Filippo Severin

Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival.

Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines. As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients.

Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells

Cortactin (CTTN) is a substrate of the Src kinase Lyn that is known to play an actin cytoskeletal regulatory role involved in cell migration and cancer progression following its phosphorylation at Y421. We recently demonstrated that Cortactin is overexpressed in patients with chronic lymphocytic leukaemia (CLL). This work was aimed at defining the functional role of Cortactin in these patients. We found that Cortactin is variably expressed in CLL patients both in the peripheral blood and lymph nodes and that its expression correlates with the release of matrix metalloproteinase 9 (MMP‐9) and the motility of neoplastic cells. Cortactin knockdown, by siRNA, induced a reduction in MMP‐9 release as well as a decrease of migration capability of leukaemic B cells in vitro, also after chemotactic stimulus. Furthermore, Cortactin phosphorylation was lowered by the Src kinase‐inhibitor PP2 with a consequent decrease of MMP‐9 release in culture medium. An impaired migration, as compared to control experiments without Cortactin knockdown, was observed following CXCL12 triggering. Reduced Cortactin expression and phosphorylation were also detected both in vivo and in vitro after treatment with Ibrutinib, a Btk inhibitor. Our results highlight the role of Cortactin in CLL as a check‐point molecule between the BCR and CXCR4 signalling pathways.

Targeting Brutons Tyrosine Kinase in Chronic Lymphocytic Leukemia at the Crossroad between Intrinsic and Extrinsic Pro-survival Signals

Chemo immunotherapies for chronic lymphocytic leukemia (CLL) showed a positive impact on clinical outcome, but many patients relapsed or become refractory to the available treatments. The main goal of the researchers in CLL is the identification of specific targets in order to develop new therapeutic strategies to cure the disease. The Bcell receptor-signalling pathway is necessary for survival of malignant B cells and its related molecules recently become new targets for therapy. Moreover, leukemic microenvironment delivers survival signals to neoplastic cells also overcoming the apoptotic effect induced by traditional drugs. In this context, the investigation of Bruton’s tyrosine kinase (Btk) is useful in: i) dissecting CLL pathogenesis; ii) finding new therapeutic approaches striking simultaneously intrinsic as well as extrinsic pro-survival signals in CLL. This paper will review these main topics.

Profiling B cell chronic lymphocytic leukemia by reverse phase protein array: Focus on apoptotic proteins

B cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B lymphocytes from proliferative activity and apoptosis resistance. The increased awareness of the importance of B cell receptor signaling in CLL has raised new opportunities for targeted intervention. Herein, we describe a study performed with the high‐throughput RPPA (reverse phase protein array) technique, which allowed us to simultaneously study different molecules in a large series of patients. We analyzed B lymphocytes from 57 patients with CLL and 11 healthy subjects. Different pathways were assessed for activation/expression of key signaling proteins. Data obtained were validated by Western blotting and confocal microscopy. The RPPA investigation and its validation, identified 3 series of proteins: 1) molecules whose expression levels reached statistically significant differences in CLL vs. healthy controls (HSP70, Smac/DIABLO, cleaved PARP, and cleaved caspase‐6); 2) proteins with a positive trend of difference in CLL vs. healthy controls (HS1, γ‐tubulin, PKC α/β‐II Thr‐638/641, p38 MAPK Thr‐180/Tyr‐182, NF‐κB Ser‐536, Bcl2 Ser‐70 and Src Tyr‐527); and 3) molecules differentially expressed in patients with IGHV mutations vs. those without mutations (ZAP70, PKC‐ζλ, Thr‐410/403, and CD45). This study identified some molecules, particularly those involved in apoptosis control, which could be considered for further studies to design new therapeutic strategies in CLL.

Abnormal regulation of BCR signalling by c-Cbl in chronic lymphocytic leukaemia

Abnormalities of molecules involved in signal transduction pathways are connected to Chronic Lymphocytic Leukemia (CLL) pathogenesis and a critical role has been already ascribed to B-Cell Receptor (BCR)-Lyn axis. E3 ubiquitin ligase c-Cbl, working together with adapter protein CIN85, controls the degradation of protein kinases involved in BCR signaling. To investigate cell homeostasis in CLL, we studied c-Cbl since in normal B cells it is involved in the ubiquitin-dependent Lyn degradation and in the down-regulation of BCR signaling. We found that c-Cbl is overexpressed and not ubiquitinated after BCR engagement. We observed that c-Cbl did not associate to CIN85 in CLL with respect to normal B cells at steady state, nor following BCR engagement. c-Cbl association to Lyn was not detectable in CLL after BCR stimulation, as it happens in normal B cells. In some CLL patients, c-Cbl is constitutively phosphorylated at Y731 and in the same subjects, it associated to regulatory subunit p85 of PI3K. Moreover, c-Cbl is constitutive associated to Cortactin in those CLL patients presenting Cortactin overexpression and bad prognosis. These results support the hypothesis that c-Cbl, rather than E3 ligase activity, could have an adaptor function in turn influencing cell homeostasis in CLL.