Evidence supporting a dominant negative mechanism for DNMT3A hotspot mutation-mediated leukemic cell transformation
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PROVIDER: PRJNA533827 | ENA |
REPOSITORIES: ENA
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Project description:Mutation of DNA methyltransferase 3A at arginine 882 (DNMT3AR882mut) is prevalent in various hematological cancers. DNMT3AR882mut was recently shown to carry partially defective, dominant-negative or gain-of-function activities under different in vitro contexts. However, the causal roles for such a multifaceted effect of DNMT3AR882mut on leukemogenesis remain undefined. Here we report TF-1 leukemia cells as a robust system for modeling DNMT3AR882mut-dependent cell transformation phenotypes and for performing structure-function relationship studies of DNMT3AR882mut. We show that expression of DNMT3AR882mut and not its wildtype counterpart promotes TF-1 cell transformation and induces CpG hypomethylation predominantly at enhancers. Such effect by DNMT3AR882mut is dose-dependent, acts synergistically with that of IDH1 mutation, and resembles what was seen in human leukemia patients with DNMT3AR882mut. Both transformation- and hypomethylation-inducing capacities of DNMT3AR882mut rely on a motif involved in heterodimerization whereas its various chromatin-binding and enzymatic domains were dispensable. We also show bromodomain inhibition as a therapeutic means for treatment of murine leukemia carrying DNMT3AR882mut. Collectively, this study describes a useful model system for studying DNMT3AR882mut and supports a requirement for the dominant-negative effect of DNMT3AR882mut during leukemogenesis.
2019-06-12 | GSE130094 | GEO
Project description:DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote macro-oligomer formation, leading to aberrant DNA methylation that in turn contributes to pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the hotspot mutation-induced functional mis-regulation of DNMT3A remains unclear. Here, we report the crystal structure of DNMT3A methyltransferase (MTase) domain, revealing a molecular basis for its DNMT3B-distinct oligomerization behavior. Introducing DNMT3B-converting mutations to DNMT3A R882 mutants also led to structure determination of R882H- and R882C-mutated DNMT3A, which show enhanced intermolecular contacts than wild-type DNMT3A. Consistently, our in vitro and genomic DNA methylation analyses reveal that the DNMT3B-converting mutations eliminate the gain-of-function effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.
2023-12-31 | GSE225828 | GEO
Project description:Somatic mutations in DNA Methyltransferase 3A (DNMT3A) with a hotspot in exon 23 at Arginine 882 (DNMT3AR882mut) are the most frequent single mutations in clonal hematopoiesis. Here we analyze the expression of genes and endogenous retrovirus (ERV) sequences in a murine model carrying human DNMT3A-R882H mutation in one allele of the endogenous DNMT3A with respect to normal condition and azacitidine treatment.
2021-01-26 | GSE146907 | GEO
Project description:DNA Methyltransferase 3A (DNMT3A) is frequently mutated in various hematopoietic malignancies. We report that DNMT3A mutational ‘hotspot’ at Arg882 (i.e., DNMT3A-R882H) cooperates with constitutively activated RAS in transforming murine hematopoietic stem/progenitor cells (HSPCs) ex vivo and inducing acute leukemias in vivo. Mechanistically, DNMT3A-R882H induced DNA hypomethylation facilitates gene enhancer/promoter activation. In this dataset, we provided microarray data showing effect of BRD4 inhibitor I-BET151 on gene expression in leukemic stem cells transformed by DNMT3A R882H and NRAS G12D.
2019-06-12 | GSE130634 | GEO
Project description:Structure-guided functional suppression of AML-associated DNMT3A hotspot mutations
| PRJNA937683 | ENA
Project description:DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 & 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7-23% of acute myeloid leukemia (AML). Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five EBV-B cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in DNMT3A or IDH1/2. Peptides eluted from HLA class I alleles on these EBV-B cell lines were analyzed by tandem mass spec-trometry. We identified an HLA-A*01:01-binding DNMT3AR882H peptide and HLA-B*07:02-binding IDH2R140Q peptide, for which we searched for specific T cells in healthy individuals using pep-tide-HLA tetramers. Various T-cell clones were isolated showing specific reactivity against cell lines transduced with full-length DNMT3AR882H or IDH2R140Q genes, while cell lines transduced with wildtype genes were not recognized. One T-cell clone for DNMT3AR882H also reacted against patient AML cells with the mutation, while AML cells without the mutation were not recognized, thereby validating surface presentation of a DNMT3AR882H neoantigen that can potentially be targeted on HLA-A*01:01 positive AML by immunotherapy.
2024-06-23 | PXD050560 | Pride
Project description:Targeting of DNMT3A hotspot mutations in Clonal Hematopoiesis and Acute Myeloid Leukemia
| PRJNA612341 | ENA
Project description:DNA methyltransferases DNMT3A- and DNMT3B-mediated de novo DNA methylation critically regulates epigenomic and transcriptomic patterning during development. The hotspot DNMT3A mutations at the site of Arg822 (R882) promote macro-oligomer formation, leading to aberrant DNA methylation that in turn contributes to pathogenesis of acute myeloid leukemia (AML). However, the molecular basis underlying the hotspot mutation-induced functional mis-regulation of DNMT3A remains unclear. Here, we report the crystal structure of DNMT3A methyltransferase (MTase) domain, revealing a molecular basis for its DNMT3B-distinct oligomerization behavior. Introducing DNMT3B-converting mutations to DNMT3A R882 mutants also led to structure determination of R882H- and R882C-mutated DNMT3A, which show enhanced intermolecular contacts than wild-type DNMT3A. Consistently, our in vitro and genomic DNA methylation analyses reveal that the DNMT3B-converting mutations eliminate the gain-of-function effect of the DNMT3A R882 mutations in cells. Together, this study provides mechanistic insights into DNMT3A R882 mutation-triggered aberrant oligomerization and DNA hypomethylation in AML, with important implications in cancer therapy.
2023-12-31 | GSE226062 | GEO
Project description:To identify early events involved in pre-leukemic transformation, we have performed single-cell RNA-sequencing (scRNA-seq) in WT and Dnmt3a KO mice. Flow cytometry sorted wild-type and pre-leukemic Dnmt3a KO HSPC cells were captured using 10X genomics chromium platform.
2021-12-01 | GSE154624 | GEO
Project description:The DNA methylation program is at the bottom layer of the epigenetic regulatory cascade of vertebrate development. While the methylation at C-5 position of the cytosine (C) residues on the vertebrate genomes is achieved through the catalytic activities of the DNA methyltransferases (DNMTs), the conversion of the methylated cytosine (5mC) could be accomplished by the combined actions of the TET enzyme and DNA repair. Interestingly, it has been found recently that the mouse and human DNMTs also possess active DNA demethylation activity in vitro in a Ca2+- and redox condition-dependent manner. We report here the study of tracking down the fate of the methyl group removed from 5mC on DNA during in vitro demethylation reaction by mouse de novo DNMTs, i.e. DNMT3A and DNMT3B. Remarkably, the methyl group becomes covalently linked to the catalytic cysteines utilized by the two de novo DNMTs in their DNA methylation reactions. Thus, the forward and reverse reactions of DNA methylations by the DNMTs may utilize the same cysteine residue(s) as the active site despite of their distinctive pathways. Secondly, we demonstrate that active DNA demethylation of a heavily methylated GFP reporter plasmid by ectopically expressed DNMT3A or DNMT3B occurs in vivo in transfected human HEK 293 cells in culture. Furthermore, the extent of DNA demethylation by the DNMTs in this cell-based system is affected by Ca2+ homeostasis as well as by mutation of their putative active cysteines. These findings substantiate the roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation in vitro as well as in a cellular context.
2022-02-24 | PXD003401 | Pride