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TET1 is a tumor suppressor of hematopoietic malignancy

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An Erratum to this article was published on 21 July 2015

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Abstract

The methylcytosine dioxygenase TET1 (‘ten-eleven translocation 1’) is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.

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Figure 1: TET1 deficiency drives B cell malignancy in mice of advanced age.
Figure 2: Whole-exome sequencing of TET1-deficient tumors reveals mutations of B-NHL.
Figure 3: TET1 is hypermethylated and transcriptionally downregulated in B-NHL.
Figure 4: TET1-deficient HSCs display increased self-renewal in vivo with a bias toward B cell differentiation.
Figure 5: Loss of Tet1 in HSCs promotes differentiation with a lymphoid bias.
Figure 6: Aberrant hydroxymethylation of DNA in TET1-deficient stem and progenitor cells.
Figure 7: Enhanced colony formation and accumulation of DNA damage in Tet1-deficient progenitor B cells.

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Change history

  • 17 June 2015

    In the version of this article initially published, labels reading "5hmC gain" were incorrectly included below the plots in Figure 6e, and the plot at right was mislabeled above (as "loss"). The plot at left should have a single label above reading "5hmC loss" and the plot at right should have a single label above reading "5hmC gain." The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank members of the Aifantis laboratory for discussions; M. Xu and F. Yang for help and advice; L. Pasqualucci (Columbia University) for control antibodies and control tissue sections for immunohistochemistry of mouse B cell lymphomas and expertise; K. Ganz, R. Flannery, D. Fu and T. Adil for help with animal husbandry and tissue collection; A. Heguy and I. Dolgalev for assistance with microarray experiments, RNA-seq and exome sequencing; the NYU Flow Cytometry facility for cell sorting; the NYU Histology Core; and E. Oricchio for assistance with analysis of data from patients with FL. Supported by the Damon Runyon Cancer Research Foundation (M.M.D.), the US National Institutes of Health (5RO1HD045022 and 5R37CA084198 for work in the laboratory of R.J.; and 1R01CA169784, 1R01CA133379, 1R01CA105129, 1R01CA149655 and 5R01CA173636 for the Aifantis laboratory), the Simons Foundation (for work in the laboratory of R.J.), the William Lawrence and Blanche Hughes Foundation, The Leukemia & Lymphoma Society (TRP#6340-11 and LLS#6373-13), The Chemotherapy Foundation, The V Foundation for Cancer Research, the Alex's Lemonade Stand Foundation for Childhood Cancer, the St. Baldrick's Cancer Research Foundation, the National Health and Medical Research Council (L.C.) and the Howard Hughes Medical Institute (I.A.).

Author information

Authors and Affiliations

Authors

Contributions

L.C., M.M.D., R.J. and I.A. designed and performed experiments; D.N.-L., S.Ba. and J.M. provided technical assistance; Y.S.Y. performed bisulfite-sequencing experiments; Y.Y., S.Bh. and A.K.V. performed, analyzed and provided bioinformatics support for HELP-GT assays; R.S. and H.G. provided bioinformatics support for patient HELP assays and RNA-seq; and R.S. performed MassArray experiments; C.Lo., B.K., T.T., B.A.-O. and S.S. participated in bioinformatics analysis; C.Li. provided histopathology advice; and L.C. and I.A. wrote the manuscript.

Corresponding authors

Correspondence to Rudolf Jaenisch or Iannis Aifantis.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Peripheral blood analysis and organ histology of sick Tet1-deficient mice.

a) Hemavet quantification of whole blood cell numbers from Tet1+/+, Tet1+/– and Tet1–/– mice aged 18-24 months. WBC = white blood cells, RBC = red blood cells, NS = not significant, * P = <0.05. (unpaired t-test) Small horizontal lines indicate the mean; Tet1+/+ (n = 20), Tet1+/– (n = 20) and Tet1–/– (n = 32). b-c) Peripheral blood smears stained with Wright-Giemsa and d) flow cytometric analysis of peripheral blood from sick Tet1-deficient mice compared to age-matched Tet1+/+ controls. Data are representative of 3 independent experiments, n = 8-10 mice per genotype. e) Histological analysis of spleens from sick Tet1+/– and Tet1–/– mice compared to age-matched Tet1+/+ controls. Sections were stained with H&E and Ki67 as indicated. RP = Red pulp, F = Follicle. f) H&E staining of liver, lung and kidney sections from sick Tet1+/– showing diffuse lymphocytic infiltration compared to age-matched Tet1+/+ mice. g) H&E and Ki67 staining of hyperproliferative infiltrating cells in liver, lung and kidney of Tet1-deficient mice. Histology is representative of Tet1+/+, Tet1+/– and Tet1–/– mice (n = 6-8 mice per genotype). Scale bar = 100μm in all panels.

Supplementary Figure 2 Tet1-deficient B cell lymphomas display both IgM and IgM+ phenotypes and are transplantable.

a-b) Examples of flow cytometric analysis of lymph nodes from sick Tet1+/– and Tet1–/– mice compared to age-matched Tet1+/+controls, displaying IgM, IgM+ and CD11b+ staining patterns. c-d) H&E stained sections of liver, kidney, spleen and lymph nodes from sick Tet1+/– and Tet1–/– mice with multinucleated giant cells and histiocytic sarcoma. Scale bar = 100μm in all panels. e) White blood cell (WBC) and lymphocyte cell counts in the peripheral blood of Tet1-deficient tumor recipient mice 8 and 12 weeks post-transplant. f) Recipient mice 12 weeks post-transplant. Upper left panel; example of gross-anatomy of recipient mice with white patchy liver and enlarged spleen. Upper middle and right and lower panels; H&E staining of recipient mouse tissue histological sections, with spleen and liver infiltration, and histiocytic sarcoma in the liver. Scale bar = 100μm in all panels. g) Representative flow cytometric analysis of spleen cells from recipient mice gated on CD45.2+ donor cells co-stained for B cell (B220) and surface Ig (IgD and IgM) expression.

Supplementary Figure 3 Mutational analysis and base-substitution frequency in Tet1-deficient lymphomas.

Exome sequencing data for thirteen Tet1-deficient tumors (T1-13) were divided into 2 groups; Group 1 = low mutation frequency (<50 total exonic variations), Group 2 = high mutation frequency (50-1200 total exonic variations). Low (Group1) and high-grade (Group 2) mutated tumors were compared for a) average number of indels and nsSNVs, b) frequency of mutation type and c) frequency of transversion or transition base substitutions. A, T, C or G base substitution frequencies were calculated d) overall and e) in the context of transversion or transition mutation;(e) pairs of nucleotides indicate transversion mutations targeting T or A (TG: T-to-G, TA: T-to-A, AC: A-to-T, AT: A-to-T) and C or G (CA: C-to-A, CG: C-to-G, GT: G-to-T, GC: G-to-C) and transition mutations targeting T or A (TC: T-to-C, AG: A-to-G) and C or G (CT: C-to-T, GA: G-to-A). Average mutation frequency of base substitutions in f) low (Group1) and g) high-grade (Group 2) mutated Tet1-deficient tumors according to trinucleotide context with examples of individual high-grade mutated tumors; h) T12 and i) T11. Mean ± SEM (Group 1, n = 8; Group 2, n = 5).

Supplementary Figure 4 Overlapping mutations in IgM+ and IgM Tet1-deficient tumors, hypermethylation of TET1 in mature B cell lymphomas and contrasting disease spectra of Tet1 and Tet2 deficiency in mice.

a) Frequency of IgM+ and IgM lymphomas in Tet1-deficient mice. b) V(D)J-rearrangements in DNA of exome sequenced tumor samples. DNA isolated from total splenic cells (SP) was used as a control for the amplification of the constant heavy chain (Cμ), and for the rearrangements of D-Jh4, V7183-Jh4 and V558-Jh4, Vκ-Jκ and Vλ-Jλ. c) Venn Diagram of overlapping recurrently mutated genes in IgM+ and IgM tumors. d) HELP assay for methylation in patient samples are shown; human naïve B (NB), centroblast B (CB), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), multiple myeloma (MM), activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL, precursor B (Pre-B), B-acute lymphoblastic leukemia (B-ALL) subtypes – BCR-ABL, E2A-PBX1, MLL-rearranged (MLLr) and other, normal T, T-acute lymphoblastic leukemia (T-ALL), CD34+ progenitor cells and acute myeloid leukemia (AML) * P ≤ 0.05, ** P ≤ 0.005 and ***P ≤ 0.0005 (Wilcoxon test). e) EpiGram of an amplicon targeting CpGs in the first intron of TET1 used in Sequenom-Targeted Methylation Analysis by Sequenom MassARRAY. Two normal human germinal center B (GCB) samples are displayed compared to 5 FL patient samples. Circles depict increasing CpG methylation status from 0-100% as indicated. f) Kaplan-Meier survival curve of Tet2-deficient mice with heterozygous (Tet2+/–) and homozygous (Tet2–/–) deletion compared to wild-type mice (Tet2+/+). * P = <0.0005 (Mantel-Cox test). Frequency of diseases; acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML), myeloid dysplasia (MDS), B cell lymphoma (BCL) and T-cell lymphoma (TCL) observed in g) Tet2- and h) Tet1-deficient mice.

Supplementary Figure 5 Tet1 deficiency causes a decrease in the frequency of mature B cells in the bone marrow and spleen.

Summary of flow cytometric analysis of the frequency of lineage positive (Lin+) cells in the a-b) bone marrow and c-d) spleen of Tet1+/+, Tet1+/– and Tet1–/– mice. B cell (B220+), T cell (CD3+), granulocyte (Gr1+), neutrophil (Gr1+CD11b+), monocyte (Gr1CD11b+), progenitor (CD71+Ter119), precursor (CD71+Ter119+) and mature nucleated erythroid cell (CD71 Ter119+) frequencies are shown. e-f) Summary of flow cytometric analysis to assess the frequency of B220+ B cell subsets in the bone marrow stained with IgM and IgD for progenitor and precursor B (Pro/PreB), immature (ImmB), transitional (TransB) and mature B cells (MatB). g) Representative flow cytometric analysis of the frequency of B cell subsets in the bone marrow of Tet1+/+ and moribund Tet1–/– mice. All bar graphs display the mean ± SEM (3 months, n = 4 mice per genotype; Moribund, n = 6-8 mice per genotype); * P = <0.01, ** P = <0.001, *** = P <0.0001 (unpaired t-test).

Supplementary Figure 6 Tet1-deficient HSCs display increased self-renewal in vivo with a deficiency in bone marrow mature B cells and expression of genes in histone cluster 1 in hematopoietic cell lineages.

a) Frequency of CD45.2+ competitive donor cells in total LSKs, LT-HSCs and MPPs from the bone marrow of primary transplanted mice 20-weeks post-reconstitution (mean ± SEM, n = 6 mice per genotype); * P = <0.001, ** P = <0.0001) (unpaired t-test). b) Representative flow cytometry of CD45.2+ cells from the bone marrow of primary transplanted mice. Upper panel; HSC subsets of CD45.2+ LSK cells stained for LT-HSC, ST-HSC, MPP1 and MPP2 with CD150 and CD48 as previously described. Lower panel; CD45.2+ B220+ cells stained for B cell subsets with IgM and IgD. c) Sorting strategy for microarray and RNA-seq analysis of total LSK, LT-HSC and MPP populations. d) Heat map of Histone cluster 1 gene expression in hematopoietic stem, progenitor and mature cells.

Supplementary Figure 7 5hmC losses and 5mC gains in Tet1-deficient LSK cells target genes encoding molecules involved in DNA repair, G protein– coupled receptor signaling and tumor-suppressor pathways.

a) Percent of 5hmC losses and gains across genomic regions. b) Overlap of 5hmC losses and gains with enhancer histone marks in ST-HSCs and MPPs. c) Coverage per base and d) sample clustering of RRBS data from Tet1+/+ and Tet1–/– LSKs. e) Representative frequency of CpG sites with 0-100 percent CpG methylation in Tet1+/+ and Tet1–/– LSKs. f) Ingenuity Pathway Analysis (IPA) software was used to generate schematic representations of genes that lose 5hmC and gain 5mC in Tet1–/– LSK cells. Signaling pathways displayed include genes pathways involved in tumor suppression (TGF-β, WNT/β-Catenin, p53 and PTEN), DNA repair (BER) and B cell function (RhoA, G-protein coupled). Log adjusted P-value for significance is shown along the x axis. Red lines indicate threshold of significance (P = 0.05).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–11 (PDF 1660 kb)

Supplementary Data Set 1

Somatic non-synonymous SNVs identified by whole exome sequencing in hematopoietic tumors from Tet1-deficient mice. (XLS 512 kb)

Supplementary Data Set 2

Somatic indels identified by whole exome sequencing in hematopoietic tumors from Tet1-deficient mice. (XLS 82 kb)

Supplementary Data Set 3

Recurrent somatic mutations identified by whole exome sequencing in hematopoietic tumors from Tet1- deficient mice. (XLS 140 kb)

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Cimmino, L., Dawlaty, M., Ndiaye-Lobry, D. et al. TET1 is a tumor suppressor of hematopoietic malignancy. Nat Immunol 16, 653–662 (2015). https://doi.org/10.1038/ni.3148

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