Acute T-cell lymphoblastic leukemia (T-ALL) is a childhood cancer, characterized by the infiltration of immature T-cells in the bone marrow. Aberrant Notch signaling has been implicated in the development of different human cancers, including T-ALL. T-ALL cases very frequently bear somatic gain-of-function gene mutations in Notch1, as well as overexpression of Notch3. Moreover, Notch3 gene activating mutations have been recently described in T-ALL patients. Although, Notch hyperactivation is known to be a major driver of T-ALL development, the nature of malignant lymphoblasts expressing immature T-cells markers, but capable to escape thymus retention and infiltrate the bone marrow are still unclear. These cells could possibly represent ¿pre-leukemic¿ cells and the mechanisms underlying their dissemination need to be clarified.
The aim of our study is the analysis of the early intrathymic progenitors in a Notch3 transgenic mouse model, which recapitulates some of the features of human T-ALL. We want to focus our attention on double negative T-cells (CD4-CD8-, DN) by analysing the presence of a chemokine receptor, CXCR4, known to be highly expressed in CD25+ T-cells and known to play a role in T-cell differentiation. To this end, flow cytometry analysis will be performed to analyze the expression of this chemokine receptor in all four DN subpopulations (DN1-4). To define the possible modulation of CXCR4 in these cells we will perform transcriptional and post-trascriptional analysis in order to unveil new molecular mechanisms in T-ALL biology.
Final purpose of the present research plan will be to identify early immature T-cell deregulated processes, that might influence the following progressive stages of T-cell development driving to T-cell leukemia. In so doing, we would correlate the identity of these cells with diagnosis and relapse of the disease and to open new opportunities for the treatment of T-ALL.
The purpose of the present research plan is to determine how the transduction pathways activated by two transmembrane receptors, Notch3 e CXCR4, that normally regulate differentiation and proliferation programs, could interfere in the thymic microenviroment. More precisely, our aim is to identify and to characterize the main molecular mechanisms that deregulate early T-cell maturation steps.
To achieve our goals we will use a transgenic mouse model N3-ICtg, created in our laboratory, over-expressing the intracellular active form of Notch3. This mouse model develops an aggressive T-cell leukemia, that recapitulates most features of human T-ALL. Until seven weeks of age the transgenic animals appear normal, but later they develop a rapidly evolving disease characterized by: lethargy, hunched posture and distended abdomen. Along with these observations a complete disruption of the normal architecture of the thymus is observed1. Indeed, the disorganization of the normal thymus architecture causes a deregulated differentiation of thymocytes; this occurs because the differentiation of T-cells is not autonomous but depends on multiple signals provided by the three-dimensional network created by the stromal cells. Even less studied in the development of T-ALL, is the role of the thymic stromal cells during the initial stages of T-ALL. For this purpose, experiments aimed at highlighting the expression of SDF-1 in stromal thymic cells and the migration capability of early immature T-cells, such as the DN cells, can lead to a better understanding of the lympho-stromal interactions in tumour microenviroment. We focus our attention on CXCR4 receptor in view of the fact that SDF-1/CXCR4 axis plays an important role in ß-selection together with preTCR and Notch2. Indeed, CXCR4 is expressed on the surface of all thymocytes, human and mouse, from the DN1 to the DP stage, with a peak of expression in the DN2-DN3 and DN-DP transitions, while it is completely absent in the single positive cells3,4. Of note, Notch3 governs the transitions from DN3 until DP T-cell stage. It is also known that Notch works as a regulator of DN metabolism5 and that this signaling is not sufficient to support the metabolic changes associated with ß-selection. To allow the differentiation of T-cells from the DN to the DP stage, Notch pathway has to cooperate with that of preTCR and with SDF-1/CXCR4 axis6. So, a cooperation between Notch, pre-TCR and CXCR4 in the regulation of early stages of T-cell maturation seems to be necessary. Assuming these evidences, Notch3 over-expression, through the disorganization of thymus architecture, causes a deregulated differentiation of thymocytes and a deregulated SDF-1/CXCR4 interaction between stromal and early immature developing T-cells. In so doing, Notch3 and CXCR4 interplay can interfere with normal T-cell progressive maturation and can induce T-ALL.
Further proofs sustaining that Notch drives self-renewal of thymocytes are given from the studies in Tal1/Lmo2 transgenic mouse model of T-ALL, where it is shown that the DN cells are the cells possessing self-renewal properties while retaining the potential to differentiate. Such an assumption, makes DN population possible candidate for leukemia initiating cells7. For these purposes, the expression of CXCR4, Bcl2A1, Tal1 and Lmo2 genes in DN thymocytes and Notch3-expressing leukemic cell lines could allow to unveil the molecular mechanisms that drive initial stages of T-ALL development.
Our findings could identify the precise stage of early T-cell maturation, deregulated by Notch3, that may be the start point for T-ALL development. So, the importance in this research plan lies in the fact that such findings lead to a deeper knowledge of T-ALL biology, that could open new therapeutic opportunities for a precocious treatment in T-ALL.
1 Bellavia et al., 2000; 2 Kumar et al., 2006; 3 Ara et al 2003; 4 Plotkin et al., 2003; 5 Ciofani and Zuniga-Pflucker 2005; 6 Janas and Turner 2010; 7 Tatarek et al., 2011.