Tcl1 interacts with Atm and enhances NF-κB activation in hematologic malignancies

The T-cell leukemia/lymphoma 1 (TCL1) oncogene is a target of chromosomal translocations and inversions at 14q31.2, and its rearrangement in T cells causes T-cell prolymphocytic leukemias. TCL1 dysregulation in B cells is responsible for the development of an aggressive form of chronic lymphocytic leukemia (CLL), the most common human leukemia. We have investigated the mechanisms underlying the oncogenic functions of Tcl1 protein using a mass spectrometry approach and have identified Atm (ataxia-telangiectasia mutated) as a candidate Tcl1-interacting protein. The Tcl1-Atm complex formation was validated by coimmunoprecipitation experiments. Importantly, we show that the association of Atm with Tcl1 leads to enhanced IκBα phosphorylation and ubiquitination and subsequent activation of the NF-κB pathway. Our findings reveal functional cross-talk between Atm and Tcl1 and provide evidence for a novel pathway that could be targeted in leukemias and lymphomas.     

Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression

The TCL1 gene at 14q32.1 is involved in chromosomal translocations and inversions in mature T cell leukemias. These leukemias are classified either as T prolymphocytic leukemias, which occur very late in life, or as T chronic lymphocytic leukemias, which often arise in patients with ataxia telangiectasia (AT) at a young age. In transgenic animals, the deregulated expression of TCL1 leads to mature T cell leukemia, demonstrating the role of TCL1 in the initiation of malignant transformation in T cell neoplasia. Expression of high levels of Tcl1 have also been found in a variety of human tumor-derived B cell lines ranging from pre-B cell to mature B cell. Here we describe the phenotype of transgenic mice, E mu-TCL1, established with TCL1 under the control of a V(H) promoter-Ig(H)-E mu enhancer to target TCL1 expression to immature and mature B cells. Flow cytometric analysis reveals a markedly expanded CD5(+) population in the peritoneal cavity of E mu-TCL1 mice starting at 2 mo of age that becomes evident in the spleen by 3-5 mo and in the bone marrow by 5-8 mo. Analysis of Ig gene rearrangements indicates monoclonality or oligoclonality in these populations, suggesting a preneoplastic expansion of CD5(+) B cell clones, with the elder mice eventually developing a chronic lymphocytic leukemia (CLL)-like disorder resembling human B-CLL. Our findings provide an animal model for CLL, the most common human leukemia, and demonstrate that deregulation of the Tcl1 pathway plays a crucial role in CLL pathogenesis.     

The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a

Dnmt3L is required for the establishment of maternal methylation imprints at imprinting centers (ICs). Dnmt3L, however, lacks the conserved catalytic domain common to DNA methyltransferases. In an attempt to define its function, we coexpressed DNMT3L with each of the two known de novo methyltransferases, Dnmt3a and DNMT3B, in human cells and monitored de novo methylation by using replicating minichromosomes carrying various ICs as targets. Coexpression of DNMT3L with DNMT3B led to little or no change in target methylation. However, coexpression of DNMT3L with Dnmt3a resulted in a striking stimulation of de novo methylation by Dnmt3a. Stimulation was observed at maternally methylated ICs such as small nuclear ribonucleoprotein polypeptide N (SNRPN), Snrpn, and Igf2rAir, as well as at various nonimprinted sequences present on the episomes. Stimulation of Dnmt3a by DNMT3L was also observed at endogenous sequences in the genome. Therefore, DNMT3L acts as a general stimulatory factor for de novo methylation by Dnmt3a. The implications of these findings for the function of DNMT3L and Dnmt3a in DNA methylation and genomic imprinting are discussed.     

Aging in heterozygous Dnmt1-deficient mice: effects on survival, the DNA methylation genes, and the development of amyloidosis

We previously reported that heterozygous DNA methyltransferase 1-deficient (Dnmt1(+/-)) mice maintain T-cell immune function and DNA methylation levels with aging, whereas controls develop autoimmunity, immune senescence, and DNA hypomethylation. We therefore compared survival, cause of death, and T-cell DNA methylation gene expression during aging in Dnmt1(+/-) mice and controls. No difference in longevity was observed, but greater numbers of Dnmt1(+/-) mice developed jejunal apolipoprotein AII amyloidosis. Both groups showed decreased Dnmt1 expression with aging. However, expression of the de novo methyltransferases Dnmt3a and Dnmt3b increased with aging in stimulated T cells from control mice. MeCP2, a methylcytosine binding protein that participates in maintenance DNA methylation, increased with age in Dnmt1(+/-) mice, suggesting a mechanism for the sustained DNA methylation levels. This model thus provides potential mechanisms for DNA methylation changes of aging, and suggests that changes in DNA methylation may contribute to some forms of amyloidosis that develop with aging.     

AP-1 elements and TCL1 protein regulate expression of the gene encoding protein tyrosine phosphatase PTPROt in leukemia

We previously demonstrated that the gene encoding PTPROt, the truncated form of protein tyrosine phosphatase receptor type O expressed predominantly in hematopoietic cells, is a candidate tumor suppressor and is down-regulated in chronic lymphocytic leukemia (CLL). Here, we show that PTPROt expression is significantly reduced in CD19(+) spleen B cells from Eμ-T cell leukemia 1 (TCL1) transgenic mice relative to the wild-type mice. Strikingly, as much as a 60% decrease in PTPROt expression occurs at 7 weeks independently of promoter methylation. To elucidate the potential mechanism for this early suppression of PTPROt in these mice, we explored the role of activating protein-1 (AP-1) in its expression. We first demonstrate that AP-1 activation by 12-O-tetradecanoylphorbol-13-acetate induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. The PTPROt promoter is also responsive to over- and underexpression of AP-1, confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that TCL1 can repress the PTPROt promoter by altering c-fos expression and c-jun activation state. Finally, using primary CLL cells we have shown an inverse relationship between TCL1 and PTPROt expression. These findings further substantiate the role of TCL1 in PTPROt suppression and its importance in the pathogenesis of CLL.     

DNA methyltransferase 3a limits the expression of interleukin-13 in T helper 2 cells and allergic airway inflammation

The inverse correlation between DNA methylation and lineage-specific gene expression during T helper cell development is well documented. However, the specific functions of the de novo methyltransferases Dnmt3a and Dnmt3b in cytokine gene regulation have not been defined. We demonstrate that the expression of Dnmt3a and Dnmt3b are induced to a greater extent in T helper 2 (Th2) cells than in T helper 1 cells during polarization. Using conditional mutant mice, we determined that Dnmt3a, but not Dnmt3b, regulated expression of T helper cell cytokine genes, with the Il13 gene most prominently affected. Dnmt3a deficiency was accompanied by decreases in DNA methylation and changes in the H3K27 acetylation/methylation status at the Il13 locus. Dnmt3a-dependent regulation of Il13 also occurred in vivo because Dnmt3a(fl/fl)Cd4cre mice exhibited increased lung inflammation in a murine asthma model, compared with littermate controls. Based on these observations, we conclude that Dnmt3a is required for controlling normal Il13 gene expression and functions as a rate-limiting factor to restrict T helper 2-mediated inflammation.     

TCL1 targeting miR-3676 is codeleted with tumor protein p53 in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia and dysregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease based on transgenic mouse studies. To determine a role of microRNAs on the pathogenesis of the aggressive form of CLL we studied regulation of TCL1 expression in CLL by microRNAs. We identified miR-3676 as a regulator of TCL1 expression. We demonstrated that miR-3676 targets three consecutive 28-bp repeats within 3′UTR of TCL1 and showed that miR-3676 is a powerful inhibitor of TCL1. We further showed that miR-3676 expression is significantly down-regulated in four groups of CLL carrying the 11q deletions, 13q deletions, 17p deletions, or a normal karyotype compared with normal CD19⁺ cord blood and peripheral blood B cells. In addition, the sequencing of 539 CLL samples revealed five germ-line mutations in six samples (1%) in miR-3676. Two of these mutations were loss-of-function mutations. Because miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 (Tp53), and is codeleted with Tp53, we propose that loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression.Chronic lymphocytic leukemia (CLL) is the most common adult leukemia (1). Unfavorable prognosis associates with the expression of unmutated immunoglobulin variable genes (IGVH) and high levels of 70 kD zeta-associated protein (ZAP-70). CLLs can be subgrouped into aggressive and indolent cases and chromosomal aberrations are present in more than 80% of patients (1–3). Cytogenetic abnormalities are also a prognostic parameter (3). The most common chromosomal abnormalities detectable by cytogenetics include deletion at 13q (50%), 11q (18%), trisomy 12 (15–18%), and 17p (8%) (2, 3). Prognostic categories have shown poor survival in patients harboring 17p deletion, 11q deletion, or trisomy 12, and better survival for patients with normal karyotype and 13q deletion as the sole abnormality (3).We have shown that loss of miR-15a/16-1 is the most common genetic alteration in CLL (70–80%) (4). This alteration is very common in indolent CLLs and in indolent CLLs that progress to the aggressive form (4, 5). Previously, we have observed loss of miR-181 and miR-29, which negatively regulate T-cell leukemia/lymphoma 1 (TCL1), during the progression of CLL (6, 7).Activation of the TCL1 gene is arguably one of most studied events in the pathogenesis of aggressive CLL (8). We and others have found that TCL1 overexpression occurs in a number of B-cell malignancies, including CLL (8–10). To validate that overexpression of TCL1 is oncogenic in B-cells, we have created a Eμ-TCL1 transgenic mouse model overexpressing TCL1 (11, 12). All of these transgenic mice developed the aggressive form of CLL at the average age of 12 mo with 100% penetrance (11, 12), indicating that deregulation of TCL1 is critically important in the pathogenesis of the aggressive form of CLL. Numerous publications of this CLL model have confirmed our findings (13–17). Previously we reported that Tcl1 overexpression contributes to CLL pathogenesis by coactivating Akt, and inhibiting AP-1 and de novo DNA methylation by inhibiting of the de novo DNA methyltransferases (18–21). The mechanisms responsible for overexpression of Tcl1 in CLL are not entirely clear. For this reason, we studied the regulation of TCL1 expression by microRNAs.     

Deletion of the de novo DNA methyltransferase Dnmt3a promotes lung tumor progression

Alterations in DNA methylation have been associated with genome-wide hypomethylation and regional de novo methylation in numerous cancers. De novo methylation is mediated by the de novo methyltransferases Dnmt3a and 3b, but only Dnmt3b has been implicated in promoting cancer by silencing of tumor-suppressor genes. In this study, we have analyzed the role of Dnmt3a in lung cancer by using a conditional mouse tumor model. We show that Dnmt3a deficiency significantly promotes tumor growth and progression but not initiation. Changes in gene expression show that Dnmt3a deficiency affects key steps in cancer progression, such as angiogenesis, cell adhesion, and cell motion, consistent with accelerated and more malignant growth. Our results suggest that Dnmt3a may act like a tumor-suppressor gene in lung tumor progression and may be a critical determinant of lung cancer malignancy.     

Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a

Current evidence indicates that methylation of cytosine in mammalian DNA is restricted to both strands of the symmetrical sequence CpG, although there have been sporadic reports that sequences other than CpG may also be methylated. We have used a dual-labeling nearest neighbor technique and bisulphite genomic sequencing methods to investigate the nearest neighbors of 5-methylcytosine residues in mammalian DNA. We find that embryonic stem cells, but not somatic tissues, have significant cytosine-5 methylation at CpA and, to a lesser extent, at CpT. As the expression of the de novo methyltransferase Dnmt3a correlates well with the presence of non-CpG methylation, we asked whether Dnmt3a might be responsible for this modification. Analysis of genomic methylation in transgenic Drosophila expressing Dnmt3a reveals that Dnmt3a is predominantly a CpG methylase but also is able to induce methylation at CpA and at CpT.     

Impaired T- and B-cell development in Tcl1-deficient mice

TCL1, the overexpression of which may result in T-cell leukemia, is normally expressed in early embryonic tissues, the ovary, and lymphoid lineage cells. Our analysis of mouse B-lineage cells indicates that Tcl1 expression is initiated in pro-B cells and persists in splenic marginal zone and follicular B cells. T-lineage Tcl1 expression begins in thymocyte progenitors, continues in CD4(+)CD8(+) thymocytes, and is extinguished in mature T cells. In Tcl1-deficient mice, we found B lymphopoiesis to be compromised at the pre-B cell stage and T-cell lymphopoiesis to be impaired at the CD4(+)CD8(+) thymocyte stage. A corresponding increase was observed in thymocyte susceptibility to anti-CD3epsilon-induced apoptosis. Reduced numbers of splenic follicular and germinal center B cells were accompanied by impaired production of immunoglobulin G1 (IgG1) and IgG2b antibodies in response to a T-dependent antigen. The marginal zone B cells and T-cell-independent antibody responses were also diminished in Tcl1(-/-) mice. This analysis indicates a significant role for Tcl1, a coactivator of Akt signaling, in normal T- and B-cell development and function.     

Tcl1 enhances Akt kinase activity and mediates its nuclear translocation

The TCL1 oncogene at 14q32.1 is involved in the development of human mature T-cell leukemia. The mechanism of action of Tcl1 is unknown. Because the virus containing the v-akt oncogene causes T-cell lymphoma in mice and Akt is a key player in transduction of antiapoptotic and proliferative signals in T-cells, we investigated whether Akt and Tcl1 function in the same pathway. Coimmunoprecipitation experiments showed that endogenous Akt1 and Tcl1 physically interact in the T-cell leukemia cell line SupT11; both proteins also interact when cotransfected into 293 cells. Using several AKT1 constructs in cotransfection experiments, we determined that this interaction occurs through the pleckstrin homology domain of the Akt1 protein. We further demonstrated that, in 293 cells transfected with TCL1, the endogenous Akt1 bound to Tcl1 is 5-10 times more active compared with Akt1 not bound to Tcl1. The intracellular localization of Tcl1 and Akt1 in mouse fibroblasts was investigated by immunofluorescence. When transfected alone, Akt1 was found only in cytoplasm whereas Tcl1 was localized in the cytoplasm and in the nucleus. Interestingly, Akt1 was also found in the nucleus when AKT1 was cotransfected with TCL1, suggesting that Tcl1 promotes the transport of Akt1 to the nucleus. These findings were supported by the intracellular localization of Akt1 or Tcl1 when Tcl1 or Akt1, respectively, were confined to the specific cellular compartments. Thus, we demonstrate that Tcl1 is a cofactor of Akt1 that enhances Akt1 kinase activity and promotes its nuclear transport.