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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., (© 2017 John Wiley & Sons Ltd.)

STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features. Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8±) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France). Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8± T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia., Competing Interests: CONFLICTS OF INTEREST The authors declare no competing financial interests.

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., (© Society for Leukocyte Biology.)

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.

Introduction: Several prognostic factors have been identified to predict the outcome of patients with chronic lymphocytic leukemia (CLL), but only a few studies analyzed more markers together., Patients and Methods: Taking advantage of a population of 608 patients, we identified the strongest prognostic markers of survival and, subsequently, in a cohort of 212 patients we integrated data of cytogenetic lesions, IGHV mutational status, and CD38 expression in a new and easy scoring system we called the integrated CLL scoring system (ICSS). ICSS defines 3 groups of risk: (1) low risk (patients with 13q(-) or normal fluorescence in-situ hybridization analysis results, mutated IGHV, and CD38) (2) high risk (all 11q(-) or 17p(-) patients and/or all unmutated IGHV and CD38(+) patients); and (3) intermediate risk (all remaining patients)., Results: Using only these 3 already available prognostic factors, we were able to properly redefine patients and better predict the clinical course of the disease., Conclusion: ICSS could become a useful tool for CLL patients' management., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Functional abnormalities of chronic lymphocytic leukaemia (CLL) cells may be related to the microtubular network of cell cytoskeleton; specifically tubulin involvement in cells after B-cell receptor engagement. As microtubule inhibitors could represent a therapeutic strategy for CLL, this study investigated the capability of nocodazole, a synthetic depolymerizing agent, to kill CLL leukaemic cells. We demonstrated that nocodazole was highly specific for the in vitro induction of apoptosis in leukaemic cells from 90 CLL patients, without affecting the viability of T-cells and/or mesenchymal stromal cells (MSCs) recovered from the same patients. Nocodazole was observed to overcome the pro-survival signals provided by MSCs. Competing with ATP for the nucleotide-binding site, nocodazole has been observed to turn off the high basal tyrosine phosphorylation of leukaemic cells mediated by the Src-kinase Lyn. Considering that most anti-microtubule drugs have limited clinical use because of their strong toxic effects, the high selectivity of nocodazole for leukaemic cells in CLL and its capability to bypass microenvironmental pro-survival stimuli, suggests the use of this inhibitor for designing new therapeutic strategies in CLL treatment., (© 2014 John Wiley & Sons Ltd.)

Human natural killer (NK) cells are functionally regulated by killer cell immunoglobulin-like receptors (KIRs) and their interactions with HLA class I molecules. As KIR expression in a given NK cell is stochastically established, KIR repertoire perturbations reflect a dominance of discrete NK-cell subsets as the consequence of adaptation of the NK-cell compartment to exogenous agents, more often represented by virus infection. Although inhibitory interactions between KIR and their cognate HLA class I ligands abrogate effector responses of NK cells, they are also required for the functional education of NK cell. The biology and molecular specificities of the activating KIRs are less well defined, and most interactions with presumed HLA class I ligands are weak. Interestingly, epidemiologic studies link activating KIR genes to resistance against numerous virus infections. Chronic lymphoproliferative disorder of NK cells (CLPD-NK) is an indolent NK cell disease characterized by a persistent increase of circulating NK cells (usually exceeding 500 NK cells/mm(3)). The mechanism through which NK cells are induced to proliferate during CLPD-NK pathogenesis is still a matter of debate. Accumulating data suggest that exogenous agents, in particular viruses, might play a role. The etiology of CLPD-NK, however, is largely unknown. This is likely due to the fact that not a single, specific agent is responsible for the NK cells proliferation, which perhaps represents the expression of an abnormal processing of different foreign antigens, sharing a chronic inflammatory background. Interestingly, proliferating NK cells are typically characterized by expression of a restricted pattern of KIR, which have been demonstrated to be mostly represented by the activating form. This finding indicates that these receptors may be directly involved in the priming of NK cells proliferation.

Lyn, a member of the group of tyrosine kinases named the Src family kinases (SFKs), is overexpressed, associated with an aberrant multiprotein complex and constitutively active in B-cell chronic lymphocytic leukemia (B-CLL) cells, resulting in a high level of tyrosine phosphorylation and contributing to their resistance to apoptosis. By using biochemical and bioinformatics tools, we identified procaspase-8 (procasp8), the caspase-8 zymogen, as a cytosolic target for Lyn in B-CLL cells, the phosphorylation of which at Tyr380 promotes the formation of an inactive procasp8 homodimer. This complex remains segregated in the cytosol and appears to be crucial in mediating the antiapoptotic function of Lyn in this disease. The significance of the Lyn-procasp8 axis in impairing apoptosis in B-CLL cells was further confirmed by pharmacological and genetic inhibition of procasp8, which drastically reduced the apoptosis induced by the SFK inhibitors PP2 and dasatinib. Our data highlight that Lyn's dysregulated expression, activity, and localization in B-CLLs support resistance to cell demise by inhibiting an early player of apoptotic signaling, and potentially broaden the perspectives of developing new strategies for the treatment of this disease.

Mature Large Granular lymphocytes (LGL) disorders include a spectrum of conditions, ranging from polyclonal to clonal indolent and/or overt leukemic LGL proliferations. Most cases are represented by clonal expansions of TCRα/β+ LGL displaying a CD8+ phenotype with expression of cytotoxic T-cell antigens (CD57, CD16, TIA-1, perforin and granzyme B). Proliferations of CD3-CD16+ NK cells with a restricted patter of NK receptors are less common, usually comprising 15% of the cases. Main features are cytopenias, splenomegaly and autoimmune phenomena. Morphology, immunophenotyping and molecular analyses are crucial to establish a correct diagnosis of disease. According to the 2008 WHO classification, two separate entities account for the majority of cases, T-LGL leukemia and Chronic Lymphoproliferative Disease of NK cell (this latter still provisional). Although these disorders are characterized by the expansion of different cells types i.e. T and NK cells, with specific genetic features and abnormalities, compelling evidence supports the hypothesis that a common pathogenic mechanism would be involved in both disorders. As a matter of fact, a foreign antigen driven clonal selection is considered the initial step in the mechanism ultimately leading to generation of both conditions. In this chapter we will discuss recent advances on the pathogenesis of chronic T and NK disorders of granular lymphocytes, challenging the current WHO classification on the opportunity to separate T and NK disorders, which are likely to represent two sides of the same coin.

The JAK/STAT pathway is altered in T-cell large granular lymphocytic leukemia. In all patients, leukemic LGLs display upregulation of phosphorylated STAT3 (P-STAT3) that activates expression of many antiapoptotic genes. To investigate the mechanisms maintaining STAT3 aberrantly phosphorylated using transcriptional protein and functional assays, we analyzed interleukin (IL)-6 and suppressor of cytokine signaling-3 (SOCS3), 2 key factors of the JAK/STAT pathway that induce and inhibit STAT3 activation, respectively. We showed that IL-6 was highly expressed and released by the patients' peripheral blood LGL-depleted population, accounting for a trans-signaling process. By neutralizing IL-6 or its specific receptor with specific antibodies, a significant reduction of P-STAT3 levels and, consequently, LGL survival was demonstrated. In addition, we found that SOCS3 was down-modulated in LGL and unresponsive to IL-6 stimulation. By treating neoplastic LGLs with a demethylating agent, IL-6-mediated SOCS3 expression was restored with consequent P-STAT3 and myeloid cell leukemia-1 down-modulation. Methylation in the SOCS3 promoter was not detectable, suggesting that an epigenetic inhibition mechanism occurs at a different site. Our data indicate that loss of the inhibitor SOCS3 cooperates with IL-6 to maintain JAK/STAT pathway activation, thus contributing to leukemic LGL survival, and suggest a role of demethylating agents in the treatment of this disorder.

Although intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cell-derived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival of CLL B cells by prolonging their residency in the prosurvival niche of peripheral lymphoid organs.

In B-Chronic Lymphocytic Leukemia (B-CLL) kinase Lyn is overexpressed, active, abnormally distributed, and part of a cytosolic complex involving hematopoietic lineage cell-specific protein 1 (HS1). These aberrant properties of Lyn could partially explain leukemic cells' defective apoptosis, directly or through its substrates, for example, HS1 that has been associated to apoptosis in different cell types. To verify the hypothesis of HS1 involvement in Lyn-mediated leukemic cell survival, we investigated HS1 protein in 71 untreated B-CLL patients and 26 healthy controls. We found HS1 overexpressed in leukemic as compared to normal B lymphocytes (1.38±0.54 vs 0.86±0.29, p<0.01), and when HS1 levels were correlated to clinical parameters we found a higher expression of HS1 in poor-prognosis patients. Moreover, HS1 levels significantly decreased in ex vivo leukemic cells of patients responding to a fludarabine-containing regimen. We also observed that HS1 is partially localized in the nucleus of neoplastic B cells. All these data add new information on HS1 study, hypothesizing a pivotal role of HS1 in Lyn-mediated modulation of leukemic cells' survival and focusing, one more time, the attention on the BCR-Lyn axis as a putative target for new therapeutic strategies in this disorder.

Background and Aims: Sarcoidosis is characterised by a compartmentalisation of CD4(+) T helper 1 (Th1) lymphocytes and activated macrophages in involved organs, including the lung. Recently, Th17 effector CD4(+) T cells have been claimed to be involved in the pathogenesis of granuloma formation. The objective of this study was to investigate the involvement of Th17 cells in the pathogenesis of sarcoidosis., Methods: Peripheral and pulmonary Th17 cells were evaluated by flow cytometry, real-time PCR, immunohistochemistry analyses and functional assays in patients with sarcoidosis in different phases of the disease and in control subjects., Results: Th17 cells were detected both in the peripheral blood (4.72 ± 2.27% of CD4(+) T cells) and in the bronchoalveolar lavage (BAL) (8.81 ± 2.25% of CD4(+) T lymphocytes) of patients with sarcoidosis and T cell alveolitis. Immunohistochemical analysis of lung and lymph node specimens showed that interleukin 17 (IL-17)(+)/CD4(+) T cells infiltrate sarcoid tissues surrounding the central core of the granuloma. IL-17 was expressed by macrophages infiltrating sarcoid tissue and/or forming the granuloma core (7.88 ± 2.40% of alveolar macrophages). Analysis of some lung specimens highlighted the persistence of IL-17(+)/CD4(+) T cells in relapsed patients and their absence in the recovered cases. Migratory assays demonstrated the ability of the Th17 cell to respond to the chemotactic stimulus CCL20-that is, the CCR6 ligand (74.8 ± 8.5 vs 7.6 ± 2.8 migrating BAL lymphocytes/high-powered field, with and without CCL20, respectively)., Conclusions: Th17 cells participate in the alveolitic/granuloma phase and also to the progression towards the fibrotic phase of the disease. The recruitment of this cell subset may be driven by CCL20 chemokine.

Glycogen synthase kinase (GSK)-3 modulates the production of inflammatory cytokines. Because bleomycin (BLM) causes lung injury, which is characterized by an inflammatory response followed by a fibrotic degeneration, we postulated that blocking GSK-3 activity with a specific inhibitor could affect the inflammatory and profibrotic cytokine network generated in the BLM-induced process of pulmonary inflammation and fibrosis. Thus, here we investigated the effects of the GSK-3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) on a BLM-induced lung fibrosis model in mice. SB216763 prevented lung inflammation and the subsequent fibrosis when coadministered with BLM. Bronchoalveolar lavage fluid analysis of mice treated with BLM plus SB216763 revealed a significant reduction in BLM-induced alveolitis. Furthermore, SB216763 treatment was associated with a significantly lower production of inflammatory cytokines by macrophages. BLM-treated mice that received SB216763 developed alveolar epithelial cell damage and pulmonary fibrosis to a significantly lower extent compared with BLM-treated controls. These findings suggest that GSK-3 inhibition has a protective effect on lung fibrosis induced by BLM and candidate GSK-3 as a potential therapeutic target for preventing pulmonary fibrosis.

B-chronic lymphocytic leukemia (B-CLL) is a malignant disorder characterized by the accumulation of the leukemic cells in the G0-G1 phase of the cell cycle and expressing high levels of the anti-apoptotic protein Bcl-2. Since we observed that the treatment of autoimmune complications with Cyclosporine A (CsA) determined in some CLL patients an improvement not only of the autoimmune phenomena, but also of the leukemic process, we evaluated the in vitro cytotoxicity of CsA as compared to Dexamethasone (Dex) on leukemic cells. Leukemic cells obtained from 32 B-CLL patients showed a heterogeneous pattern of spontaneous apoptosis at 24 h interval and this pattern permitted to identify: Group 1 (14/32) with high (>20%) apoptotic rate and Group 2 (18/32) with low cell death. CsA and Dex increased cell death in both groups with a different timing by an apoptotic mechanism that does not involve Bcl-2. Furthermore, in Group 2, CsA-induced apoptosis was significant higher than that observed with Dex both at 4 and 24 h. We suggest that, in B-CLL, CsA has a significant pro-apoptotic activity manifested also in patients with low spontaneous apoptosis. Our observations might be taken into account to consider new therapeutic strategies in B-CLL.

Oncogenes contribute to tumorigenesis by promoting growth and inhibiting apoptosis. Here we examine the function of Sch9, the Saccharomyces cerevisiae homologue of the mammalian Akt and S6 kinase, in DNA damage and genomic instability during aging in nondividing cells. Attenuation of age-dependent increases in base substitutions, small DNA insertions/deletions, and gross chromosomal rearrangements (GCRs) in sch9Delta mutants is associated with increased mitochondrial superoxide dismutase (MnSOD) expression, decreased DNA oxidation, reduced REV1 expression and translesion synthesis, and elevated resistance to oxidative stress-induced mutagenesis. Deletion of REV1, the lack of components of the error-prone Polzeta, or the overexpression of SOD1 or SOD2 is sufficient to reduce age-dependent point mutations in SCH9 overexpressors, but REV1 deficiency causes a major increase in GCRs. These results suggest that the proto-oncogene homologue Sch9 promotes the accumulation of superoxide-dependent DNA damage in nondividing cells, which induces error-prone DNA repair that generates point mutations to avoid GCRs and cell death during the first round of replication.

Lyn, a tyrosine kinase belonging to the Src family, plays a key role as a switch molecule that couples the B-cell receptor to downstream signaling. In B-CLL cells, Lyn is overexpressed, anomalously present in the cytosol, and displays a high constitutive activity, compared with normal B lymphocytes. The aim of this work was to gain insights into the molecular mechanisms underlying these aberrant properties of Lyn, which have already been demonstrated to be related to defective apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Herein, Lyn is described to be in an active conformation as integral component of an aberrant cytosolic 600-kDa multiprotein complex in B-CLL cells, associated with several proteins, such as Hsp90 through its catalytic domain, and HS1 and SHP-1L through its SH3 domain. In particular, Hsp90 appears tightly bound to cytosolic Lyn (CL), thus stabilizing the aberrant complex and converting individual transient interactions into stable ones. We also demonstrate that treatment of B-CLL cells with geldanamycin, an Hsp90 inhibitor already reported to induce cell death, is capable of dissociating the CL complex in the early phases of apoptosis and thus inactivating CL itself. These data identify the CL complex as a potential target for therapy in B-CLL.

Werner and Bloom syndromes are human diseases characterized by premature age-related defects including elevated cancer incidence. Using a novel Saccharomyces cerevisiae model system for aging and cancer, we show that cells lacking the RecQ helicase SGS1 (WRN and BLM homologue) undergo premature age-related changes, including reduced life span under stress and calorie restriction (CR), G1 arrest defects, dedifferentiation, elevated recombination errors, and age-dependent increase in DNA mutations. Lack of SGS1 results in a 110-fold increase in gross chromosomal rearrangement frequency during aging of nondividing cells compared with that generated during the initial population expansion. This underscores the central role of aging in genomic instability. The deletion of SCH9 (homologous to AKT and S6K), but not CR, protects against the age-dependent defects in sgs1Delta by inhibiting error-prone recombination and preventing DNA damage and dedifferentiation. The conserved function of Akt/S6k homologues in lifespan regulation raises the possibility that modulation of the IGF-I-Akt-56K pathway can protect against premature aging syndromes in mammals.

Sir2 is a conserved deacetylase that modulates life span in yeast, worms, and flies and stress response in mammals. In yeast, Sir2 is required for maintaining replicative life span, and increasing Sir2 dosage can delay replicative aging. We address the role of Sir2 in regulating chronological life span in yeast. Lack of Sir2 along with calorie restriction and/or mutations in the yeast AKT homolog, Sch9, or Ras pathways causes a dramatic chronological life-span extension. Inactivation of Sir2 causes uptake and catabolism of ethanol and upregulation of many stress-resistance and sporulation genes. These changes while sufficient to extend chronological life span in wild-type yeast require severe calorie restriction or additional mutations to extend life span of sir2Delta mutants. Our results demonstrate that effects of SIR2 on chronological life span are opposite to replicatve life span and suggest that the relevant activities of Sir2-like deacetylases may also be complex in higher eukaryotes.

Aging is believed to be a nonadaptive process that escapes the force of natural selection. Here, we challenge this dogma by showing that yeast laboratory strains and strains isolated from grapes undergo an age- and pH-dependent death with features of mammalian programmed cell death (apoptosis). After 90-99% of the population dies, a small mutant subpopulation uses the nutrients released by dead cells to grow. This adaptive regrowth is inversely correlated with protection against superoxide toxicity and life span and is associated with elevated age-dependent release of nutrients and increased mutation frequency. Computational simulations confirm that premature aging together with a relatively high mutation frequency can result in a major advantage in adaptation to changing environments. These results suggest that under conditions that model natural environments, yeast organisms undergo an altruistic and premature aging and death program, mediated in part by superoxide. The role of similar pathways in the regulation of longevity in organisms ranging from yeast to mice raises the possibility that mammals may also undergo programmed aging.