Titolo della tesi: Characterization of Atm kinase dead mouse model and cancer-associated Atm kinase mutations
RATIONALE: ATM is a key enzyme in the DNA damage response. Its deficiency is associated to Ataxia Telangiectasia (A-T) a rare, multi-system disease characterized by progressive cerebellar neurodegeneration, immunodeficiency, leukemia, and lymphoid cancer predisposition. The majority of A-T patients show null mutations in the ATM gene that result in a total loss of ATM protein. A small cohort of patients presents a severe cancer phenotype compared to classical A-T patients, probably due to missense mutations that impact the ATM kinase domain leading to the production of ATM protein without kinase activity, called “ATM Kinase Dead” (ATM KD). ATM KD mutations have been also found in oncologic patients, in different types of cancers such as leukemia, lymphomas, breast, pancreas, and gastric cancers. Based on previous studies, the hypothesis is that ATM protein may have a structural role different from its kinase activity, that deserve molecular investigation.
GENERALE OBJECTIVES: The project aims to characterize lymphoblastoid cell lines of ATM KD A-T patients and a tamoxifen inducible Atm kd transgenic mouse model, in order to elucidate the cellular and molecular mechanisms that may explain the phenotype of the A-T cohort and the oncogenic predisposition of ATM KD mutations that may have therapeutic cancer implications.
EXPERIMENTAL DESIGN AND METHODS: The tamoxifen inducible Atm-kd mouse model carries a transgenic copy of Atm with the kinase dead mutation D2899A, in Atm knockout background, AtmTgD2899AAtm-/- (AtmTgKD). The effects of Atm protein were analyzed in the short or long term on: T lymphocyte development by flow cytometry (CD4, CD8, TCRβ), VDJ recombination by PCR and B cells class switch recombination (IgG); spermatogenesis process by Hematoxylin and Eosin staining; DNA damage response by western blot analyses of Atm specific targets (KAP1, CHK1) in human and murine cells; genomic instability by metaphase FISH (chromosome and chromatid breaks). The effects of Atm-kd protein on tumorigenesis were also analyzed in TgKD mice.
RESULTS: ATM-KD compared to ATM-KO samples showed: an increase in T cells number and development in blood and thymus tissues; a slight but significant increase of B cells maturation in spleen and an elevated percentage of high expressing TCRβ cells in thymus and an improvement in V(D)J trans-rearrangement. Atm-kd protein prevents/delays thymoma development in mice at least up to the age of 6 months. Moreover, we found that ATM KD partially activates DNA-PK enzyme after DNA damage in lymphoblastoid cell lines and thymocytes. AtmTgKD mice, however, show an increase of DNA damage with a greater number of depleted seminiferous tubules and more chromosome aberrations with a higher percentage of metaphase chromatids breaks.
CONCLUSION: Our findings support the idea that Atm-kd protein has a structural role in Non-homologous end Joining (NHEJ) Recombination processes that do not necessarily require Atm kinase activity (i.e. improvement of T and B lymphocytes maturation and thymoma prevention). Atm kd might exert a structural role favoring the phosphorylation of substrates after DNA damage trough the activation of other kinases of the DNA repair family. On the other hand, AtmTgKD mice exhibit a worse phenotype in the Homologous Recombination (HR) processes that instead need Atm kinase activity (i.e. germ cells development) and that might cause genomic instability.