Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation - PubMed (original) (raw)

Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation

Allison Mayle et al. Blood. 2015.

Abstract

DNA methyltransferase 3A (DNMT3A) is mutated in hematologic malignancies affecting myeloid, mixed, and lymphoid lineages, and these mutations are associated with poor prognosis. Past studies in mice revealed Dnmt3a-knockout (KO)hematopoietic stem cells (HSCs) had increased self-renewal, but no leukemia was observed. Here, all lethally irradiated mice transplanted with Dnmt3a-deleted HSCs died within 1 year. Animals were diagnosed with a spectrum of malignancies similar to those seen in patients with DNMT3A mutations, including myelodysplastic syndrome, acute myeloid leukemia, primary myelofibrosis, and T- and B-cell acute lymphocytic leukemia. In some cases, acquired malignancies exhibited secondary mutations similar to those identified in patients. Loss of Dnmt3a led to disturbed methylation patterns that were distinct in lymphoid and myeloid disease, suggesting lineage-specific methylation aberrations promoted by Dnmt3a loss. Global hypomethylation was observed in all of the malignancies, but lymphoid malignancies also exhibited hypermethylation, particularly at promoter regions. This mouse model underscores the important role of Dnmt3a in normal hematopoietic development and demonstrates that Dnmt3a loss of function confers a preleukemic phenotype on murine HSCs. This model may serve as a tool to study DNMT3A mutation associated malignancies and for developing targeted strategies for eliminating preleukemic cells for prevention and treatment of hematologic malignancies in the future.

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Figures

Figure 1

Figure 1

Mice transplanted with _Dnmt3a_-KO HSCs have reduced survival. (A) WBC and lymphocyte (LY) count at 26 weeks posttransplant; n = 18 WT and n = 41 _Dnmt3a_-KO. Error bars represent mean plus standard deviation. (B) Kaplan-Meier survival curve of mice transplanted with WT (n = 20) or _Dnmt3a_-KO (n = 45) HSCs. Censored points (squares) represent 5 mice that were sacrificed for a nonhematopoietic phenotype. Median survival is 321 days for _Dnmt3a_-KO; undetermined for WT. The experiment was repeated with the same numbers of animals, with median survival times of 246 days for mice transplanted with _Dnmt3a_-KO HSCs and 467 days for those transplanted with WT. (C) Number of mice in the transplanted cohort that were definitively diagnosed with the indicated hematopoietic diseases. ***P < .0001. B-ALL, B-cell acute lymphocytic leukemia/lymphoma; CMML, chronic myelomonocytic leukemia; ETP, early thymic progenitor acute lymphoblastic leukemia; PMF, primary myelofibrosis.

Figure 2

Figure 2

Development of T-ALL in mice transplanted with _Dnmt3a_-KO HSCs. (A) Thymus weights of mice transplanted with WT HSCs killed as age-matched controls while healthy, and 4 mice transplanted with _Dnmt3a_-KO HSCs that presented with enlarged thymuses on necropsy. Error bars represent mean ± SEM. (B) WBC count over time for 1 mouse receiving _Dnmt3a_-KO HSCs and 3 representative WT mice. Error bars represent mean ± SEM. (C) Bone marrow touch preps stained with Wright-Geimsa from a representative mouse transplanted with WT HSCs and from a mouse with T-ALL. Bars represent 20 μm. (D) Fluorescence-activated cell sorter analysis of the same _Dnmt3a_-KO recipient and a representative WT, showing (top row) that CD45.2 (donor) cells make up nearly all of the bone marrow cells, and (bottom row) myeloid (My), B cells (indicated with letter B), and T cells (T); parent gate is live cells. (E) Further immunophenotyping of these mice, bone marrow stained with CD4 and CD8; parent gate is live; CD45.2+. (F) Chromatogram traces showing Notch1 mutations identified in 2 T-ALL mice in exon 27. The variant base is listed below the sequence in red, and corresponding amino acid changes are indicated. **P < .001.

Figure 3

Figure 3

Mice transplanted with _Dnmt3a_-KO HSCs develop myeloid malignancies. (A) WBC counts of 2 mice transplanted with _Dnmt3a_-KO HSCs and 3 representative mice transplanted with WT HSCs. Error bars represent mean ± SEM. (B) Wright-Giemsa staining of bone marrow and spleen touch preps from a mouse transplanted with WT HSCs and a mouse transplanted with _Dnmt3a-_KO HSCs diagnosed with AML. (C) Immunophenotyping confirms donor-derived cells (CD45.2) in the bone marrow express myeloid markers (Mac1/Gr1). My, myeloid; B, B-cells; T, T-cells. (D) Wright-Giemsa–stained bone marrow touch preps from a mouse transplanted with WT HSCs and from a mouse transplanted with _Dnmt3a_-KO HSCs that was diagnosed with MDS; shown is a dysplastic binucleated megakaryocyte with coarse granules in the cytosol in the MDS mouse. (E) Reticulin staining of a bone marrow section from a mouse transplanted with WT HSCs, and from a mouse transplanted with _Dnmt3a_-KO HSCs, with black fibers indicative of fibrosis. (F) Hematoxylin and eosin staining of spleen sections from the 2 mice diagnosed with PMF demonstrating extramedullary hematopoiesis. Bars represent 20 μm (B,E-F) and 10 μm (D).

Figure 4

Figure 4

Lack of Dnmt3a accelerates Nras driven malignancy. (A) Number of colonies per plate after single green fluorescent protein (GFP)-positive stem and progenitor cells were sorted into individual wells of 96-well plates (N = 3 plates per genotype/transduction). Representative of 2 independent experiments. (B) Kaplan-Meier survival curve of mice transplanted with WT HSCs transduced with MSCV-IRES-GFP (n = 7) or MSCV-NrasG12D-IRES-GFP (n = 10) and mice transplanted with _Dnmt3a_-KO HSCs transduced with MSCV-IRES-GFP (n = 7) or MSCV-NrasG12D-IRES-GFP (n = 10) (median survival of WT-NrasG12D mice: 83 days; KO-NrasG12D: 27 days; P < .0001). Representative of 2 independent experiments. Red blood cell (RBC) counts (C) and WBC counts (D) 3 weeks after transplant of WT or _Dnmt3a_-KO HSCs transduced with GFP or NrasG12D (n = 10 mice for each genotype transplanted with NrasG12D; n = 7 mice for each genotype transplanted with GFP). (E) Spleen weights of mice sacrificed at 10 weeks posttransplant. Error bars represent mean ± SEM. (F) Immunophenotyping of bone marrow from mice transplanted with WT HSCs transduced with MSCV-IRES-GFP or MSCV-NrasG12D-IRES-GFP and _Dnmt3a_-KO HSCs transduced with MSCV-IRES-GFP, stained for myeloid (My) and B cells (indicated with letter B). Parent gate is live; CD45.2+GFP+. (G) Wright-Giemsa staining of bone marrow (BM) touch preps showing dysplastic erythroid precursors with irregular nuclear contours and peripheral blood smears, and a hematoxylin and eosin (H&E)-stained bone marrow section showing dysplastic megakaryocytes with bizarre-shaped nuclei from mice transplanted with _Dnmt3a_-KO HSCs transduced with MSCV-IRES-GFP or MSCV-NrasG12D-IRES-GFP. Bars represent 20 μm. *P < .05, **P < .01, ***P < .001.

Figure 5

Figure 5

DNA methylation changes in _Dnmt3a_-null driven malignancies. (A) Distribution of absolute DNA methylation levels in all experiments for genomic regions subject to differential methylation in MDS, AML, and/or T-ALL mice. The width of the y-axis indicates the frequency with which the value is observed. (B) The top 6 classes of DNA methylation changes (relative to control samples) across the diseases are shown. Hypermethylation in T-ALL is the most frequent, and shared hypomethylation between MDS and AML is the second most frequent. (C) The methylation levels of DMRs in 1 or more diseases were analyzed by hierarchical clustering using a Jensen-Shannon distance metric of methylation specificity. (D) Pie chart showing the proportion of DMRs in T-ALL mice by gee association. Left chart summarizes region coverage of intergenic, introns, exons, and promoters. Right chart summarizes DMRs by the same categories. (E) Chromatin and transcriptional regulator enrichment analysis of MDS, AML, and T-ALL DMRs. Enrichment of DMRs in peaks or binding sites curated in publicly available mouse functional genomics data sets for the indicated factors and cell/tissue lineages was quantified using odds ratios and P values (Benjamini-Hochberg testing correction) determined by Fisher’s exact test. Dot sizes are proportionate to the odds ratio. Color intensity represents the negative-log Benjamini-Hochberg–corrected P value. Nonsignificant results are in gray. (F) T-ALL hypermethylation recapitulates functions and ontologies of human leukemia. Potential _cis_-regulatory functions of promoter-proximal (10 kb up to 1 kb downstream of transcriptional start sites) hypermethylated DMRs in T-ALL were predicted with GREAT 2.0. All enriched terms have a multiple testing corrected q < 0.005. CGI, CpG islands; LSK, Lin-Sca-1+c-Kit+; NS, nonsignificant; TALL, T-ALL; THY, thymus; UMR, undermethylated regions.

Comment in

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