Notch3 sustains CXCR4 expression in acute T cell lymphoblastic leukemia progression

Introduction: Acute T cell lymphoblastic-leukemia (T-ALL) is the most common of childhood cancers. The majority of T-ALL cases are driven by Notch deregulated signaling, that activates different downstream pathways essential for TALL cell proliferation and leukemia-initiating cell (LIC) activity. The oncogenic function of Notch3 in T-ALL was demonstrated by a murine model of our laboratory, characterized by enforced expression of the Notch3 receptor active form (N3-IC) in immature thymocytes (N3-ICtg). Aberrant proliferation and maturation at the preT/T transition phase and constitutive activation of preTCR and NF-kB signalings were observed in N3-ICtg mice. Multiple signals from stroma sustain T cell differentiation programs in the (DN) to Double Positive (DP) thymocytes. The stromal cell derived factor SDF-1 (CXCL12) and its receptor CXCR4 promote survival of DN thymocytes and regulate the migration during the DN/DP transition. The CXCR4/SDF-1 axis has been recently suggested to play a role in the pathogenesis of T-ALL. Material and Methods: CXCR4 and Notch3 cell-surface evalution was performed by FACS analysis with freshly isolated DP T cells obtained from thymus, blood, spleen and bone marrow of N3-ICtg and wt mice. Statistical analysis of the data was performed. Biochemical analysis of the modulatory effect of Notch3 overexpression in TALL1, a human leukemic CD3+/CD4+/CD8+ cell line, was performed by FACS, RealTime-PCR and Western Blot. Results and Discussion: In Notch3-ICtg mice, DP-gated thymocytes display an increased level of CXCR4 expression per cell with respect to wt. Furthermore, most of the DP thymocytes highly co-express Notch3 and CXCR4. Abnormally represented DP T cells at different ages in lymphoid organs of N3-ICtg show a combined Notch3/CXCR4 expression suggesting a crosstalk of the two receptors, possibly modulating their leukemogenicity. Through inhibition of Notch signaling (GSI treatment) or by silencing Notch3 gene expression (shRNA) we could modulate CXCR4 cell-surface expression in TALL1 (CD4+/CD8+/CD3+ leukemic cell line). Conclusion: Overall, our results are suggestive of Notch3 deregulated pathway as a molecular mechanism that may modulate DP T cells egress from thymus, in early steps of T-ALL development, by forcing CXCR4 cell-surface expression through a beta-arrestin-mediated mechanism.