Telomeres are unique terminal chromosomal structures that shorten with cell division in vitro and with increased age in vivo in human somatic cells. Telomerase, a ribonucleoprotein enzyme that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells and is absent in most normal human somatic cells.We demonstrated that telomerase is in fact highly regulated during development and activation of mouse and human lymphocytes. Expression of the two genes encoding the necessary and sufficient components of telomerase, RNA template (TR) and reverse transcriptase catalytic component (TERT), was found to be regulated during lymphocyte development and activation. Our studies of genetically engineered knockout mice have demonstrated that, in addition to the defect in telomere length maintenance observed in complete homozygous knockouts for either of the telomerase components, there is a deficiency in telomere maintenance in mice that are heterozygous for inactivation of one copy of either telomerase component. This phenomenon, termed haplo-insufficiency, has recently been observed in an increasing number of clinical entities, including dyskeratosis congenita, aplastic anemia, and pulmonary fibrosis, resulting from mutation and inactivation of telomerase components or related genes. In collaboration with the laboratory of Dr. Neal Young (NHLBI), we have assessed the impact of parental haplo-insufficiency for telomere maintenance on the telomere length and function of genetically normal or abnormal offspring in these affected families. The outcome of studies in mice and humans demonstrated that inheritance of telomere length is not determined by species-specific homeostatic mechanisms, but by stochastic factors. Telomerase activity is well documented in embryonic stem cells and the vast majority of tumor cells, but its role in somatic cells remains to be understood. In recent studies, we have identified an unexpected function of telomerase during cellular senescence and tumorigenesis. We crossed Tert heterozygous knockout mice (mTert +/-) for 26 generations, during which time there was progressive shortening of telomeres, and obtained primary skin fibroblasts from mTert +/+ and mTert -/- progeny of the 26th cross. As a consequence of insufficient telomerase activities in prior generations, both mTert +/+ and mTert -/- fibroblasts showed comparable and extremely short telomere length. However, mTert -/- cells approached cellular senescence faster and exhibited a significantly higher rate of malignant transformation than mTert +/+ cells. Furthermore, an evident upregulation of TERT expression was detected in mTert +/+ cells at the pre-senescence stage. Moreover, removal or downregulation of TERT expression in mTert +/+ and human primary fibroblast cells via CRISPR/Cas9 or shRNA recapitulated mTert -/- phenotypes of accelerated senescence and transformation, and overexpression of TERT in mTert -/- cells rescued these phenotypes. Together, this study suggests that TERT has a previously under-appreciated, protective role in buffering senescence stresses due to short, dysfunctional telomeres, and preventing malignant transformation. Cell survival and proliferation are also regulated by the p53 tumor suppressor molecule. Ataxia-telangiectasia mutated (ATM) is a kinase that plays a central role in maintaining genomic integrity. In both humans and mice, ATM deficiency is associated with an increased incidence of lymphoid cancers. We asked if ATM plays a more general role in preventing non-T cell malignancies by breeding mice that were both ATM-and T cell-deficient. This model removes T cells as targets for lymphomagenesis as well as eliminating T cell-dependent immune surveillance. These mice exclusively develop early onset IgM+ B cell lymphomas that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B cell lymphomas (DLBCL). Tumors express clonal as wellas emerging IghV hypermutation, and express AID, but B lymphoma development is independent of AID, occurring at equal frequency in AID knockout mice. These ATM-deficient lymphomas show considerable chromosomal instability with a recurrent amplification of a 4.48Mb region on chromosome 18 (MMU18), orthologous to a region amplified in some cases of human ABC-DLBCL, and containing Malt1 in the region of highest amplification. Importantly, these lymphomas also depend on NF-kB, MALT1, and BCR signaling for survival. Gene expression analysis revealed strong similarities between these mouse lymphomas and human ABC-DLBCL. This study reveals that ATM is required to prevent the development of B cell lymphomas that model human ABC-DLBCL and identifies an unappreciated role of T cells in preventing the emergence of these tumors. Studies now in progress are characterizing the pathways that mediate transformation of the T cell thymic lymphomas in ATM-deficient mice. We had previously reported that thymic T cell lymphomas develop in mice that are deficient in RAG-1 or RAG-2 recombinase in addition to ATM deficiency. These findings contrast to the absence of T cell lymphomas in mice double deficient in ATM and CD3e. This suggests the possibility that CD3e-containing complex, independent of rearranged TCR gene products, is required for lymphomagenesis. Additional studies now in progress are testing the requirement for components of the prototypic TCR/pre-TCR signal pathways, such as LAT, in development of lymphomas. The requirements for survival and proliferation of ATM-deficient T cell lymphomas have in fact not been fully elucidated. We have therefore initiated studies of the genetic and epigenetic changes in these lymphomas, and their dependence on defined signaling pathways. Initial findings have included a universal defect in the phosphatase PTEN, through genetic/non-genetic mechanisms that vary from tumor to tumor. Consistent with loss of PTEN activity, we have observed activation of the AKT pathway and concomitant susceptibility of lymphomas to inhibitors of AKT activity. These findings will inform the underlying biology of T cell transformation with potential relevance to clinical approaches to human T cell malignancies. Lentiviral shRNA knockdown of targets including CD3e and LAT are in progress to test the requirements for TCR signaling pathways in survival of ATM-deficient T cell lymphomas.
