INCREASED SURVIVAL OF CLL B CELLS IN THE PRESENCE OF MARROW MESENCHYMAL STROMAL CELLS: A NOVEL MODEL TO DEFINE NEW TARGETS FOR THERAPY

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western World, accounting for about 30% of adult leukemia, and it is characterized by the clonal expansion and accumulation of mature CD19+/CD5+/CD23+ B lymphocytes in the peripheral blood, bone marrow and secondary lymphoid organs. Despite their apparent longevity in patients, in vitro CLL leukemic B cells rapidly undergo spontaneous apoptosis. The selective survival advantage is due both to intrinsic defects on apoptosis mechanism and to signals delivered by accessory cells at the active site of the disease. Previous studies demonstrated that mesenchymal stromal cells (MSCs), derived from bone marrow, and CD68+ nurse-like cells, derived from peripheral blood, are involved in CLL clone longevity and migration, suggesting a crucial role of MSCs on favouring disease progression. Therefore, in this thesis we evaluated the effect of MSCs, the main stromal population in the bone marrow of CLL patients, on the survival of leukemic B cells and their role in drug resistance. MSCs were isolated from the bone marrow of 46 CLL patients; their immunophenotypic characterization was based on the expression of CD105, CD73 and CD90 and the negativity of CD14, CD34, CD45 and CD31. Co-culturing MSCs and CLL B cells, we confirmed that MSCs are able to support malignant B cell survival, providing an in vitro culture system that closely approximates CLL microenvironment in vivo. We observed that different leukemic clones demonstrated a large variety in the pro-survival effect. Evaluating the cleavage pattern of PARP, we revealed two subsets of CLL clones with different sensitivity to MSCs pro-survival signals. Our results indicate that both cell-cell contact and soluble molecules are actors in the relationship between malignant B cells and the MSCs, promoting CLL B cell survival and migration. Later, we evaluated the role of the MSCs on CLL B cells during the most common cytotoxic therapy used in clinical practice. Our data demonstrate that MSCs are able to protect leukemic B cells from apoptosis during Fludarabine and Cyclophosphamide treatment, both in vitro and in vivo. We tested MSCs protective role also during CLL B cells treatment with Ibrutinib, a novel inhibitor of Btk involved in the BCR signaling pathway, and we found that the treatment counteracts the MSC pro-survival effect. To better understand the effect of Ibrutinib on the cross-talk between CLL B cells and MSCs, we evaluated its role on leukemic B cell migration, also analyzing the expression levels of CCR7 and CXCR4, two chemokine receptors that are central in the homing of the neoplastic clone. We demonstrated that malignant B cell migration is not significantly affected by the Btk inhibitor; since cell-cell contact with MSC is crucial for CLL B cell survival, we analyzed the adhesion of leukemic B cells to MSCs after treatment with Ibrutinib. We found a significant reduction in leukemic B cells and MSCs interactions mediated by the CD49d integrin. In this thesis, we demonstrate that MSCs enhance the survival of leukemic B cells through the release of soluble factors and cell-cell direct contact and that each CLL clone reveals a peculiar response to the anti-apoptotic signals delivered by MSCs. These observations could be relevant to identify patients more responsive to druggable targets on marrow microenvironment and also to find putative new strategies for CLL therapy. A better understanding on the complexity of the cross-talk between CLL cells and their microenvironment during CLL therapy could also help to define mechanisms of drug resistance and treatment failure, as well to plan randomized clinical trials comparing new compounds and their combinations with standard chemo-immunotherapy