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Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1− Tαβ-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1− Tαβ-LGL ranged between 0.36 and 571 cells/μL (3.2–91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/μL (96% TRBC1+ cells). Among the distinct TCRVβ families, the CD28− effector-memory and terminal-effector polyclonal Tαβ cells ranged between 0 and 25 TRBC1+ or TRBC1− cells/μL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1− cells/μL, representing 98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations., This work was supported by the CB16/12/00400 (CIBERONC) and PI20-01346 grants from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (Madrid, Spain) and FONDOS FEDER; the 0639-IDIAL-NET-3-3 grant (INTERREG POCTEP Spain-Portugal) from Fondo Europeo de Desarrollo Regional, and by the EuroFlow Foundation (Leiden, The Netherlands). N.M.-G. was supported by a pre-doctoral grant (Ref. IBPredoc17/00012) from IBSAL (Salamanca, Spain). M.L.,N.V., J.J.M.v.D., A.O. and J.A. are members of the EuroFlow Consortium

Significance Determination of macromolecular crystal structures by molecular replacement (MR) uses a related structure to generate approximate phases for the unknown crystal. MR fails if the search model is too dissimilar to the unknown structure. In an accompanying paper, we described a method that generates many ensembles of possible solutions and refines them against the diffraction data without human intervention, and demonstrated its ability to solve test cases of known structure. Here, we apply this method to crystals of the epithelial cell polarity protein lethal giant larvae, which had resisted structure solution using conventional MR or experimental phasing. The results show that our method of unsupervised protein structure solution can be applied to a very large and difficult phasing problem., In the companion paper by Ufimtsev and Levitt [Ufimtsev IS, Levitt M (2019) Proc Natl Acad Sci USA, 10.1073/pnas.1821512116], we presented a method for unsupervised solution of protein crystal structures and demonstrated its utility by solving several test cases of known structure in the 2.9- to 3.45-Å resolution range. Here we apply this method to solve the crystal structure of a 966-amino acid construct of human lethal giant larvae protein (Lgl2) that resisted years of structure determination efforts, at 3.2-Å resolution. The structure was determined starting with a molecular replacement (MR) model identified by unsupervised refinement of a pool of 50 candidate MR models. This initial model had 2.8-Å RMSD from the solution. The solved structure was validated by comparison with a model subsequently derived from an alternative crystal form diffracting to higher resolution. This model could phase an anomalous difference Fourier map from an Hg derivative, and a single-wavelength anomalous dispersion phased density map made from these sites aligned with the refined structure.

Shosaku Nomura, Tomoki Ito, Atsushi Satake, Kazuyoshi Ishii First Department of Internal Medicine, Kansai Medical University, Hirakata, Osaka, Japan Background: The outcome for chronic myelogenous leukemia (CML) patients presented in the chronic phase has changed dramatically since the introduction of tyrosine kinase inhibitor (TKI) therapy. Notably, an increased incidence of large granular lymphocytes (LGLs), which is related to immunological conditions, appears to be predictive of a favorable outcome for dasatinib therapy. We therefore examined the immunological characteristics of CML patients during dasatinib therapy by determining the plasma concentrations of five different biomarkers. Methods: The plasma levels of biomarkers, specifically interleukin-6, platelet-derived microparticles (PDMPs), soluble vascular cell adhesion molecule 1 (sVCAM-1), transforming growth factor (TGF) β1, and soluble cytotoxic T lymphocyte-associated antigen-4 (sCTLA-4), were measured by ELISA at baseline and after 2 and 6 months of TKI treatment. The incidence of LGLs was estimated by microscopic examination. Results: The levels of PDMPs, sVCAM-1, and TGFβ1 were significantly elevated in patients with CML. Dasatinib treatment was associated with a significant reduction in the levels of these markers and with an increased incidence of LGLs compared with imatinib or nilotinib treatment. In addition, an increased incidence of LGLs was significantly correlated with a decreased sCTLA-4 level during dasatinib therapy. Conclusion: The assessment of the levels of specific biomarkers may be beneficial to understand the immunological conditions of patients with CML during dasatinib treatment. Keywords: CML, TKI, LGL, PDMP, TGFβ1, sCTLA-4

Background The outcome for chronic myelogenous leukemia (CML) patients presented in the chronic phase has changed dramatically since the introduction of tyrosine kinase inhibitor (TKI) therapy. Notably, an increased incidence of large granular lymphocytes (LGLs), which is related to immunological conditions, appears to be predictive of a favorable outcome for dasatinib therapy. We therefore examined the immunological characteristics of CML patients during dasatinib therapy by determining the plasma concentrations of five different biomarkers. Methods The plasma levels of biomarkers, specifically interleukin-6, platelet-derived microparticles (PDMPs), soluble vascular cell adhesion molecule 1 (sVCAM-1), transforming growth factor (TGF) β1, and soluble cytotoxic T lymphocyte-associated antigen-4 (sCTLA-4), were measured by ELISA at baseline and after 2 and 6 months of TKI treatment. The incidence of LGLs was estimated by microscopic examination. Results The levels of PDMPs, sVCAM-1, and TGFβ1 were significantly elevated in patients with CML. Dasatinib treatment was associated with a significant reduction in the levels of these markers and with an increased incidence of LGLs compared with imatinib or nilotinib treatment. In addition, an increased incidence of LGLs was significantly correlated with a decreased sCTLA-4 level during dasatinib therapy. Conclusion The assessment of the levels of specific biomarkers may be beneficial to understand the immunological conditions of patients with CML during dasatinib treatment.

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell-cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apico-basolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment, because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.

Large granular lymphocytes (LGLs) are large lymphocytes with azurophilic granules in their cytoplasm. LGLs are either natural killer (NK) cells or T lymphocytes. Expansions of the LGLs in the peripheral blood are seen in various conditions, including three clonal disorders: T-cell LGL (T-LGL) leukemia, chronic lymphoproliferative disorders of NK cells (CLPD-NK), and aggressive NK-cell leukemia (ANKL). However, the monoclonal and polyclonal expansion of LGLs has been associated with many other conditions. The present article describes these LGL disorders, with special emphasis on the clinical features, pathogenesis, and treatments of the three above-mentioned clonal disorders.

Apical-basal polarity is a common trait that underlies epithelial function. Although the asymmetric distribution of cortical polarity proteins works in a functioning equilibrium, it also retains plasticity to accommodate cell division, during which the basolateral determinant Lgl is released from the cortex. Here, we investigated how Lgl restores its cortical localization to maintain the integrity of dividing epithelia. We show that cytoplasmic Lgl is reloaded to the cortex at mitotic exit in Drosophila epithelia. Lgl cortical localization depends on protein phosphatase 1, which dephosphorylates Lgl on the serines phosphorylated by aPKC and Aurora A kinases through a mechanism that relies on the regulatory subunit Sds22 and a PP1-interacting RVxF motif of Lgl. This mechanism maintains epithelial polarity and is of particular importance at mitotic exit to couple Lgl cortical reloading with the polarization of the apical domain. Hence, PP1-mediated dephosphorylation of Lgl preserves the apicalbasal organization of proliferative epithelia. We thank Daniel St Johnston, François Schweisguth, Guilles Hickson, Jürgen Knoblich, Torcato Martins, Yang Hong, and the Bloomington Drosophila Stock Center for providing plasmids and fly stocks. This work was funded by national funds through Fundação para a Ciência e a Tecnologia (FCT) under project PTDC/BEX-BCM/0432/2014 . This work has also received funding from the project Norte-01-0145-FEDER-000029 , supported by Norte Portugal Regional Operational Program (NORTE 2020). E.M. holds an FCT Investigator position. S.M. and M.G. are supported by FCT PhD grants. M.O. is supported by a fellowship from FCT and the GABBA PhD program from the University of Porto .

Aggressive NK cell leukemia (ANKL) is a rare malignant lymphoproliferative disorder of mature NK cells closely associated with Epstein-Barr virus (EBV) and more common in East Asia than in other areas. Significant variations exist in the morphology of ANKL tumor cells, from typical large granular lymphocyte morphology to highly atypical features with basophilic cytoplasm containing azurophilc granules. The main involved sites are hepatosplenic lesions, bone marrow and peripheral blood, and nasal or skin lesions are infrequent. A fever and liver dysfunction with an often rapidly progressive course are the main clinical symptoms, including hemophagocytic syndrome and disseminated intravascular coagulation. Although the outcome had been dismal for decades, with a median survival of less than three months, the introduction of combined chemotherapy including L-asparaginase and allogeneic hematopoietic cell transplantation has helped achieve a complete response and potential cure for some patients. With the advent of next-generation sequencing technologies, molecular alterations of ANKL have been elucidated, and dysfunctions in several signaling pathways, including the JAK/STAT pathway, have been identified. Novel target approaches to managing these abnormalities might help improve the prognosis of patients with ANKL.

Oncogenic potential of Notch signaling and its cooperation with other factors to affect proliferation are widely established. Notch exhibits a cooperative effect with loss of a cell polarity gene, scribble to induce neoplastic overgrowth. Oncogenic Ras also show cooperative effect with loss of cell polarity genes such as scribble (scrib), lethal giant larvae (lgl) and discs large to induce neoplastic overgrowth and invasion. Our study aims at assessing the cooperation of activated Notch with loss of function of lgl in tumor overgrowth, and the mode of JNK signaling activation in this context. In the present study, we use Drosophila as an in vivo model to show the synergy between activated Notch (N act ) and loss of function of lgl (lgl-IR) in tumor progression. Coexpression of N act and lgl-IR results in massive tumor overgrowth and displays hallmarks of cancer, such as MMP1 upregulation and loss of epithelial integrity. We further show activation of JNK signaling and upregulation of its receptor, Grindelwald in N act /lgl-IR tumor. In contrast to previously described Notch act /scrib−/− tumor, our experiments in N act /lgl-IR tumor showed the presence of dying cells along with tumorous overgrowth.

Feline large granular lymphocyte (LGL) lymphoma is an uncommon subtype of lymphoma characterized by a grave prognosis and scarce response to chemotherapy. There are limited reports on clinico-pathological and prognostic factors. One-hundred and 9 cats with newly diagnosed LGL lymphoma that underwent initial staging (including hematology, serum biochemistry, thoracic radiographs and abdominal ultrasound), and followed-up were retrospectively evaluated. LGL lymphoma was localized within the gastrointestinal tract with or without extra-intestinal involvement in 91.7% of the cases, and at extra-gastrointestinal sites in 8.3%. Symptoms were frequent. Anemia (31.2%) and neutrophilia (26.6%) were commonly observed, and 14 (12.8%) cats had neoplastic circulating cells. Frequent biochemistry abnormalities included elevated ALT (39.4%) and hypoalbuminemia (28.4%). Twenty (54.1%) of 37 cats had elevated serum LDH. Treatment varied among cats, and included surgery (11%), chemotherapy (23%), corticosteroids (38.5%) and no treatment (27.5%). Median time to progression (MTTP) was 5 days, and median survival time (MST) 21 days. MST was significantly shorter in the case of substage b, circulating neoplastic cells, lack of chemotherapy administration, and lack of treatment response. A small subset of cats (7.3%) survived more than 6 months, suggesting that a more favorable clinical course can be found among LGL lymphoma patients.

Despite their emergence as an important class of noncoding RNAs involved in cancer cell transformation, invasion, and migration, the precise role of microRNAs (miRNAs) in tumorigenesis remains elusive. To gain insights into how miRNAs contribute to primary tumor formation, we conducted an RNA sequencing (RNA-Seq) analysis of Drosophila wing disc epithelial tumors induced by knockdown of a neoplastic tumor-suppressor gene (nTSG) lethal giant larvae (lgl), combined with overexpression of an active form of oncogene Ras (RasV12), and identified 51 mature miRNAs that changed significantly in tumorous discs. Followed by in vivo tumor enhancer and suppressor screens in sensitized genetic backgrounds, we identified 10 tumor-enhancing (TE) miRNAs and 11 tumor-suppressing (TS) miRNAs that contributed to the nTSG defect-induced tumorigenesis. Among these, four TE and three TS miRNAs have human homologs. From this study, we also identified 29 miRNAs that individually had no obvious role in enhancing or alleviating tumorigenesis despite their changed expression levels in nTSG tumors. This systematic analysis, which includes both RNA-Seq and in vivo functional studies, helps to categorize miRNAs into different groups based on their expression profile and functional relevance in epithelial tumorigenesis, whereas the evolutionarily conserved TE and TS miRNAs provide potential therapeutic targets for epithelial tumor treatment.

// Alexander Gont 1, 2 , Jennifer E.L. Hanson 1 , Sylvie J. Lavictoire 1 , Manijeh Daneshmand 1, 3 , Garth Nicholas 1, 5 , John Woulfe 1, 2, 3 , Amin Kassam 4, 6 , Vasco F. Da Silva 4 , Ian A.J. Lorimer 1, 2, 5 1 Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, K1H 8L6, Canada 2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada 3 Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada 4 Department of Surgery, University of Ottawa, Ottawa, Ontario, Canada 5 Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada 6 Aurora St. Luke’s Medical Center, Aurora Health Care, Milwaukee, WI 53215, USA Correspondence to: Ian A. J. Lorimer, e-mail: ilorimer@ohri.ca Keywords: Glioblastoma, glioma, Lgl, Lgl1, PTEN, invasion Received: July 04, 2014 Accepted: October 08, 2014 Published: October 15, 2014 ABSTRACT lethal giant larvae ( lgl ) was first identified as a tumor suppressor in Drosophila , where its loss repressed the differentiation and promoted the invasion of neuroblasts, the Drosophila equivalent of the neural stem cell. Recently we have shown that a human homolog of Lgl, Lgl1 (LLGL1), is constitutively phosphorylated and inactivated in glioblastoma cells; this occurs as a downstream consequence of PTEN loss, one of the most frequent genetic events in glioblastoma. Here we have investigated the consequences of this loss of functional Lgl1 in glioblastoma in vivo . We used a doxycycline-inducible system to express a non-phosphorylatable, constitutively active version of Lgl1 (Lgl3SA) in either a glioblastoma cell line or primary glioblastoma cells isolated under neural stem cell culture conditions from patients. In both types of cells, expression of Lgl3SA, but not wild type Lgl1, inhibited cell motility in vitro . Induction of Lgl3SA in intracerebral xenografts markedly reduced the in vivo invasion of primary glioblastoma cells. Lgl3SA expression also induced the differentiation of glioblastoma cells in vitro and in vivo along the neuronal lineage. Thus the central features of Lgl function as a tumor suppressor in Drosophila are conserved in human glioblastoma.

In both Drosophila and mammalian systems, the Hippo (Hpo) signalling pathway controls tissue growth by inhibiting cell proliferation and promoting apoptosis. The core pathway consists of a protein kinase Hpo (MST1/2 in mammals) that is regulated by a number of upstream inputs including Drosophila Ras Association Factor, dRASSF. We have previously shown in the developing Drosophila eye epithelium that loss of the apico-basal cell polarity regulator lethal-(2)-giant-larvae (lgl), and the concomitant increase in aPKC activity, results in ectopic proliferation and suppression of developmental cell death by blocking Hpo pathway signalling. Here, we further explore how Lgl/aPKC interacts with the Hpo pathway. Deregulation of the Hpo pathway by Lgl depletion is associated with the mislocalization of Hpo and dRASSF. We demonstrate that Lgl/aPKC regulate the Hpo pathway independently of upstream inputs from Fat/Dachs and the Kibra/Expanded/Merlin complex. We show depletion of Lgl also results in accumulation and mislocalization of components of the dSTRIPAK complex, a major phosphatase complex that directly binds to dRASSF and represses Hpo activity. However, depleting dSTRIPAK components, or removal of dRASSF did not rescue the lgl-/-or aPKC overexpression phenotypes. Thus, Lgl/aPKC regulate Hpo activity by a novel mechanism, independently of dRASSF and dSTRIPAK. Surprisingly, removal of dRASSF in tissue with increased aPKC activity results in mild tissue overgrowth, indicating that in this context dRASSF acts as a tumor suppressor. This effect was independent of the Hpo and Ras Mitogen Activated Protein Kinase (MAPK) pathways, suggesting that dRASSF regulates a novel pathway to control tissue growth. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Glioblastoma is the most aggressive and invasive adult brain cancer. In glioblastoma, the loss of the tumour suppressor PTEN is the most common genetic alteration resulting in aberrant activation of the PI3-kinase pathway. In Drosophila, the loss of tumour suppressor Lgl results in massive overgrowth of brain tissue that is highly invasive when transplanted into wildtype hosts. Subsequent study of Lgl protein function revealed that it is important for maintenance of cell polarity and neuroblast differentiation through asymmetric cell divisions. It is unclear if inactivation of Lgl occurs in human brain cancers and what role it plays in glioblastoma malignancy. Firstly, this study demonstrated that the loss of PTEN leads to inactivation of Lgl1 via phosphorylation by atypical protein kinase C iota (PKCι). In primary glioblastoma cultures, preventing Lgl1 inactivation by either PTEN expression, PKCι knockdown or expression of non-phosphorylatable Lgl (Lgl3SA) promoted differentiation. In a follow-up study, the effect of active Lgl1 in glioblastoma invasion was investigated. Lgl3SA expression inhibited invasion in vitro through decreased motility. In an orthotopic xenograft mouse model using primary glioblastoma cells, Lgl3SA expression promoted differentiation and decreased invasion. Therefore, PTEN loss, acting via PKCι and Lgl1, has a key role in maintaining glioblastoma in an undifferentiated, highly invasive state similar to what is observed following Lgl loss in Drosophila. PREX1 was investigated as a potential link between PTEN loss and activation of PKCι. PREX1, a Rac activator, is synergistically activated by the PI3-kinase product PIP3 and G protein-coupled receptor (GPCR) βγ subunits. PREX1 expression was detected in primary glioblastoma cell cultures as well as the majority of patient tumour samples. Both PI3-kinase and GPCR βγ subunit activity is required for PREX1 to promote invasion in glioblastoma. In primary glioblastoma cells, Rac1 preferentially associated with Par6a leading to activation of PKCι. Knockdown of PREX1 decreased activation of PKCι. Thus, PREX1 stimulates PKCι activity in glioblastoma likely by modulating the Rac1/Par6a/PKCι complex. The PI3-kinase pathway is activated by mutation in most glioblastomas and these results show this requires a context of GPCR signalling to promote invasion.

Loss of function of the neoplastic tumors suppressors, lgl, scrib and dlg or overexpression of the apical polarity components, Crumbs and atypical protein kinase C (aPKC), are associated with polarity loss and tissue overgrowth, however, the mechanism behind these effects is poorly understood. In our recent study, we showed that Lgl, aPKC and Crumbs mediate their effects on proliferation and survival via the Salvador/Warts/Hippo (SWH) tumor suppressor pathway. Loss of lgl can lead to substantial overgrowth, however the lgl mutant phenotype can be quite variable and the amount of overgrowth of the mutant tissue, its survival and ultimate fate is strongly determined by context and competition. In this extra-view we present a more detailed description of the lgl mutant phenotype and highlight the phenotypic differences between lgl and SWH pathway mutant phenotypes. In addition, we explore the role for the Jun kinase (JNK) pathway in the development of the lgl mutant phenotype.

Mutant larvae for the Drosophila gene lethal giant larva (lgl) develop neoplastic tumors in imaginal discs. However, lgl mutant clones do not form tumors when surrounded by wild-type tissue, suggesting the existence of a tumor-suppressing mechanism. We have investigated the tumorigenic potential of lgl mutant cells by generating wing compartments that are entirely mutant for lgl and also inducing clones of various genetic combinations of lgl− cells. We find that lgl− compartments can grow indefinitely but lgl− clones are eliminated by cell competition. lgl mutant cells may form tumors if they acquire constitutive activity of the Ras pathway (lgl− UAS-rasV12), which confers proliferation advantage through inhibition of the Hippo pathway. Yet, the majority of lgl− UAS-rasV12 clones are eliminated in spite of their high proliferation rate. The formation of a tumor requires in addition the formation of a microenvironment that allows mutant cells to evade cell competition., This work has been supported by grants from the Ministerio de Ciencia e Innovación (Consolider and BFU-02427), from the Comunidad de Madrid, and from an institutional grant from the Fundación Ramón Areces.

Lethal giant larvae (Lgl) plays a critical role in establishment of cell polarity in epithelial cells. While Frizzled/Dsh signaling has been implicated in the regulation of the localization and activity of Lgl, it remains unclear whether specific Wnt ligands are involved. Here we show that Wnt5a triggers the release of Lgl from the cell cortex into the cytoplasm with the concomitant decrease in Lgl stability. The observed changes in Lgl localization were independent of atypical PKC (aPKC), which is known to influence Lgl distribution. In ectodermal cells, both Wnt5a and Lgl triggered morphological and molecular changes characteristic of apical constriction, whereas depletion of their functions prevented endogenous and ectopic bottle cell formation. Furthermore, Lgl RNA partially rescued bottle cell formation in embryos injected with a dominant negative Wnt5a construct. These results suggest a molecular link between Wnt5a and Lgl that is essential for apical constriction during vertebrate gastrulation.

The evolutionarily conserved neoplastic tumor suppressor protein, Lethal (2) giant larvae (Lgl), plays roles in cell polarity and tissue growth via regulation of the Hippo pathway. In our recent study, we showed that in the developing Drosophila eye epithelium, depletion of Lgl leads to increased ligand-dependent Notch signaling. lgl mutant tissue also exhibits an accumulation of early endosomes, recycling endosomes, early-multivesicular body markers and acidic vesicles. We showed that elevated Notch signaling in lgl(-) tissue can be rescued by feeding larvae the vesicle de-acidifying drug chloroquine, revealing that Lgl attenuates Notch signaling by limiting vesicle acidification. Strikingly, chloroquine also rescued the lgl(-) overgrowth phenotype, suggesting that the Hippo pathway defects were also rescued. In this extraview, we provide additional data on the regulation of Notch signaling and endocytosis by Lgl, and discuss possible mechanisms by which Lgl depletion contributes to signaling pathway defects and tumorigenesis.

Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes.

Large granular lymphocyte leukemia (LGLL) is a rare and heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal large granular lymphocytes (LGLs) with cytotoxic activity. Two subtypes of LGL proliferations are distinguished: the most common type (~85% of cases), T-LGL Leukemia (T-LGLL), is sustained by T-cells and the rarer type (~15% of cases), Chronic Lymphoproliferative Disorder of Natural Killer cells (CLPD-NK), sustained by NK-cells. This PhD will focus on the T-LGL Leukemia. The etiology of T-LGLL still remains matter of debate. The main hypothesis suggests an antigenic stimulation as initial step activating LGLs, which undergo a clonal expansion then maintained by the abnormal release of cytokines (mainly IL-6 and IL-15) and by an impairment of the apoptotic machinery due to the activation of several survival pathways. The project developed in this PhD was aimed to better define this etiopathogenetic hypothesis and to find new therapeutic targets. First of all, we have taken into account the JAK/STAT pathway, reported to be deregulated in T-LGLL. We focused on STAT3, constitutively activated in LGLs, and its specific inhibitor SOCS3, that we found to be down-expressed and unresponsive to IL-6 triggering. The pathophysiology of this mechanism is unclear, since we found no methylation in SOCS3 promoter region; anyway we still hypothesize an epigenetic mechanism, since the use of a demethylating agent restored SOCS3 responsiveness. We then focused our attention on the main activator of STAT3/SOCS3 axis, IL-6, and we observed that it enhances LGL survival through STAT3 phosphorylation. We also provided evidence that IL-6 is mainly expressed by LGLs-depleted PBMC population of T-LGLL patients characterized by low levels of circulating LGLs (55% ), accounting for 40% of all LGL cohort.

Glioblastoma multiforme is an aggressive and incurable type of brain tumor. A subset of undifferentiated glioblastoma cells, known as glioblastoma tumor initiating cells (GTICs), has an essential role in the malignancy of this disease and also appears to mediate resistance to radiation therapy and chemotherapy. GTICs retain the ability to differentiate into cells with reduced malignant potential, but the signaling pathways controlling differentiation are not fully understood at this time. PTEN loss is a very common in glioblastoma multiforme and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Increased signalling through this pathway leads to activation of multiple protein kinases, including atypical protein kinase C. In Drosophila, active atypical protein kinase C has been shown to promote the self-renewal of neuroblasts, inhibiting their differentiation along a neuronal lineage. This effect is mediated by atypical protein kinase c-mediated phosphorylation and inactivation of Lgl, a protein that was first characterized as a tumour suppressor in Drosophila. The effects of the atypical protein kinase C/Lgl pathway on the differentiation status of GTICs, and its potential link to PTEN loss, have not been assessed previously. Here we show that PTEN loss leads to the phosphorylation and inactivation of Lgl by atypical protein kinase C in glioblastoma cells. Re-expression of PTEN in GTICs promoted their differentiation along a neuronal lineage. This effect was also seen when atypical protein kinase C was knocked down using RNA interference, and when a non-phosphorylatable, constitutively active form of Lgl was expressed in GTICs. Thus PTEN loss, acting via atypical protein kinase C activation and Lgl inactivation, helps to maintain GTICs in an undifferentiated state.

Loss of cell polarity is a prominent feature of epithelial cancers. Several tumour-suppressor genes are indeed involved in establishing and maintaining a correct apical-basal polarity suggesting that a link exists between disruption of epithelial polarity and the control of cell proliferation. Nevertheless, the molecular basis of this link is only beginning to be unveiled. In Drosophila, the tumour suppressor gene lethal giant larvae (lgl) is widely used as a genetic tool in cancer modelling: its loss of function causes neoplastic growth of the imaginal tissues, larval epithelial organs from which adult structures originate. These mutant epithelia are characterised by loss of cell polarity and tissue architecture as well as hyperproliferation. We observed that in a clonal context, the ability of lgl mutant cells to express their neoplastic potential correlates with the levels of the oncoprotein Myc, a master regulator of cell growth and proliferation. Malignant, polarity-deficient mutant cells upregulate Myc and are able to overcome the tumour-suppressive defences imposed by the surrounding wild-type tissue. How does the loss of lgl function induce an increase in Myc levels? The answer to this question came from the finding that Lgl is an upstream regulator of the Hippo pathway, a highly conserved signalling network that controls proliferation of epithelial cells and organ size. The core of this pathway responds to several upstream regulators and converges on the inhibition of a transcriptional co-factor, Yorkie, which, as we and others have shown, is a direct regulator of the myc promoter. In this review we discuss the key findings that contributed to the identification of this regulatory network that links cell polarity to cell proliferation control.

The CD94/NKG2 complex is expressed on T and NK lymphocytes. CD94 molecules covalently associate to activating or inhibitory NKG2 molecules, and their expression finely tunes cell responses. Human γδ T cells express several NKRs. Expression of these receptors is confined to the cytolytic Vδ2 subset, which coexpresses the FcγRIII CD16 and CD45RA and has been defined as Vγ9Vδ2 TEMRA cells. We show that the CD94/NKG2C complex, associated with KARAP/DAP12, is fully functional in γδ T cells, as determined by measuring IFN-γ production, T cell proliferation, and cytolytic activity by γδ lymphocytes. In contrast, NKG2A expression was found on all γδ T cell memory subsets, suggesting a crucial role of the inhibitory signal provided by this receptor on γδ T cell responses. Moreover, we found Vγ9Vδ2 TEMRA, NK, and CD8+ αβ T cells coexpressing NKG2A and NKG2C receptors. Functional experiments showed that the inhibitory signal mediated by the NKG2A receptor prevails when double-positive cells are activated. Finally, NKG2A expression on γδ LDGL correlates with asymptomatic pathology, even in the presence of NKG2C coexpression, whereas in symptomatic patients affected by severe disease, the inhibitory NKG2A receptor is absent, and a variety of activatory NKRs was found. We propose that the silent behavior of γδ cells in LDGL patients is a result of effective inhibitory HLA class I receptors.

Exocytosis is the major mechanism by which new membrane components are delivered to the cell surface. In most, if not all, eukaryotic cells this is also a highly spatially regulated process that is tightly coordinated with the overall polarity of a cell. The Rho/Cdc42 family of GTPases and the lethal giant larvae/Sro7 family are two highly conserved families of proteins which appear to have dual functions both in cell polarity and exocytosis. Analysis of their functions has begun to unravel the coordination between these processes and propose a model for polarized vesicle docking and fusion at the site of asymmetric cell growth.

In homozygous mutants of Drosophila lethal-2-giant larvae (lgl), tissues lose apico-basal cell polarity and exhibit ectopic proliferation. Here, we use clonal analysis in the developing eye to investigate the effect of lgl null mutations in the context of surrounding wild-type tissue. lgl− clones in the larval eye disc exhibit ectopic expression of the G1–S regulator, Cyclin E, and ectopic proliferation, but do not lose apico-basal cell polarity. Decreasing the perdurance of Lgl protein in larval eye disc clones, by forcing extra proliferation of lgl− tissue (using a Minute background), leads to a loss in cell polarity and to more extreme ectopic cell proliferation. Later in development at the pupal stage, lgl mutant photoreceptor cells show aberrant apico-basal cell polarity, but this is not associated with ectopic proliferation, presumably because cells are differentiated. Thus in a clonal context, the ectopic proliferation and cell polarity defects of lgl− mutants are separable. Furthermore, lgl− mosaic eye discs have alterations in the normal patterns of apoptosis: in larval discs some lgl− and wild-type cells at the clonal boundary undergo apoptosis and are excluded from the epithelia, but apoptosis is decreased elsewhere in the disc, and in pupal retinas lgl− tissue shows less apoptosis.

We describe a 9-year-old boy with Wiskott-Aldrich syndrome and IgM-rheumatoid factor-positive arthritis who presented expansion of two distinct subsets (one CD8dim and the other CD8-) of large granular lymphocytes. Natural killer activity against the K-562 cell line was absent. An increased percentage of CD5+ B cells was also observed. Since patients with Wiskott-Aldrich syndrome are at risk of developing autoimmune disorders - conditions in which increased CD5+ B cells have been observed - the high percentage of CD5+ B cells together with the presence of IgM-rheumatoid factor and anti-platelet antibodies may represent an early manifestation of an autoimmune process. The possible relationship between CD5+ B cells and large granular lymphocyte expansion is discussed.