Telomeres are unique terminal chromosomal structures that shorten with cell division in vitro and with increased age in vivo for 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. However, we demonstrated that telomerase is in fect 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. To further study the regulation of telomerase at a transcriptional level, a series of genetically engineered mice have been constructed including: mouse TERT (mTERT) cDNA transgenics, green fluorescent protein (GFP) knock-in as a reporter for mTERT transcriptional activity (also resulting in mTERT knock-out inactivation), and a human TERT (hTERT) bacterial artificial chromosome (BAC) transgenic that allowed study of human TERT regulation in human versus mouse cellular environments. In transgenic mice, hTERT transgene expression was similar to endogenous hTERT in humans, and different from endogenous mTERT (mouse TERT). Reporter constructs and mutational analysis of the hTERT and mTERT promoters revealed that a non-conserved GC-box within the hTERT promoter was responsible for the human-specific repression. These results revealed that a difference in cis-regulation of transcription, rather than trans-acting transcription factors, is critical to the difference in TERT tissue-specific expression between species. This insight into species-specific regulation of telomerase and telomeres provides an experimental basis for generating mice humanized for telomerase enzyme and its pattern of expression, a model that may be useful in studying both normal human biology and pathologic processes, including malignant transformation. To translate these findings into models of clinical relevance, we studied the significance of telomere length maintenance in clinical cancer immunotherapy, in tumor-specific populations of tumor-infiltrating lymphocytes (TILs) that are isolated from human tumors, expanded in vitro, and used to treat the donor patients. We found that the telomere length of administered TILs is highly correlated with persistence of TILs after administration to patients, and is correlated as well with survival of patients treated for malignant melanoma. TILs fail to express significant telomerase activity either in vitro or after in vivo administration to patients. These findings provide a means for predicting the effectiveness of treatment in individual patients as well as a basis for enhancing therapeutic effect by interventions designed to maintain telomere length. In addition to studies of telomerase function, the functions of the telomere-associated proteins TIN-2, tankyrase-2, and tankyrase-1 have been analyzed through construction of both constitutive and conditional knockouts for each of the corresponding mouse genes. Initial studies demonstrated that constitutive inactivation of TIN-2 results in early embryonic lethality. Conditional knockouts have been generated and will be used to analyze the mechanism of these effects. Tankyrase-2 and tankyrase-1 single knockout mice are viable. In contrast, tankyrase-1/tankyrase-2 double knockouts reveal embryonic lethality, indicating redundancy as well as a critical role for these two related poly ADP-ribosylases. Conditional knock-outs have been generated to further assess the functions of these proteins. T cell lineage-specific deletion of tankyrase results in defects in T cell development at both DN-DP early differentiation and at differentiaion of DP to mature SP thymocytes. Ongoing studiess are analyzing the mechanism of tankyrase during these distinct developmental stages. Collectively, these studies will identify targets for intervention to modulate the replicative capacity of malignant as well as normal cell populations. Our studies of genetically engineerd 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 appears to be determined largely by stochastic factors. Regulation of cell survival and proliferation is also regulated by the p53 molecule. Although the function of p53 in preventing malignant transformation and tumorigenesis is well established, there is much less understanding of the role of p53 in normal cell function. Our recent studies of p53 function in antigen-specific murine T cells have revealed a striking role for p53 in integrating the outcome of signaling through the antigen-specific T-cell receptor and the receptor for interleukin-2. Our studies of p53 expression have also revealed a previously unappreciated complexity in transcriptional regulation of p53, and the impact of this complexity on tumor susceptibility. Introduction of a reversible stop in the first exon of p53, using BAC recombineering, has resulted in expression of p53 with a unique tissue specificity, leading to a tissue-specific tumor phenotype. These studies reveal the role of p53 in tissue-specifc tumor suppression. Analysis of possible human parallels is in progress. Another critical regulator of genomic stability and DNA damage response is ATM. We have previously characterized the thymic T cell lymphomas that arise in ATM-deficient mice, paralleling the susceptibility of patients with mutations in the corresponding gene. Most recently, we have probed the effect of ATM deficiency on other cell types by generating ATM KO mice that are also deficient in T cells. Strikingly, these mice develop splenic B cell lymphomas with characteristic chromosomal abnormalities and a common copy number amplicon that contains the MALT1 gene as well as a consistent and distinct pattern of gene expression assessed by microarray and principle componenet analysis. These B cell lymphomas have activated NFkB signaling and growth is suppressed by inhibitors of NFkB, syk, and btk. These lymphomas thus resemble the ABC subset of human DLCL B lymphomas and provide a model for intervention. Ongoing studies are also characterizing the T cell surveillance that prevents or rejects these lymphomas in mice with intact T cell function.
