Genome-wide methylation profiling of Peripheral T-cell lymphomas identifies TRIP13 as a critical driver of tumor proliferation and survival.

Cytosine methylation of genomic DNA contributes to the regulation of gene expression and is involved in normal development including hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases (DNMTs) that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent a diverse group of aggressive mature T-cell malignancies accounting for approximately 10-15% of non-Hodgkin lymphoma cases in the US. PTCLs exhibit a broad spectrum of clinical, histological, and immunophenotypic features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we used high-resolution Whole Genome Bisulfite Sequencing (WGBS) and RNA-seq to profile DNA methylation and gene expression of PTCLs and normal T-cells. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose both genetic and pharmacologic inactivation, inhibited cellular growth of PTCL cell lines by inducing G2-M arrest accompanied by apoptosis suggesting that such an approach might be beneficial in human lymphoma treatment. Altogether we show that human PTCLs are characterized by a large number of recurrent methylation alterations, and demonstrated that TRIP13 is critical for PTCL maintenance in vitro.

Decreases in different Dnmt3b activities drive distinct development of hematologic malignancies in mice.

DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low-frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, hairy cell leukemia, and diffuse large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/- mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/- mice developed T-cell lymphomas, chronic lymphocytic leukemia, and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/- mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1, and Trip13, and p53 downregulation were major events contributing to Dnmt3b+/- lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo, whereas accessory and methylation-independent repressive functions are important to prevent T-cell transformation.

Dnmt3b catalytic activity is critical for its tumour suppressor function in lymphomagenesis and is associated with c-Met oncogenic signalling.

BACKGROUND: DNA methylation regulates gene transcription in many physiological processes in mammals including development and haematopoiesis. It is catalysed by several DNA methyltransferases, including Dnmt3b that mediates both methylation-dependant and independent gene repression. Dnmt3b is critical for mouse embryogenesis and functions as a tumour suppressor in haematologic malignancies in mice. However, the extent to which Dnmt3b's catalytic activity (CA) is involved in development and cancer is unclear. METHODS: We used a mouse model expressing catalytically inactive Dnmt3b (Dnmt3bCI) to study a role of Dnmt3b's CA in development and cancer. We utilized global approaches including Whole-genome Bisulfite sequencing and RNA-seq to analyse DNA methylation and gene expression to identify putative targets of Dnmt3b's CA. To analyse postnatal development and haematopoiesis, we used tissue staining, histological and FACS analysis. To determine potential involvement of selected genes in lymphomagenesis, we used overexpression and knock down approaches followed by in vitro growth assays. FINDINGS: We show that mice expressing Dnmt3bCI only, survive postnatal development and develop ICF (the immunodeficiency-centromeric instability-facial anomalies) -like syndrome. The lack of Dnmt3b's CA promoted fibroblasts transformation in vitro, accelerated MLL-AF9 driven Acute Myeloid Leukaemia and MYC-induced T-cell lymphomagenesis in vivo. The elimination of Dnmt3b's CA resulted in decreased methylation of c-Met promoter and its upregulation, activated oncogenic Met signalling, Stat3 phosphorylation and up-regulation of Lin28b promoting lymphomagenesis. INTERPRETATION: Our data demonstrates that Dnmt3b's CA is largely dispensable for mouse development but critical to prevent tumourigenesis by controlling events involved in cellular transformation. FUNDING: This study was supported by Department of Anatomy and Cell Biology and Cancer Centre at the University of Florida start-up funds, NIH/NCI grant 1R01CA188561-01A1 (R.O.).

Catalytically inactive Dnmt3b rescues mouse embryonic development by accessory and repressive functions.

DNA methylation regulates gene expression in a variety of processes, including mouse embryonic development. Four catalytically active enzymes function in mice as DNA methyltransferases (Dnmts) and as transcriptional regulators. Inactivation of Dnmt3b results in mouse embryonic lethality, but which activities are involved is unclear. Here we show that catalytically inactive Dnmt3b restores a majority of methylation and expression changes deregulated in the absence of Dnmt3b, and as a result, mice survive embryonic development. Thus, Dnmt3b functions as an accessory cofactor supporting catalytic activities performed by other Dnmts. We further demonstrate that Dnmt3b is linked to a control of major developmental pathways, including Wnt and hedgehog signaling. Dnmt3b directly represses Wnt9b whose aberrant up-regulation contributes to embryonic lethality of Dnmt3b knockout embryos. Our results highlight that Dnmt3b is a multifaceted protein that serves as an enzyme, an accessory factor for other methyltransferases, and as a transcriptional repressor in mouse embryogenesis.

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

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 Dnmt3a+/- 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 Dnmt3a+/- and Dnmt3aΔ/Δ lymphomas were largely conserved and significantly overlapped with those of human tumors. Importantly, we observed downregulation of tumor suppressor p53 in Dnmt3a+/- and Dnmt3aΔ/Δ lymphomas as well as in pre-tumor thymocytes from 9 months old but not 6 weeks old Dnmt3a+/- 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 bona fide tumor suppressor of T cell malignancies p53.

Loss of Dnmt3a induces CLL and PTCL with distinct methylomes and transcriptomes in mice.

Cytosine methylation of DNA is an epigenetic modification involved in the repression of genes that affect biological processes including hematopoiesis. It is catalyzed by DNA methyltransferases, one of which -DNMT3A- is frequently mutated in human hematologic malignancies. We have previously reported that Dnmt3a inactivation in hematopoietic stem cells results in chronic lymphocytic leukemia (CLL) and CD8-positive peripheral T cell lymphomas (PTCL) in EµSRα-tTA;Teto-Cre;Dnmt3afl/fl; Rosa26LOXPEGFP/EGFP (Dnmt3aΔ/Δ) mice. The extent to which molecular changes overlap between these diseases is not clear. Using high resolution global methylation and expression analysis we show that whereas patterns of methylation and transcription in normal B-1a cells and CD8-positive T cells are similar, methylomes and transcriptomes in malignant B-1a and CD8+ T cells are remarkably distinct, suggesting a cell-type specific function for Dnmt3a in cellular transformation. Promoter hypomethylation in tumors was 10 times more frequent than hypermethylation, three times more frequent in CLL than PTCL and correlated better with gene expression than hypermethylation. Cross-species molecular comparison of mouse and human CLL and PTCL reveals significant overlaps and identifies putative oncogenic drivers of disease. Thus, Dnmt3aΔ/Δ mice can serve as a new mouse model to study CLL and PTCL in relevant physiological settings.

Promoter Hypomethylation and Expression Is Conserved in Mouse Chronic Lymphocytic Leukemia Induced by Decreased or Inactivated Dnmt3a.

DNA methyltransferase 3a (DNMT3A) catalyzes the formation of 5-methyl-cytosine in mammalian genomic DNA, and it is frequently mutated in human hematologic malignancies. Bi-allelic loss of Dnmt3a in mice results in leukemia and lymphoma, including chronic lymphocytic leukemia (CLL). Here, we investigate whether mono-allelic loss of Dnmt3a is sufficient to induce disease. We show that, by 16 months of age, 65% of Dnmt3a(+/-) mice develop a CLL-like disease, and 15% of mice develop non-malignant myeloproliferation. Genome-wide methylation analysis reveals that reduced Dnmt3a levels induce promoter hypomethylation at similar loci in Dnmt3a(+/-) and Dnmt3a(Δ/Δ) CLL, suggesting that promoters are particularly sensitive to Dnmt3a levels. Gene expression analysis identified 26 hypomethylated and overexpressed genes common to both Dnmt3a(+/-) and Dnmt3a(Δ/Δ) CLL as putative oncogenic drivers. Our data provide evidence that Dnmt3a is a haplo-insufficient tumor suppressor in CLL and highlights the importance of deregulated molecular events in disease pathogenesis.

Methylation-independent repression of Dnmt3b contributes to oncogenic activity of Dnmt3a in mouse MYC-induced T-cell lymphomagenesis.

DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood. Here we analyzed the functional consequences of Dnmt3a inactivation in a mouse model of MYC-induced T-cell lymphomagenesis (MTCL). Loss of Dnmt3a delayed tumorigenesis by suppressing cellular proliferation during disease progression. Gene expression profiling and pathway analysis identified upregulation of 17 putative tumor suppressor genes, including DNA methyltransferase Dnmt3b, in Dnmt3a-deficient lymphomas as molecular events potentially responsible for the delayed lymphomagenesis in Dnmt3a(Δ/Δ) mice. Interestingly, promoter and gene body methylation of these genes was not substantially changed between control and Dnmt3a-deficient lymphomas, suggesting that Dnmt3a may inhibit their expression in a methylation-independent manner. Re-expression of both wild type and catalytically inactive Dnmt3a in Dnmt3a(Δ/Δ) lymphoma cells in vitro inhibited Dnmt3b expression, indicating that Dnmt3b upregulation may be directly repressed by Dnmt3a. Importantly, genetic inactivation of Dnmt3b accelerated lymphomagenesis in Dnmt3a(Δ/Δ) mice, demonstrating that upregulation of Dnmt3b is a relevant molecular change in Dnmt3a-deficient lymphomas that inhibits disease progression. Collectively, our data demonstrate an unexpected oncogenic role for Dnmt3a in MTCL through methylation-independent repression of Dnmt3b and possibly other tumor suppressor genes.

Essential role for Dnmt1 in the prevention and maintenance of MYC-induced T-cell lymphomas.

DNA cytosine methylation is an epigenetic modification involved in the transcriptional repression of genes controlling a variety of physiological processes, including hematopoiesis. DNA methyltransferase 1 (Dnmt1) is a key enzyme involved in the somatic inheritance of DNA methylation and thus plays a critical role in epigenomic stability. Aberrant methylation contributes to the pathogenesis of human cancer and of hematologic malignancies in particular. To gain deeper insight into the function of Dnmt1 in lymphoid malignancies, we genetically inactivated Dnmt1 in a mouse model of MYC-induced T-cell lymphomagenesis. We show that loss of Dnmt1 delays lymphomagenesis by suppressing normal hematopoiesis and impairing tumor cell proliferation. Acute inactivation of Dnmt1 in primary lymphoma cells rapidly induced apoptosis, indicating that Dnmt1 is required to sustain T-cell lymphomas. Using high-resolution genome-wide profiling, we identified differentially methylated regions between control and Dnmt1-deficient lymphomas, demonstrating a locus-specific function for Dnmt1 in both maintenance and de novo promoter methylation. Dnmt1 activity is independent of the presence of Dnmt3a or Dnmt3b in de novo promoter methylation of the H2-Ab1 gene. Collectively, these data show for the first time that Dnmt1 is critical for the prevention and maintenance of T-cell lymphomas and contributes to aberrant methylation by both de novo and maintenance methylation.

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.

Mouse development with a single E2F activator.

The E2F family is conserved from Caenorhabditis elegans to mammals, with some family members having transcription activation functions and others having repressor functions. Whereas C. elegans and Drosophila melanogaster have a single E2F activator protein and repressor protein, mammals have at least three activator and five repressor proteins. Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a(3bki) or E2f3a(1ki), respectively) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development.

Specific tumor suppressor function for E2F2 in Myc-induced T cell lymphomagenesis.

Deregulation of the Myc pathway and deregulation of the Rb pathway are two of the most common abnormalities in human malignancies. Recent in vitro experiments suggest a complex cross-regulatory relationship between Myc and Rb that is mediated through the control of E2F. To evaluate the functional connection between Myc and E2Fs in vivo, we used a bitransgenic mouse model of Myc-induced T cell lymphomagenesis and analyzed tumor progression in mice deficient for E2f1, E2f2, or E2f3. Whereas the targeted inactivation of E2f1 or E2f3 had no significant effect on tumor progression, loss of E2f2 accelerated lymphomagenesis. Interestingly, loss of a single copy of E2f2 also accelerated tumorigenesis, albeit to a lesser extent, suggesting a haploinsufficient function for this locus. The combined ablation of E2f1 or E2f3, along with E2f2, did not further accelerate tumorigenesis. Myc-overexpressing T cells were more resistant to apoptosis in the absence of E2f2, and the reintroduction of E2F2 into these tumor cells resulted in an increase of apoptosis and inhibition of tumorigenesis. These results identify the E2f2 locus as a tumor suppressor through its ability to modulate apoptosis.

Extra-embryonic function of Rb is essential for embryonic development and viability.

The retinoblastoma (Rb) gene was the first tumour suppressor identified. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5 (refs 2-4). However, the actual cause of the embryonic lethality has not been fully investigated. Here we show that loss of Rb leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. We used two complementary techniques-tetraploid aggregation and conditional knockout strategies-to demonstrate that Rb-deficient embryos supplied with a wild-type placenta can be carried to term, but die soon after birth. Most of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-null animals were virtually absent in rescued Rb-null pups. These findings identify and define a key function of Rb in extra-embryonic cell lineages that is required for embryonic development and viability, and provide a mechanism for the cell autonomous versus non-cell autonomous roles of Rb in development.