Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b–/– lymphomas, but not in Dnmt3b–/– pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b–/– lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies

Dnmt3a Is a Haploinsufficient Tumor Suppressor in CD8+ Peripheral T Cell Lymphoma

<div><p>DNA methyltransferase 3A (DNMT3A) is an enzyme involved in DNA methylation that is frequently mutated in human hematologic malignancies. We have previously shown that inactivation of Dnmt3a in hematopoietic cells results in chronic lymphocytic leukemia in mice. Here we show that 12% of Dnmt3a-deficient mice develop CD8+ mature peripheral T cell lymphomas (PTCL) and 29% of mice are affected by both diseases. 10% of <i>Dnmt3a</i>^<i>+/-</i> mice develop lymphomas, suggesting that Dnmt3a is a haploinsufficient tumor suppressor in PTCL. DNA methylation was deregulated genome-wide with 10-fold more hypo- than hypermethylated promoters and enhancers, demonstrating that hypomethylation is a major event in the development of PTCL. Hypomethylated promoters were enriched for binding sites of transcription factors AML1, NF-κB and OCT1, implying the transcription factors potential involvement in Dnmt3a-associated methylation. Whereas 71 hypomethylated genes showed an increased expression in PTCL, only 3 hypermethylated genes were silenced, suggesting that cancer-specific hypomethylation has broader effects on the transcriptome of cancer cells than hypermethylation. Interestingly, transcriptomes of <i>Dnmt3a</i>^<i>+/-</i> and <i>Dnmt3a</i>^<i>Δ/Δ</i> lymphomas were largely conserved and significantly overlapped with those of human tumors. Importantly, we observed downregulation of tumor suppressor p53 in <i>Dnmt3a</i>^<i>+/-</i> and <i>Dnmt3a</i>^<i>Δ/Δ</i> lymphomas as well as in pre-tumor thymocytes from 9 months old but not 6 weeks old <i>Dnmt3a</i>^<i>+/-</i> tumor-free mice, suggesting that p53 downregulation is chronologically an intermediate event in tumorigenesis. Decrease in p53 is likely an important event in tumorigenesis because its overexpression inhibited proliferation in mouse PTCL cell lines, suggesting that low levels of p53 are important for tumor maintenance. Altogether, our data link the haploinsufficient tumor suppressor function of Dnmt3a in the prevention of mouse mature CD8+ PTCL indirectly to a <i>bona fide</i> tumor suppressor of T cell malignancies p53.</p></div

P53 is downregulated in pretumor thymocytes and in <i>Dnmt3a</i>^<i>+/-</i> and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCLs.

<p>(A) Immunoblot showing p53 protein levels in <i>Dnmt3a</i>^<i>+/+</i> control spleen (SP), <i>Dnmt3a</i>^<i>+/-</i> PTCL (+/-) <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL (Δ/Δ) samples. HDAC1 is shown as a loading control. (B) Heatmap showing fold change in gene expression data derived from RNA-seq of 181 p53 pathway genes identified through GSEA for <i>Dnmt3a</i>^<i>+/-</i> PTCL and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL relative to CD8+ controls. (C) RNA-seq expression profiles on <i>Dnmt3a</i>^<i>+/-</i> PTCL, and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL were subjected to GSEA to identify enriched signatures. Each group was run as a pairwise comparison to normal CD8+ cells. In both tumor groups, the p53 pathway was identified as being significantly downregulated relative to controls. Normalized enrichment scores (NES), false discovery rate (FDR) and P-values are shown for each analysis. Black bars indicate individual genes within the pathway. Red indicates genes with high expression and blue indicates low expression in tumors relative to controls. (D) Immunoblot showing p53 protein levels in <i>Dnmt3a</i>^<i>+/+</i> (+/+) and <i>Dnmt3a</i>^<i>+/-</i> (+/-) cells isolated from the spleen (SP), thymus (TH), and lymph node (LN) of 9 month old mice with no signs of lymphoma development. HSC-70 is shown as a loading control. (E) Immunoblot showing p53 protein levels in <i>Dnmt3a</i>^<i>+/+</i> (+/+) and <i>Dnmt3a</i>^<i>+/-</i> (+/-) cells isolated from the spleen (SP) and thymus (TH) of 6 week and 9 month old mice with no signs of lymphoma development. HSC-70 is shown as a loading control. (F) Relative percentage of EGFP positive cells as determined by FACS for <i>Dnmt3a</i>^<i>+/+</i> (+/+) and <i>Dnmt3a</i>^<i>-/-</i> (-/-) lymphoma cell lines infected with either MSCV-IRES-EGFP (Vec) or MSCV-IRES-p53-EGFP (p53). EGFP measurements were taken at multiples time points and normalized by the percentage of EGFP positive cells observed 2 days post-transduction.</p

<i>Dnmt3a</i>^<i>Δ/Δ</i> mice develop B220+CD19+CD5+ CLL and CD8+ PTCL.

<p>(A) Genetic setting used to conditionally delete Dnmt3a. The tetracycline activator protein (tTA) is expressed in ~40% of all hematopoietic cells (including stem cells). tTA promotes expression of the <i>Teto-Cre</i> transgene. Expression of Cre results in the excision of the stop cassette located upstream of the <i>Rosa26LOXP</i>^<i>EGFP</i> reporter locus and deletion of Dnmt3a within the same subpopulation of cells. Thus, inclusion of the EGFP transgene allows for monitoring cells expressing tTA, and Cre, as well as to identify cells deleted for Dnmt3a. (B) PCR-based genotyping of the Dnmt3a locus using DNA isolated from FACS-sorted EGFP-positive (+) and EGFP-negative (−) populations on cells obtained from the bone marrow (BM), spleen (SP) and thymus (TH) of 6-week-old <i>Dnmt3a</i>^{<i>−/−</i>} mice. PCR reactions were performed on samples in the following order, BM: (1) -LSK, (2) +LSK, SP: (1) -B2, (2) +B2, (3) -B1, (4) +B1, (5) -myeloid, (6) +myeloid, (7) -erythroid, (8) +erythroid, TH: (1) -DP, (2) +DP, (3) -CD4, (4) +CD4, (5) -CD8, (6) +CD8. Immunophenotypes of sorted populations are as follows: LSK (Lineage-, Sca-1+, ckit+), B2 (B220+, CD5-), B1 (B220+, CD5+), Myeloid (CD11b+), erythroid (Ter119+), DP (CD4+, CD8+), CD4 (CD4+, CD8-), CD8 (CD4-, CD8+). Fragments from floxed (F), <i>wild-type</i> (WT) and knockout (KO) alleles are shown. DNA from conventional Dnmt3a heterozygous (F/- and +/-) mice were used as controls. (C) Kaplan Meyer survival curve for <i>Dnmt3a</i>^<i>Δ/Δ</i> mice. Moribund mice were classified at the time of terminal harvest with CLL (red), PTCL (yellow), or mixed CLL/PTCL (green) based on the presence of B-1a or CD8+ T cells tumors, as determined by FACS. <i>Dnmt3a</i>^<i>+/+</i> <i>wild-type</i> (+/+) controls are shown in blue. (D) A terminally ill <i>Dnmt3a</i>^<i>Δ/Δ</i> mouse with PTCL. Generalized lymphadenopathy is denoted by arrows. (E) H&E stained sections of <i>Dnmt3a</i>^<i>+/+</i> (normal) and <i>Dnmt3a</i>^<i>Δ/Δ</i> (PTCL) spleens (40X and 200X). (F) CD8 and CD4 expression in cells isolated from the lymph nodes (LN) and spleens (SP) of <i>Dnmt3a</i>^<i>+/+</i> control (+/+) and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL (Δ/Δ) mice as determined by FACS. Percentages of cells in each quadrant are shown in the top right in red. (G) CD3, CD8, TCR-β and TCR-γδ expression in cell isolated from the spleens (SP) of <i>Dnmt3a</i>^<i>+/+</i> control (+/+) and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL (Δ/Δ) mice, as determined by FACS. (H) Immunoblot showing Dnmt3a protein levels in <i>Dnmt3a</i>^<i>+/+</i> normal (N) controls (+/+) and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL (Δ/Δ) lymph node samples. γ-tubulin is shown as a loading control. (I) Time to tumor for primary (1), secondary (2) and tertiary (3) sub-lethally irradiated FVB-recipient mice serially transplanted with CD8+ PTCL tumors isolated from the lymph nodes of <i>Dnmt3a</i> ^<i>Δ/Δ</i> terminally sick mice. Data are presented as average time to tumor development. Three PTCL lines are shown.</p

Dnmt3a is a haploinsufficient tumor suppressor in the prevention of CD8+ PTCL in mice.

<p>(A) Percentage of conventional heterozygous <i>Dnmt3a</i>^<i>+/-</i> mice developing PTCL (yellow), MPD (green), CLL (red), or no disease (blue) at time of harvest, as determined FACS. (N = 30). (B) FACS analysis of CD8 and CD4 expression in cells isolated from the spleens (SP) and lymph nodes (LN) of <i>Dnmt3a</i>^<i>+/+</i> control (+/+) and <i>Dnmt3a</i>^<i>+/-</i> PTCL (+/-) mice. Percentages of cells in each quadrant are shown in the top right in red. (C) H&E stained sections of <i>Dnmt3a</i>^<i>+/+</i> (normal) and <i>Dnmt3a</i>^<i>+/-</i> (PTCL) spleens (40X and 200X). (D) The time to tumor development for primary (1), secondary (2) and tertiary (3) sub-lethally irradiated FVB-recipient mice serially transplanted with CD8+ PTCL tumors isolated from the lymph nodes of a <i>Dnmt3a</i>^<i>+/-</i> terminally sick mice. Data are presented as average time to tumor development. Three PTCL lines are shown. (E) Immunoblot showing Dnmt3a protein levels in <i>Dnmt3a</i>^<i>+/+</i> controls (+/+) and <i>Dnmt3a</i>^<i>+/-</i> PTCL (+/-) samples. <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL (Δ/Δ) is shown as a negative control. γ-tubulin is shown as a loading control. (F) Representative flow diagram showing CD8 and TCR-Vβ 8.1/8.2 expression in <i>Dnmt3a</i>^<i>+/+</i> controls (+/+) and <i>Dnmt3a</i>^<i>+/-</i> PTCL LN samples.</p

Promoter hypomethylation is conserved across multiple <i>Dnmt3a</i>^<i>Δ/Δ</i> and <i>Dnmt3a</i>^<i>+/-</i> mouse lymphomas.

<p>COBRA analysis of promoter methylation for <i>Coro2a</i>, <i>Cxcr5</i>, <i>Ikzf3</i>, <i>IL2Rβ</i>, <i>Jdp2</i>, <i>Lpar5</i>, <i>Oas3</i>, <i>Ppil1</i>, <i>Pvt1</i>, <i>Racgap1</i> and <i>Wnt8a</i> in <i>wild-type</i> CD8+, <i>Dnmt3a</i>^<i>Δ/Δ</i> pre-tumor CD8+, <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL, <i>Dnmt3a</i>^<i>+/-</i> pre-tumor CD8+, and <i>Dnmt3a</i>^<i>+/-</i> PTCL samples. PCR fragments amplified from bisulfite-treated genomic DNA were digested with the restriction enzymes <i>BstU</i>I, <i>Taq</i>1 or <i>Tai</i>I. Undigested (U) and digested (D) fragments correspond to unmethylated and methylated DNA, respectively. Control PCR fragments generated from fully methylated mouse genomic DNA that is undigested (M) or digested (CpG) are shown.</p

Jdp2 is hypomethylated and overexpressed in human and mouse PTCL.

<p>(A) COBRA analysis of mouse Jdp2 promoter methylation in three independent <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL samples. Undigested (U) and digested (D) fragments correspond to unmethylated and methylated DNA, respectively. Control PCR fragments generated from fully methylated mouse genomic DNA that is undigested (M) or digested (CpG) are shown. (B) Normalized gene expression of Jdp2 in mouse CD8+ control, <i>Dnmt3a</i>^<i>+/-</i> PTCL, and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL samples by qRT-PCR. Data presented are the average of two independent experiments. Error bars show standard deviation and an asterisk (*) denotes a p<0.05 (student t-test). (C) Bisulfite sequencing of the JDP2 promoter in normal human CD3+ T cells and in two independent human PTCL samples. Circles represent individual CpGs within the promoter. Black and white areas denote the relative portion of methylated and un-methylated sequence reads at a CpG, respectively. (D) Normalized gene expression of JDP2 in normal human CD3+ T cells and human PTCL samples, by qRT-PCR. Data presented are the average of two independent experiments. Error bars show standard deviation and an asterisk (*) denotes a p<0.05 (student t-test).</p

A majority of promoters are methylated and inactive in normal mouse CD8+ T cells.

<p>(A) CpG methylation in <i>wild-type</i> CD8+ cells, as determined by WGBS. Individual CpGs were placed into quartiles based on percent methylation (0–25%, 26–50%, 51–75%, and 76–100%). (B) Percent methylation shown by quartiles for core promoter regions (-300bp to +150bp relative to the TSS) in <i>wild-type</i> CD8+ cells. Methylation percentages for all CpGs across the 450bp region were averaged to give a mean methylation value for each gene’s core promoter. (C) A heat map displaying methylation status of 21,712 promoters in <i>wild-type</i> CD8+ as determined by WGBS. Methylation percentage for individual CpGs were annotated to the promoter regions −300bp to +150bp relative to the transcription start site (TSS). Methylation percentages for all CpGs across the 450bp region were averaged to give a mean methylation value for each gene promoter. Lowly methylated promoters are shown in yellow and highly methylated promoters in blue (D) Heat map presentation of gene-matched promoter methylation (as analyzed in panel C) and corresponding transcriptional expression (averaged FPKM values) in <i>wild-type</i> CD8+ cells, as determined by WGBS and RNA-seq for 15,732 genes. Highly expressed genes are denoted in red and lowly expressed genes are denoted in green. (E) Ingenuity Pathway analysis (IPA) of highly expressed genes (FPKM ≥ 10) in <i>wild-type</i> CD8+ cells. The top subcategories obtained in Physiological System, Development and Functions are displayed (P<0.05, for all subcategories).</p

Gene expression changes are partially conserved between mouse and human PTCL.

<p>(A) Heat maps derived from global expression profiling for genes differentially expressed in both human PTCL (PTCL) relative to normal tonsil T cells (T) and in <i>Dnmt3a</i>^<i>+/-</i> PTCL (+/-) and/or <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL (Δ/Δ) relative to normal <i>Dnmt3a</i>^<i>+/+</i> CD8+ cells. 182 genes were commonly overexpressed in human PTCL, <i>Dnmt3a</i>^<i>+/-</i> PTCL, and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL, while 210 genes were commonly underexpressed in all three tumor types. 134 overexpressed and 205 underexpressed genes were specific to human PTCL and <i>Dnmt3a</i>^<i>+/-</i> PTCL, whereas 70 overexpressed and 29 underexpressed genes were only observed in human PTCL and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL. For microarray data (human samples), genes with a fold change >1.5 and a P-value <0.05 were considered significant. For RNA-seq data (mouse samples), genes with a fold change >2 and a p-value <0.05 were considered significant. A color bar displays fold change in gene expression with overexpressed shown in red and underexpressed in green. (B) Venn diagrams showing overlaps in gene expression between human PTCL (black) and mouse <i>Dnmt3a</i>^<i>+/-</i> PTCL (blue) and <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL (green), as determined in panel A of the figure.</p

Promoter and gene-body hypomethylation is present throughout the genome of <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCLs.

<p>Circos plot of DMRS annotated to promoters and gene bodies in <i>Dnmt3a</i>^<i>Δ/Δ</i> PTCL relative to <i>wild-type</i> CD8+ cells. DMRs aligning to promoter (outer circle) and gene body (inner circles) are displayed in relation to its chromosomal position in the mouse genome. Hypomethylated DMRS are indicated by yellow lines and hypermethylated DMRS are indicated by blue lines.</p