage, we identified that DNA methylation-associated LEP expression was correlated with prognosis among obesity-related genes from the databases of The Cancer Genome Atlas. In the validation stage, we verified that LEP hypermethylation was a frequent event in AML by both targeted bisulfite sequencing and real-time quantitative methylation-specific PCR. Moreover, LEP hypermethylation, correlated with reduced LEP expression, was found to be associated with higher bone marrow blasts, lower platelets, and lower complete remission (CR) rate in AML. Importantly, survival analysis showed that LEP hypermethylation was significantly associated with shorter overall survival (OS) in AML. Moreover, multivariate analysis disclosed that LEP hypermethylation was an independent risk factor affecting CR and OS among non-M3 AML. By clinical and bioinformatics analysis, LEP may be also regulated by miR-517a/b expression in AML. Conclusions Our findings indicated that the obesity-related gene LEP methylation is associated with LEP inactivation, and acts as an independent prognostic predictor in AML.

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Decitabine induces very early in vivo DNA methylation changes in blasts from patients with acute myeloid leukemia

Leukemia Research ◽

10.1016/j.leukres.2012.10.015 ◽

2013 ◽

Vol 37 (2) ◽

pp. 190-196 ◽

Cited By ~ 20

Author(s):

Rainer Claus ◽

Dietmar Pfeifer ◽

Maika Almstedt ◽

Manuela Zucknick ◽

Björn Hackanson ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Acute Myeloid

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Investigation of measurable residual disease in acute myeloid leukemia by DNA methylation patterns

Leukemia ◽

10.1038/s41375-021-01316-z ◽

2021 ◽

Author(s):

Tanja Božić ◽

Chao-Chung Kuo ◽

Jan Hapala ◽

Julia Franzen ◽

Monika Eipel ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Residual Disease ◽

Amplicon Sequencing ◽

Multiparametric Flow Cytometry ◽

Methylation Patterns ◽

Cg Dinucleotides ◽

Measurable Residual Disease ◽

Acute Myeloid

AbstractAssessment of measurable residual disease (MRD) upon treatment of acute myeloid leukemia (AML) remains challenging. It is usually addressed by highly sensitive PCR- or sequencing-based screening of specific mutations, or by multiparametric flow cytometry. However, not all patients have suitable mutations and heterogeneity of surface markers hampers standardization in clinical routine. In this study, we propose an alternative approach to estimate MRD based on AML-associated DNA methylation (DNAm) patterns. We identified four CG dinucleotides (CpGs) that commonly reveal aberrant DNAm in AML and their combination could reliably discern healthy and AML samples. Interestingly, bisulfite amplicon sequencing demonstrated that aberrant DNAm patterns were symmetric on both alleles, indicating that there is epigenetic crosstalk between homologous chromosomes. We trained shallow-learning and deep-learning algorithms to identify anomalous DNAm patterns. The method was then tested on follow-up samples with and without MRD. Notably, even samples that were classified as MRD negative often revealed higher anomaly ratios than healthy controls, which may reflect clonal hematopoiesis. Our results demonstrate that targeted DNAm analysis facilitates reliable discrimination of malignant and healthy samples. However, since healthy samples also comprise few abnormal-classified DNAm reads the approach does not yet reliably discriminate MRD positive and negative samples.

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Tritsomy 8 Acute Myeloid Leukemia Analysis Reveals New Insights of DNA Methylome with Identification of HHEX as Potential Diagnostic Marker

Biomarkers in Cancer ◽

10.4137/bic.s19614 ◽

2015 ◽

Vol 7 ◽

pp. BIC.S19614 ◽

Cited By ~ 4

Author(s):

Marwa H. Saied ◽

Jacek Marzec ◽

Sabah Khalid ◽

Paul Smith ◽

Gael Molloy ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Diagnostic Marker ◽

Cpg Island ◽

Global Analysis ◽

Extra Chromosome ◽

Chromosome 8 ◽

Trisomy 8 ◽

Acute Myeloid

Trisomy 8 acute myeloid leukemia (AML) is the commonest numerical aberration in AML. Here we present a global analysis of trisomy 8 AML using methylated DNA immunoprecipitation-sequencing (MeDIP-seq). The study is based on three diagnostic trisomy 8 AML and their parallel relapse status in addition to nine non-trisomic AML and four normal bone marrows (NBMs). In contrast to non-trisomic DNA samples, trisomy 8 AML showed a characteristic DNA methylation distribution pattern because an increase in the frequency of the hypermethylation signals in chromosome 8 was associated with an increase in the hypomethylation signals in the rest of the chromosomes. Chromosome 8 hypermethylation signals were found mainly in the CpG island (CGI) shores and interspersed repeats. Validating the most significant differentially methylated CGI ( P = 7.88 · 10–11identified in trisomy 8 AML demonstrated a specific core region within the gene body of HHEX, which was significantly correlated with HHEX expression in both diagnostic and relapse trisomy 8 AMLs. Overall, the existence of extra chromosome 8 was associated with a global impact on the DNA methylation distribution with identification of HHEX gene methylation as a potential diagnostic marker for trisomy 8 AML.

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