Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: The role of the microRNAs in CXCR4-dependent maturation of thymocytes in a Notch3-induced Acute Lymphoblastic Leukemia model

Considering the crucial role of Notch3 in human T-ALL pathogenesis, the aim of our work was to investigate the effects of Notch3 signaling on the expression of the chemokine receptor CXCR4, in a transgenic model of T-cell Acute Lymphoblastic Leukemia (T-ALL) induced by the over-expression of the active intracellular form (IC) of Notch3 under the control of an Lck-promoter (N3-ICtg). Previous studies have shown that overactive Notch3 increases the membrane expression of CXCR4 as T-ALL progresses. It is observed that Double-Positive thymocytes (CD4⁺ CD8⁺; DP) have an increased ability to migrate and early infiltrate lymphoid organs, suggesting their potential role as ‘pre-leukemic’ cells. In human T-ALL cell lines, it was observed that high membrane expression of CXCR4 is closely linked to the ability to migrate in vitro in response to chemotactic stimuli possibly regulated by Notch3 through a β-Arrestin-dependent mechanism (F. Ferrandino et al.,2018). Given the importance of Notch3 signaling in regulating CXCR4 expression, this new study in comparison to WT, investigates the correlation between the chemokine receptor and the abnormal expansion of immature DN cells in N3-ICtg mice as T-ALL progresses. The early expression of the transgene in immature thymocytes made it possible to investigate subpopulations not yet expressing the CD4 co-receptor and CD8 termed Double Negative (CD4-CD8-; DN). Studies of the immunophenotype and epigenetic regulation of CXCR4 by microRNAs investigated the role of a hyperactive Notch3 in the regulation of CXCR4 expression and biological parameters in DN thymocytes during T-ALL progression. The techniques mainly used in the work were flow cytometry (FacsCalibur) for the characterization of specific T-cell subpopulations, FACSAria selection to isolate the cell population of interest from the thymus (DN cells), qRT-PCR, protein analysis methods, chromatin immunoprecipitation (ChIP) and mimic and AntagomiR transfection.