CARATTERIZZAZIONE DEI MECCANISMI FISIOPATOLOGICI ALLA BASE DELLA REGOLAZIONE DEI LINFOCITI B: IMPLICAZIONI NELLA LEUCEMIA LINFATICA CRONICA E POTENZIALI FATTORI PROGNOSTICI.

In Chronic Lymphocytic Leukemia (CLL) the relationships between cell proliferation and accumulation within lymphoid organs are unclear, as are the rules that control CLL cell migration and re-circulation. We previous demonstrated that Hematopoietic cell specific Lyn substrate 1 (HS1) is involved in the natural history of CLL (Scielzo et al JCI 2005). Furthermore, we have shown that HS1 interacts with distinct cytoskeleton adapters and is likely involved in cytoskeleton reorganization (Muzio et al, Leukemia 2007). Given the role played by the cytoskeleton in controlling cellular shape, mobility and homing, we hypothesized that HS1 could be potentially relevant in the regulation of CLL cells infiltration into lymphoid tissues and re-circulation between peripheral blood and tissues. To study HS1 function in vitro, we silenced its expression in a CLL cell line (MEC1), and we tested its capacity to migrate in an in vitro and in vivo assay. We also analysed B cells from HS1 KO mice (Fukuda et al, EMBO J. 1995) and we showed that in both cellular systems HS1-deficient B cells are severely impaired in their spontaneous migration capacity. A decrease in F-actin polymerization and an increased aggregation ability was also evident in cells lacking HS1. We then observed that HS1 is central for the initiation and maintenance of B-Cell Receptor (BCR) signalling events. Indeed in the absence of HS1, cells failed to form actin-myosin complexes thereby leading to an instability of the cell signalling complex, in particular of the signalosome complex proximal to the BCR. Lyn, VAV, HIP-55, SHP-1 and RAC1/2 were directly affected by the absence of HS1 while Syk, PLC-γ2 and Blnk appear to be unaffected. To test in vivo HS1 function, we injected MEC1 cells silenced for HS1 subcutaneously in RAG2-/-γc-/- mice and observed that cells lacking HS1 spread and localized preferentially in the BM and in the lymph nodes as compared to control cells. To further investigate its role played in the onset and progression of CLL, we crossed HS1 KO mice with Eµ-TCL1 transgenic animals (Bichi R et al, PNAS 2002). The double HS1 KO/TCL-1 interestingly show a preferential accumulation of monoclonal CD19+CD5+ cells, beside all other lymphoid organs, in the bone marrow where they are usually observed at low frequencies in the Eµ-TCL1 mouse. These findings suggest that HS1 has a crucial role in controlling cell migration and tissue invasion by leukemic B cells, likely through its involvement in cytoskeleton organization. This points at HS1 as a target for development of novel cancer treatments, aiming at interfering with the lymphoid tissue infiltration and invasion which is characteristic of the disease.