ALBERT

Description: Key Points Lyn’s overexpression mediates resistance to apoptosis by promoting phosphorylation and dimerization of procaspase 8 in B-CLL cells.

Description: Key PointsIn T-LGLL, autologous LGL-depleted PBMCs release high levels of IL-6 contributing to the constitutive STAT3 activation in leukemic LGL. Leukemic LGLs show SOCS3 down-modulation, which is responsible for lack of the negative feedback mechanism controlling STAT3 activation.

Description: INTRODUCTION Cortactin is an actin-binding protein involved in several cell functions, i.e. the assembly and the organization of cytoskeleton. Its overexpression was observed in several human cancers and experimental data support the role of cortactin in metastatic capability through the regulation of cell motility and the release of matrix metalloproteinase-9 (MMP-9). The activity of this protein is regulated by its phosphorylation in Tyr residues by Src kinase family. We previously demonstrated that in leukemic cells from B-CLL patients the Src kinase Lyn is overexpressed, activated and involved in the resistance to apoptosis. Recently, we found that cortactin is overexpressed in patients with B-CLL. Here we investigated the involvement of cortactin in the release of MMP-9 and, therefore, in the progression of B-CLL. METHODS Blood samples were collected from 10 controls and 34 B-CLL patients. Informed consent was obtained according to the Declaration of Helsinki. Untouched peripheral blood B cells were purified using the RosetteSep for human B cells isolation kit. The samples that were used had at least 95% of normal CD19+ or neoplastic CD5+/CD19+ cells, as assessed by flow-cytometry (FC). The purified B cells (2×106 cells/ml) were cultured in RPMI medium with or without CXCL12 (100ng/ml) and Ibrutinib (5μM) for the evaluation of MMP-9 production using ELISA. MMP-9 release by neoplastic B cells was also investigated after cortactin silencing in 4 patients which expressed high level of cortactin. The protein was silenced by SMARTpool siRNA collection (Dharmacon, Thermo Scientific), according to the manufacturer's instructions. Immunohistochemical (IHC) staining was performed on formalin-fixed, paraffin-embedded tissue sections using a fully automated platform. RESULTS By IHC and FC we confirmed the increased expression of cortactin in patients with respect to controls, moreover we defined three groups of patients according to different expression levels of Cortactin. We found that the release of MMP-9 by neoplastic B cells correlated to the expression of cortactin after 5 and 24-hrs culture. To investigate whether cortactin was involved in MMP-9 secretion in B-CLL, a cortactin-targeted siRNA silencing system was used to knockdown this protein in 4 patients with high cortactin expression. We found that following cortactin knockdown, leukemic cells showed a defect in MMP-9 secretion, as assessed by ELISA test. This protease also showed a decreased gelatinolytic activity in culture medium, supporting the hypothesis that cortactin is involved in the regulation of MMP-9 secretion in B-CLL malignant cells. We also demonstrated that the incubation of leukemic cells with PP2 or Btk decreased Tyr phosphorylation level of cortactin and shut down the release of MMP-9 in culture medium, also following CXCL12 triggering. Interestingly, we found that cortactin levels were significantly reduced following 3 months of Ibrutinib treatment and remained lower till the last clinical control, at 6 months of treatment. Consistent with these data we demonstrated i) a significant reduction in MMP-9 levels after in vivotreatment with Ibrutinib ii) a strong correlation between Cortactin expression and MMP-9 levels in CLL patients before and following Ibrutinib therapy. Finally, we also observed a redistribution of lymphocytes from the tissues to the periphery and a consequent reduction of lymphadenopathy in 4 cases where the levels of cortactin were higher than those observed in the patient who did not show enlarged lymph nodes at the time of diagnosis. CONCLUSIONS The overexpression of cortactin in neoplastic B cells and the correlation between cortactin levels, activity and MMP-9 release suggest a role of this protein in metastatic invasion and in the B-CLL aggressiveness. In addition, cortactin might represent a biomarker for diagnosis and prognosis and a target for new therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION We recently found that the Heat Shock Protein of 70kDa (HSP70), an ATP-dependent chaperone that is induced by cellular stress and protects cells against various apoptotic stimuli, was particularly overexpressed in neoplastic B cells from Chronic Lymphocytic Leukemia (CLL) vs normal B lymphocytes. HSP70 responds to a wide variety of physiological and environmental stress signals, thus allowing cells to survive to lethal conditions. The primary responsible for the transcription of HSP70 is the heat shock factor 1 (HSF1), being the major regulator of HSP70 expression. In response to stress, HSF1 becomes phosphorylated, forms homotrimers, binds DNA and activates heat shock gene transcription. Considering that the search for molecules involved in the apoptosis resistance and increased survival of B cells from CLL is still ongoing, with this as a background, we were aimed at studying and targeting HSP70 or players related to it (i.e. HSF1) in view of their clinical, prognostic and therapeutical relevance in CLL. METHODS HSP70/HSF1 axis was analysed in freshly isolated leukemic B cells from CLL patients. Expression levels of HSP70, HSF1 and HSF1-Ser326 were assessed by Western blotting analysis with specific antibodies and the obtained expression data have been correlated with clinical features of the patients. HSP70 subcellular localization has been determined by confocal microscopy and cell fractionation. HSP70 expression and localization was also assessed by immunohistochemistry in lymph nodes from CLL patients. Leukemic B cells from 15 CLL therapy-free patients were treated with different concentrations of: i) Zafirlukast, an oral leukotriene receptor antagonist used to prevent asthma symptoms and acting also as HSP70 inhibitor and ii) Fisetin, a dietary flavonoid acting as anti-inflammatory and anti-carcinogen, that inhibits HSF1 activity through the block of its binding to the HSP70 promoter. Apoptosis induction in CLL cells was evaluated by Annexin V/Propidium Iodide flow cytometry test and by the presence of cleaved PARP observed in Western blotting. RESULTS We found that HSP70 and HSF1 proteins were overexpressed in leukemic vs normal B cells and correlated to poor prognosis. In particular, IGHV unmutated or ZAP70 positive patients presented higher levels of HSP70 and HSF1 with respect to patients with a favorable prognosis. Moreover, the two proteins presented a positive correlation (p

Description: INTRODUCTION The Heat Shock Protein of 70kDa (HSP70) and its transcription factor, the Heat Shock Factor 1 (HSF1), are two cytoprotective molecules that we found overexpressed and correlated to poor prognosis in Chronic Lymphocytic Leukemia (CLL) B cells. We focused on RAS-signalling pathways, which involve proteins known to regulate HSF1 activity, such as RAS/RAF/MEK/ERK and RAS/PI3K/AKT. Taking advantage from a previous RPPA analysis (Frezzato et al., 2016), we already developed a model according to which patients with high HSP70 protein levels are characterized by the activation of RAS/PI3K/AKT pathway, while patients with low levels of HSP70 have the RAS/RAF/MEK/ERK pathway activated. The dissection of these aberrant pathways in CLL would help in providing new information on the pathobiology of CLL B cells, in order to define innovative prognostic factors and/or new therapeutic targets. METHODS Freshly isolated leukemic B cells from 20 therapy-free CLL patients were cultured in RPMI 1640 supplemented with antibiotics and 2% FBS and treated with: 10, 20 and 30µM Pterostilbene, a natural analogue of Resveratrol, which upregulates ERK and downmodulates AKT with the final effect of inhibiting HSF1 activity. Apoptosis was evaluated after 24 hours by Annexin V/Propidium iodide flow cytometry test and by the presence of cleaved PARP in Western blotting (WB). HSP70 and HSF1 expression levels were evaluated by WB analysis in leukemic B cells after in vitro and in vivo inhibition with anti-PI3K. RESULTS We previously found that molecules acting as Resveratrol on RAS signaling pathways (inhibiting HSF1 by upregulating ERK and downmodulating AKT), induce apoptosis of CLL B cells in a dose-dependent manner. Particularly, we already observed apoptosis after treatment with Triacetyl Resveratrol and Honokiol. We recently extended our preliminary data on Pterostilbene to a large cohort of patients, obtaining the following results starting from 10µM to 20µM and 30µM: 63.50 ± 14.47%, 48.50 ± 21.59% and 24.88 ± 22.03% of living cells vs untreated cells, 74.63 ± 11.77% respectively, with p value p

Description: Abstract 4571 Background. The accumulation of CD19+/CD5+/CD23+ B cells with a prolonged lifespan in peripheral blood, secondary lymphoid organs and bone marrow (BM) is a peculiar feature of B-cell chronic lymphocytic leukemia (B-CLL). Since CLL cells removed from the in vivo microenvironment and in vitro cultured rapidly undergo spontaneous apoptosis, bidirectional interactions between malignant and by-stander cells may lead to an abnormal microenvironment that confers growth advantages to neoplastic clone. Mesenchymal Stromal Cells (MSCs) are the dominant marrow stromal population in indolent subtype of CLL/small lymphocytic leukemia (SLL) and follicular lymphoma (FL), rather than other aggressive B-cell lymphomas, and are involved in B-CLL cell survival. Despite the phenotypic and cytologic homogeneity, CLL is characterized by extremely variable clinical courses, suggesting that malignant B-cells hold variable degrees of dependency on pro-survival signals coming from the microenvironment. The aim of this study was to assess the role of MSCs in CLL B-cell localization and survival, defining the degree of dependency of leukemic B-cells from external pro-survival signals, with the ultimate goal of identifying patients that mostly benefit microenvironment-targeted therapies. Methods. MSCs isolated from the BM of 47 B-CLL patients were expanded ex vivo and characterized through flow cytometry analysis and differentiation cultures. Fresh isolated CLL peripheral blood mononuclear cells were co-cultured with CLL-MSCs or stromal cells and apoptosis were measured by Annexin V test and western blotting analysis (PARP-1 detection). Chemotactic assays were performed. Results. The survival of neoplastic cells ranged from 13.3% (±13.2) in leukemic cells cultured in medium alone to 58.5% (±17.2) when leukemic cells were cultured in presence of CLL-MSCs (p

Description: Abstract 4561 Background. Cortactin is an ubiquitous actin-binding protein, encoded by EMS1 gene and localized in chromosome 11q13 region. This protein is expressed in nearly all mammalian tissues and mostly localizes to dynamic actin structures. As a consequence, cortactin is involved in regulating several actin-dependent processes, including lamellipodium protrusion, trafficking of the key invadopodia proteases and intracellular transducer downstream to kinase-mediated cell signalling upon phosphorylation by Src and Syk tyrosine kinase families. Cortactin is over-expressed in several tumors, such as esophageal squamous cell carcinoma, head and neck squamous cell carcinoma and gastric carcinoma, and so tied to tumor aggressiveness by the promotion of cell invasion and metastasis. We previously showed that cortactin is over-expressed also in neoplastic B cells of patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL). In the present study we investigated the regulation of cortactin activity by the Src kinase Lyn in neoplastic B-CLL cells and the biological implications of cortactin overexpression in this leukemia. Methods. Twenty patients and ten normal controls were enrolled. Informed consent was obtained from all patients according to the Declaration of Helsinki. CD19+ lymphocytes were purified from peripheral blood by negative selection using the RosetteSep cells isolation kit (StemCell Technologies). Cortactin localization was performed by Confocal Microscope analysis at basal condition, in presence of the chemotactic stimulus CXCL12 and Src kinase inhibitor PP2. Lyn kinase and cortactin phosphorylation levels were evaluated by western blotting analysis. Migration assay was performed using 3 μm pore filters (Transwell Permeable Supports) in the presence of CXCL12, Src kinase inhibitor PP2 and after cortactin silencing. Results. We found that cortactin localization is regulated by Lyn kinase through its phosphorylation. In CLL cells, cortactin was over-phosphorylated and it did not co-localize with actin, as compared to normal cells. PP2-inhibition of Lyn decreased cortactin phosphorylation and triggered its co-localization with actin. Cortactin over-expression proved to be associated to the increased B-CLL spreading through Lyn activity. In fact, not only cortactin over-expression correlated with leukemic cell increased response to CXCL12 (r = 0.9), but also the inhibition of cortactin activity, through PP2 inhibitor or cortactin silencing, drastically reduced neoplastic cell migration after chemotactic triggering. Conclusions. These results suggest that cortactin is involved in aggressiveness and spreading of B-CLL cells and that Lyn-cortactin axis could represent an alternative target for the development of new therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

Description: INTRODUCTION Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment. Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus kinases)-STAT (Signal Transducers and Activators of Transcription) pathways. We particularly focused on the JAK2/STAT3 axis since Interleukin-6 (IL-6), one of the most abundant cytokines released in the CLL microenvironment, is the key ligand of the receptor triggering this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. METHODS B cells were collected from 12 controls and 46 CLL patients. Purified cells (2x106cells/ml) were cultured, and treated with AG490 (10, 50 and 100μM), AZD1480 (1, 4 and 10μM), Fedratinib (1, 5 and 10μM), and Ruxolitinib (0.313, 2.5 and 10μM) (which are JAK2 inhibitors), and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs. STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test. RESULTS We demonstrated that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. As far as STAT3 phosphorylation at Tyr705, that is an essential step for STAT3 activation, we demonstrated a constitutive phosphorylation in CLL cells by FC and WB analyses, although in some patients STAT3 Tyr705 phosphorylation is barely detected. We also pointed out that the in vitroincubation of leukemic B cells with AG490 and Stattic and Fedratinib, induces a dose-dependent apoptosis of CLL B cells. However, the tested doses of Ruxolitinib and AZD1480 did not seem to CLL B cell viability but only STAT3 phosphorylation. Both AG490 and Stattic were able to bypass the microenvironmental protection when neoplastic B cells were co-cultured with MSCs. STAT3 Tyr705 localization was analyzed in normal and leukemic B cells by a subcellular protein fractionation. We separated nuclei from cytosol, detecting STAT3 Tyr705 both in the cytosolic and in the nuclear fractions of CLL B cells. We showed that AG490 and Fedratinib treatment on CLL cells can mediate other effects: i) SHP-1 activity is turned on by JAK2 inhibition, decreasing its phosphorylation at Ser591; ii) AG490 administration inactivates protein Lyn, reducing the phosphorylation in its active site at Tyr396. Lyn, a Tyr-kinase, and SH2-domain containing Tyrosine Phosphatase (SHP-1), a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells. To confirm the link between JAK2 inhibition by AG490 and Lyn dephosphorylation, we added sodium orthovanadate (Na3VO4, 100 μM), a phosphatase inhibitor, to cell culture to restore Lyn activation (resulting from the inactivation of SHP-1 phosphatase); as a result, Lyn Tyr396 phosphorylation was restored. On the contrary, the treatment of CLL cells with Stattic did not induce any change in SHP-1 status with respect to untreated cells since Stattic is effective on STAT3, that is a downstream protein with respect to JAK2. Since Stattic did not affect SHP-1 activation, it does not impact on Lyn activation/phosphorylation. CONCLUSIONS The ability of AG490 and Stattic to induce apoptosis in leukemic B cells bypassing the pro-survival stimuli provided by the tumor microenvironment and the Fedratinib effectiveness at low doses, represents a starting point for the development of new therapeutic strategies in CLL. This study also provides new insights for the investigation of the pathogenesis of CLL focusing the attention on the cross-talk between JAK/STAT and BCR/Lyn axes. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND Chronic Lymphocytic Leukemia (CLL) is one of the most common hematological malignancies in Western countries. The disease is characterized by heterogeneous clinical course and outcome. During the last 15 years several clinical, biological and molecular prognostic factors have been identified, validated and some of them are currently used in patients' and treatment management. To improve the predictive accuracy of these markers, they have been combined into prognostic indexes (W. Wierda, JCO 2011, D. Rossi, Blood 2012, J. Bahlo, Haematologica 2015). Werecently proposed the Integrated CLL Scoring System (ICSS) based on cytogenetic abnormalities by FISH, IGHV mutational status and CD38 expression from 212 patients (A. Visentin et al, Clin Lymph Myeloma & Leuk 2015). The aim of this study was to validate the prognostic power of our index into a larger series of 420 CLL patients. METHODS 420 CLL patients referred to the Hematology Unit of Padua University Hospital from 1989 to 2015 were recruited in this study. According to ICSS, patients were classified as: low-risk, those patients with 13q deletion or normal FISH, IGVH mutated and CD3830%; intermediate-risk, all remaining patients. Treatment free survival (TFS) was calculated as time from diagnosis to treatment (event), death or last known follow-up (censored). Overall survival (OS) was calculated from the date of diagnosis to death for any cause (event) or last known follow-up (censored). TFS and OS were compared with log-rank test and plotted using Kaplan-Meier method. The predictive accuracy of ICSS was evaluated by the Harrel's concordance index (c-index); a value 〉0.5 implies a good predictive ability. RESULTS The median age of our cohort was 62 years; 64% were male and 85% were Binet stage A at diagnosis. Cytogenetic analysis by FISH showed that 41 patients harbored 17p deletion, 50 11q deletion, 236 13q deletion, 44 trisomy 12 and 49 had normal FISH. 236 (56%) patients had IGHV gene homology 〉98% (i.e. mutated IGHV) and 103 (25%) expressed more then 30% of CD38. According to ICSS 202 (48%) subjects were classified as low-risk, 83 (20%) intermediate-risk and 135 (32%) high-risk. After a median follow-up of 81 months, the median TFS for ICSS classes of risk were 211, 70 and 27 months (log-rank test, p