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The tumour suppressor, Lethal (2) giant larvae [Lgl; also known as L(2)gl], is an evolutionarily conserved protein that was discovered in the vinegar fly Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity. Lgl links cell polarity and tissue growth through regulation of the Notch and the Hippo signalling pathways. Lgl regulates the Notch pathway by inhibiting V-ATPase activity via Vap33. How Lgl regulates the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Vap33 physically and genetically interacts with the actin cytoskeletal regulators RtGEF (Pix) and Git, which also bind to the Hippo protein (Hpo) and are involved in the activation of the Hippo pathway. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether, our data suggest that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism: (1) through interaction with RtGEF, Git and Arf79F, and (2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth. [ABSTRACT FROM AUTHOR]

Abstract Background Large granular lymphocytic leukemia is a rare lymphocytic neoplasm that can pose a treatment challenge in patients with severe neutropenia in whom conventional therapies fail. We report one of the first cases in which allogeneic stem cell therapy was used as treatment for large granular lymphocytic leukemia. We report and discuss the case of a 42-year-old white Caucasian female who, despite multiple therapies including methotrexate, cyclophosphamide, prednisone, cyclosporine, and pentostatin, continued to show severe neutropenia and recurrent infections. The patient was treated successfully and cured by allogeneic stem cell transplant without any major complications. Conclusions The significant importance of this case report is the introduction of a new treatment algorithm for challenging cases of T-cell large granular lymphocytic leukemia in which standard care fails. We hope that this case report will raise awareness of the potential benefits of allogeneic stem cell transplant in the treatment of aggressive forms of T-cell large granular lymphocytic leukemia.

Background: Large granular lymphocytic leukemia is a rare lymphocytic neoplasm that can pose a treatment challenge in patients with severe neutropenia in whom conventional therapies fail. We report one of the first cases in which allogeneic stem cell therapy was used as treatment for large granular lymphocytic leukemia. We report and discuss the case of a 42-year-old white Caucasian female who, despite multiple therapies including methotrexate, cyclophosphamide, prednisone, cyclosporine, and pentostatin, continued to show severe neutropenia and recurrent infections. The patient was treated successfully and cured by allogeneic stem cell transplant without any major complications.Conclusions: The significant importance of this case report is the introduction of a new treatment algorithm for challenging cases of T-cell large granular lymphocytic leukemia in which standard care fails. We hope that this case report will raise awareness of the potential benefits of allogeneic stem cell transplant in the treatment of aggressive forms of T-cell large granular lymphocytic leukemia. [ABSTRACT FROM AUTHOR]

A complex relationship exists between rheumatic diseases and cancer. This delicate balance between chronic inflammation and malignant cell transformation in hematologic neoplasms has been observed, but is not well defined. Large Granular Lymphocyte (LGL) leukemia is at the intersection of a clonal lymphoproliferative disease, chronic inflammation, and autoimmunity. The association between rheumatoid arthritis (RA) and the spectrum of Felty's Syndrome is well-known. Other rheumatic disorders have been reported including systemic lupus erythematosus (SLE), Sjogren's Syndrome (SS), vasculitis, Behcet's Disease (BD) and systemic sclerosis. The association between T-LGLL and rheumatic disease pathogenesis has been hypothesized, but has not yet been fully understood. Components of a shared pathogenesis includes chronic antigen stimulation, JAK-STAT pathway activation and overlap of various cytokines. We will summarize current knowledge on the molecular understanding between T-LGLL and rheumatic disease. There are many potential areas of research to help meet this need and lead to development of targeted therapeutic options. [ABSTRACT FROM AUTHOR]

A complex relationship exists between rheumatic diseases and cancer. This delicate balance between chronic inflammation and malignant cell transformation in hematologic neoplasms has been observed, but is not well defined. Large Granular Lymphocyte (LGL) leukemia is at the intersection of a clonal lymphoproliferative disease, chronic inflammation, and autoimmunity. The association between rheumatoid arthritis (RA) and the spectrum of Felty’s Syndrome is well-known. Other rheumatic disorders have been reported including systemic lupus erythematosus (SLE), Sjogren’s Syndrome (SS), vasculitis, Behcet’s Disease (BD) and systemic sclerosis. The association between T-LGLL and rheumatic disease pathogenesis has been hypothesized, but has not yet been fully understood. Components of a shared pathogenesis includes chronic antigen stimulation, JAK-STAT pathway activation and overlap of various cytokines. We will summarize current knowledge on the molecular understanding between T-LGLL and rheumatic disease. There are many potential areas of research to help meet this need and lead to development of targeted therapeutic options.

Discs large (Dlg) is an essential polarity protein and a tumor suppressor originally characterized in Drosophila but also well conserved in vertebrates. Like the majority of polarity proteins, plasma membrane (PM)/cortical localization of Dlg is required for its function in polarity and tumorigenesis, but the exact mechanisms targeting Dlg to the PM remain to be fully elucidated. Here, we show that, similar to recently discovered polybasic polarity proteins such as Lgl and aPKC, Dlg also contains a positively charged polybasic domain that electrostatically binds the PM phosphoinositides PI4P and PI(4,5)P2. Electrostatic targeting by the polybasic domain contributes significantly to the PM localization of Dlg in follicular and early embryonic epithelial cells, and is crucial for Dlg to regulate both polarity and tumorigenesis. The electrostatic PM targeting of Dlg is controlled by a potential phosphorylation-dependent allosteric regulation of its polybasic domain, and is specifically enhanced by the interactions between Dlg and another basolateral polarity protein and tumor suppressor, Scrib. Our studies highlight an increasingly significant role of electrostatic PM targeting of polarity proteins in regulating cell polarity. [ABSTRACT FROM AUTHOR]

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

Abstract Objectives 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. Results 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.

Dorsal closure during Drosophila embryogenesis provides a robust genetic platform to study the basic cellular mechanisms that govern epithelial wound healing and morphogenesis. As dorsal closure proceeds, the lateral epithelial tissue (LE) adjacent to the dorsal opening advance contra-laterally, with a simultaneous retraction of the amnioserosa. The process involves a fair degree of coordinated cell shape changes in the dorsal most epithelial (DME) cells as well as a few penultimate rows of lateral epithelial (LE) cells (collectively referred here as Dorsolateral Epithelial (DLE) cells), lining the periphery of the amnioserosa, which in due course of time extend contra-laterally and ultimately fuse over the dorsal hole, giving rise to a dorsal epithelial continuum. The JNK-Dpp signaling in the dorsolateral epidermis, plays an instrumental role in guiding their fate during this process. A large array of genes have been reported to be involved in the regulation of this core signaling pathway, yet the mechanisms by which they do so is hitherto unclear, which forms the objective of our present study. Here we show a probable mechanism via which lgl , a conserved tumour suppressor gene, regulates the JNK–Dpp pathway during dorsal closure and epithelial morphogenesis. A conditional/targeted knock-down of lgl in the dorsolateral epithelium of embryos results in failure of dorsal closure. Interestingly, we also observed a similar phenotype in a Rab11 knockdown condition. Our experiment suggests Rab11 to be interacting with lgl as they seem to synergize in order to regulate the core JNK-Dpp signaling pathway during dorsal closure and also during adult thorax closure process. • Rab11 or lgl knockdown in the dorso-lateral epidermis results in the failure of dorsal closure in Drosophila embryos. • A targeted knockdown of lgl or Rab11 in the dorsolateral epidermal cells results in an improper morphology. • Rab11 expression increases significantly in the dorso-lateral embryonic epidermis where lgl is knocked down. • A simultaneous knockdown of Rab11 in an lgl depleted background in the dorsolateral epidermis results in a rescue. • Rab11 and lgl interact to regulate the JNK-Dpp pathway by maintenance of cell junctions and cortical cytoskeleton. [ABSTRACT FROM AUTHOR]

Apical-basal polarity underpins the formation of epithelial barriers that are crucial for metazoan physiology. Although apical-basal polarity is long known to require the basolateral determinants Lethal Giant Larvae (Lgl), Discs Large (Dlg) and Scribble (Scrib), mechanistic understanding of their function is limited. Lgl plays a role as an aPKC inhibitor, but it remains unclear whether Lgl also forms complexes with Dlg or Scrib. Using fluorescence recovery after photobleaching, we show that Lgl does not form immobile complexes at the lateral domain of Drosophila follicle cells. Optogenetic depletion of plasma membrane PIP2 or dlg mutants accelerate Lgl cortical dynamics. However, Dlg and Scrib are required only for Lgl localization and dynamic behavior in the presence of aPKC function. Furthermore, light-induced oligomerization of basolateral proteins indicates that Lgl is not part of the Scrib-Dlg complex in the follicular epithelium. Thus, Scrib and Dlg are necessary to repress aPKC activity in the lateral domain but do not provide cortical binding sites for Lgl. Our work therefore highlights that Lgl does not act in a complex but in parallel with Scrib-Dlg to antagonize apical determinants. [ABSTRACT FROM AUTHOR]

The Hedgehog (Hh) morphogen regulates growth and patterning. Since Hh signaling is also implicated in carcinogenesis, it is conceivable that de novo Hh-secreting organizers, if formed in association with oncogenic hit could be tumor-cooperative. Here we validate this hypothesis using the Drosophila model of cooperative epithelial carcinogenesis. We generate somatic clones with simultaneous loss of tumor suppressor, Lgl, and gain of the posterior compartment selector, Engrailed (En), known to induce synthesis of Hh. We show that lgl UAS-en clones in the anterior wing compartment trigger Hh signaling cascade via cross-talk with their Ci-expressing wild type cell neighbors. Hh-Dpp signaling from clone boundaries of such ectopically formed de novo organizers in turn drive lgl carcinogenesis. By contrast, Ci-expressing lgl clones transform by autocrine and/or juxtracine activation of Hh signaling in only the posterior compartment. We further show that sequestration of the Hh ligand or loss of Dpp receptor, Tkv, in these Hh-sending or –receiving lgl clones arrested their carcinogenesis. Our results therefore reveal a hitherto unrecognized mechanism of tumor cooperation by developmental organizers, which are induced fortuitously by oncogenic hits. • Gain of En in cells with loss of Lgl tumor suppressor trigger Hh signaling. • Their Ci expressing cell neighbors then display Hh-Dpp signaling cascade. • lgl mutant clones transform in response to reverse paracrine Dpp signaling. • Gain of Ci in En-expressing lgl clones transform by autocrine Hh-Dpp signaling. • Cells-of-origin of Hh-driven lgl tumors are thus developmental context-dependent. [ABSTRACT FROM AUTHOR]

Genomic analysis of cancer offers the hope of identifying new treatments or aiding in the selection of existing treatments. Rare leukemias pose additional challenges in this regard as samples may be hard to acquire and when found the underlying pathway may not be attractive to drug development since so few individuals are affected. In this case, it can be useful to identify common mutational overlap among subsets of rare leukemias to increase the number of individuals that may benefit from a targeted therapy. This chapter examines the current mutational landscape of large granular lymphocyte (LGL) leukemia with a focus on STAT3 mutations, the most common mutation in LGL leukemia to date. We examined the linkage between these mutations and autoimmune symptoms and disorders, in cases of obvious and suspected LGL leukemia. We then summarized and compared mutations in a set of other rare leukemias that also have JAK/STAT signaling pathway activation brought about by genomic changes. These include T-cell acute lymphoblastic leukemia (T-ALL), T-cell prolymphocytic leukemia (T-PLL), cutaneous T-cell lymphoma (CTCL), select peripheral T-cell lymphoma (PTCL), and adult T-cell leukemia/lymphoma (ATLL). Though STAT3 activation is common in these leukemias, the way in which it is achieved, such as the activating cytokine pathway and/or the co-mutational background, is quite diverse. [ABSTRACT FROM AUTHOR]

The Par polarity complex, consisting of Par3, Par6 and atypical protein kinase C (aPKC), plays a crucial role in the establishment and maintenance of cell polarity. Although activation of aPKC is critical for polarity, how this is achieved is unclear. The developing zebrafish epidermis, along with its apical actin-based projections, called microridges, offers a genetically tractable system for unraveling the mechanisms of the cell polarity control. The zebrafish aPKC regulates elongation of microridges by controlling levels of apical Lgl, which acts as a pro-elongation factor. Here, we show that the nucleoporin Nup358 (also known as RanBP2) - a component of the nuclear pore complex and a part of cytoplasmic annulate lamellae (AL) - SUMOylates zebrafish aPKC. Nup358-mediated SUMOylation controls aPKC activity to regulate Lgl-dependent microridge elongation. Our data further suggest that cytoplasmic AL structures are the possible site for Nup358-mediated aPKC SUMOylation. We have unraveled a hitherto unappreciated contribution of Nup358- mediated aPKC SUMOylation in cell polarity regulation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Metazoan cell polarity is controlled by a set of highly conserved proteins. Lethal giant larvae (Lgl) functions in apical-basal polarity through phosphorylation-dependent interactions with several other proteins as well as the plasma membrane. Phosphorylation of Lgl by atypical protein kinase C (aPKC), a component of the partitioning-defective (Par) complex in epithelial cells, excludes Lgl from the apical membrane, a crucial step in the establishment of epithelial cell polarity. We present the crystal structures of human Lgl2 in both its unphosphorylated and aPKC-phosphorylated states. Lgl2 adopts a double β-propeller structure that is unchanged by aPKC phosphorylation of an unstructured loop in its second β-propeller, ruling out models of phosphorylation-dependent conformational change. We demonstrate that phosphorylation controls the direct binding of purified Lgl2 to negative phospholipids in vitro. We also show that a coil-helix transition of this region that is promoted by phosphatidylinositol 4,5-bisphosphate (PIP2) is also phosphorylationdependent, implying a highly effective phosphorylative switch for membrane association. [ABSTRACT FROM AUTHOR]

A 12‐year‐old castrated male mixed breed dog was presented with anorexia, lethargy, intermittent vomiting, diarrhea, and weight loss. Clinicopathologic and imaging abnormalities included pancytopenia, icterus, and splenomegaly with multiple minute hypoechogenic nodules. Bone marrow (BM) smears revealed 2.5% hemophagocytic macrophages. In addition, an increased number of small to intermediate lymphocytes (16.3%) and plasma cells (3.2%) were recognized in the BM smears. More than 80% of the lymphocytes contained multiple small intracytoplasmic magenta granules. Histopathologic findings of the spleen revealed hemophagocytosis. Large granular lymphocytes (LGLs) were not found on the liver cytology or splenic histopathology at this time. PCR for antigen receptor rearrangement (PARR) analysis showed a clonal reaction in the T‐cell receptor ɤ (TCRɤ) gene in the BM sample. The dog was diagnosed with hemophagocytic syndrome (HPS). The dog was maintained in good condition with immunosuppressive therapy. However, the dog developed hepatic LGL lymphoma 7 months later. At this time, PARR analysis showed a clonal TCRɤ gene rearrangement in the hepatic LGL lymphoma samples. The BM and liver sample clonal rearrangements showed 100% homology, indicating that the small to intermediate granular lymphocytes in the BM at the HPS stage had progressed to hepatic LGL lymphoma. To our knowledge, this is the first report of canine secondary HPS caused by the occurrence of a BM LGL lymphoma clone that progressed to hepatic LGL lymphoma. [ABSTRACT FROM AUTHOR]

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.

Since the first identification of the Scribble polarity module proteins as a new class of tumour suppressors that regulate both cell polarity and proliferation, an increasing amount of evidence has uncovered a broader role for Scribble, Dlg and Lgl in the control of fundamental cellular functions and their signalling pathways. Here, we review these findings as well as discuss more specifically the role of the Scribble module in PCP signalling. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Many organs form by invaginating and rolling flat epithelial cell sheets into tubes. Invagination of the ventral midline of the neural plate forms the median hinge point (MHP), an event that elevates the neural folds and is essential for neural tube closure (NTC). MHP formation involves dynamic spatiotemporal modulations of cell shape, but how these are achieved is not understood. Here, we show that cellcycle-dependent BMP and TGFβ antagonism elicits MHP formation by dynamically regulating interactions between apical (PAR complex) and basolateral (LGL) polarity proteins. TGFβ and BMPactivated receptor (r)-SMADs [phosphorylated SMAD2 or SMAD3 (pSMAD2,3), or phosphorylated SMAD1, SMAD5 or SMAD8 (pSMAD1,5,8)] undergo cell-cycle-dependent modulations and nucleo-cytosolic shuttling along the apicobasal axis of the neural plate. Non-canonical TGFβ and BMP activity in the cytosol determines whether pSMAD2,3 or pSMAD1,5,8 associates with the tight junction (PAR complex) or with LGL, and whether cell shape changes can occur at the MHP. Thus, the interactions of BMP and TGFβ with polarity proteins dynamically modulate MHP formation by regulating r-SMAD competition for tight junctions and r-SMAD sequestration by LGL. [ABSTRACT FROM AUTHOR]

Large granular lymphocyte leukemia (LGL-L) is defined morphologically as a group of lymphoproliferative disorders, including T-cell large granular lymphocytic leukemia (T-LGL-L), chronic lymphoproliferative disorders of NK cells (CLPD-NK), and aggressive NK cell leukemia. We investigated the morphological features of LGL leukemic cells in LGL-L patients to identify screening methods best suited to application in daily clinical practice. LGL leukemic cells were mostly indistinguishable from normal LGL; however, we developed a simplified approach to distinguishing among these cells, in which lymphocyte  % and the proportion of granular lymphocytes among lymphocytes (GL %) can serve as parameters at the cut-off values of 52 and 50 %, respectively. We confirmed the accuracy of these methods in validation groups. It may be useful to evaluate GL % to screen for LGL-L using a cut-off threshold of 50 when Lym % exceeds 52 in the peripheral blood examination in cases in which LGL-L is suspected in daily practice while keeping clinical context and the coefficient of variation of these parameters under consideration. [ABSTRACT FROM AUTHOR]

Large granular lymphocyte leukemia (LGL-L) has been morphologically defined as a group of lymphoproliferative disorders, including T-cell large granular lymphocytic leukemia (T-LGL-L), chronic lymphoproliferative disorders of NK cells (CLPD-NK) and aggressive NK cell leukemia. We investigated the morphological features of LGL leukemic cells in 26 LGL-L patients in order to elucidate relationships with current classifications and molecular backgrounds. LGL-L cells were mostly indistinguishable from normal LGL. Patients with STAT3 SH2 domain mutations showed significantly smaller cells compared with patients without STAT3 mutations. Four patients with T-LGL-L showed smaller granular lymphocytes with a median diameter of less than 13 μm, which were rarely seen in normal subjects. This small subtype of T-LGL-L was recognized among rather young patients and was associated with D661Y mutations in the STAT3 gene SH2 domain. In addition, all of them showed anemia including two cases with pure red cell aplasia. These results suggest the heterogeneity of T-LGL-L and a specific subtype with small variants of T-LGL-L. [ABSTRACT FROM AUTHOR]

Dasatinib, a 2nd-generation tyrosine kinase inhibitor (TKI), can specifically induce large granular lymphocytes (LGL) in some patients with Philadelphia chromosome (Ph)-positive leukemia. To investigate the properties of the induced LGLs, we performed prospective and longitudinal analyses. From Feb 2011 to Jan 2013, a total of 17 patients with Ph-positive leukemia who were previously untreated or refractory to imatinib were enrolled. T cell receptor (TCR)-γ/δ gene rearrangements and phenotypic profiles of lymphocytes were examined before and during administration of dasatinib. LGL lymphocytosis was observed in half of the dasatinib-treated cases (LGL+ group), showing a relation to increased achievement of complete cytogenetic response within 6 months. The phenotypes of the increased lymphocytes were revealed to be mostly natural killer cells. In the LGL+ group, clonal TCR-γ gene rearrangements were frequently detected at diagnosis (six of nine cases) and persisted during therapy, compared with only two of eight in the LGL- group. The proportion of regulatory T cells to CD4+ T cells at diagnosis was lower in the LGL+ compared with the LGL- group (median 4.2 vs. 6.6 %), and this disparity was sustained throughout the therapeutic period. These results demonstrate that immunological condition at diagnosis may affect LGL lymphocytosis in some dasatinib-treated patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Large granular lymphocytic (LGL) leukemia is an incurable chronic disease, characterized by clonal expansion of cytotoxic T- or NK-cells in blood and bone marrow. Cytopenias (anemia, neutropenia) and autoimmune disorders such as rheumatoid arthritis are the most common clinical manifestations of LGL leukemia. Recently, somatic activating STAT3 gene mutations were shown to be specific for LGL leukemia with a prevalence of up to 70%. Analogous mutations in the STAT5b gene were seen in a smaller proportion of patients. These gain-of-function mutations are located in the SH2 domain of STAT3 and affect the phosphotyrosine-SH2 interaction required for dimerization of STAT3. The mutations increase the phosphorylation of STAT3 and STAT5b and enhance the transcriptional activity of the mutated proteins. STAT3 and STAT5b mutations can be used as molecular markers for LGL leukemia diagnostics, and they present novel therapeutic targets for STAT3 and STAT5b inhibitors, which currently are in development for treatment of cancer and autoimmune disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain. [ABSTRACT FROM AUTHOR]

Abstract: T-cell large granular lymphocyte leukemia (T-LGLL) is a rare chronic lymphoproliferative disorder. Available reported data on the treatment regimens of this disease are variable and limited due to low number of patients. We analyzed the efficiency of cyclosporine A (CsA) in the treatment of 28 patients with T-LGLL. The overall response rate (ORR) was 82.1% with hematologic complete remission (HCR) rate of 57.1%. The median time to response (TTR) was 1.8 months and treatment duration with CsA-based regimens was 34.5 months. CsA shows low and manageable toxicity during treatment. Twenty-one patients survived with a median follow-up time of 42.0 months. Our results indicate that CsA is efficacious and safe in the treatment of T-LGLL. [Copyright &y& Elsevier]

Determinants of cell polarity orient the behaviour of many cell types during development. Pioneering genetic screens in yeast, worms and flies have identified key polarity determinants that are evolutionarily conserved across the animal kingdom. Recent work in these three model organisms has combined computer modelling with experimental analysis to reveal the molecular mechanisms that drive the polarisation of determinants. Two key principles have emerged: the first is the requirement for a positive-feedback loop to drive self-recruitment of determinants to the plasma membrane; the second is the requirement for mutual antagonism between determinants that localise to opposite ends of the cell. [ABSTRACT FROM AUTHOR]

Interest in the mechanism leading to the formation of the germline and its differentiation during Drosophila development, initiated even as soon as the first ever cloned tumour suppressor gene in Drosophila, the lethal (2) giant larvae (lgl), had been identified. Further work has shown that the lgl, as well as discs large-1 (dlg) and scribble (scrib) tumor suppressor genes code for scaffolding proteins associated with either the cytoskeletal matrix or the septate junctions that act in common pathways in various tissues. This study analysed the role of Dlg, Scrib and Lgl in the embryonic gonads and testis of Drosophila melanogaster. Loss of scrib, dlg and lgl had no effect on gonad formation, but Dlg and Scrib in the gonadal mesoderm acted critically in the somatic wrapping of the pole cells and the internal structure of the Drosophila embryonic gonads. Dlg also affected the incorporation of the male-specific Sox100B positive mesodermal cells into the male embryonic gonads, yet Sox100B expression in dlg testis remained unaffected. Analysis at later stages revealed that scrib and lgl expression in the somatic lineage of the Drosophila testis, similar to what was previously shown for dlg, was indispensable for testis development and homeostasis, as depletion of these genes resulted in extensive testes defects. The data presented here emphasize the somatic requirement of Scrib, Dlg and Lgl in embryonic gonads, as well as in the Drosophila testis that underlines the importance of the somatic lineage in the establishment and maintenance of testis formation throughout successive developmental stages. [ABSTRACT FROM AUTHOR]

Expansions of CD3+ large granular lymphocytes (LGLs) after allogeneic hematopoietic stem cell transplantation (HSCT) have been described. We sought to evaluate incidence, characteristics, and clinical significance of persistent T cell (T-)LGL after HSCT. Fourteen of 215 recipients (7%) were diagnosed with LGL expansions. Thirteen showed a CD3+/CD8+ immunophenotype, 5 of them with clonal TCR-γ rearrangement. The lymphocytes appeared at a median of 16 months (range, 3-58 months) after HSCT and lasted for a median time of 31 months (range, 2-179 months). Cytomegalovirus (CMV) reactivation (P = .001) and acute graft-versus-host disease (aGVHD) were associated with LGL expansion (P = .02). In the multivariate analysis, only CMV reactivation showed a significant association with T-LGL expansion (relative risk [RR]: 5.063; 95% confidence interval [CI]: 1.586-16.160; P = .006). The observed posttransplantation LGL expansions, even if monoclonal, showed a chronic, indolent course. Our data indicate that such expansions may be considered as an expression of chronic stimulation, triggered by CMV reactivation rather than a malignant transformation. [ABSTRACT FROM AUTHOR]

Background To evaluate the efficacy of extracorporeal photopheresis (ECP) in noncutaneous T-cell lymphoma and large granular lymphocytes leukemia (LGL). Patients and methods We have treated 12 refractory/relapsed patients. Six peripheral T-cell lymphoma (PTCL), one T-lymphoblastic lymphoma and five LGL with blood involvement received six biweekly leukapheresis as induction phase, followed by one course a week for 4 weeks as consolidation and one course of maintenance per month for responders until progression/relapse or disappearance of the peripheral clone. Results Six patients responded to phototherapy. Two PTCL and two LGL achieved a complete response (CR) and two other PTCL a partial response. The median duration of CR was 117 months (45–150 months) for these four patients. The peripheral clone followed by flow cytometry decreased in all six responders. Two patients with a complete disappearance of the peripheral clone have not relapsed. Conclusions As for cutaneous T-cell lymphoma, ECP therefore to be efficient for PTCL and LGL. Early decrease and disappearance of the peripheral clone were the indicators of clinical response and nonrelapse, respectively. [ABSTRACT FROM PUBLISHER]

We retrospectively investigated increases in large granular lymphocytes (LGL) in peripheral blood during dasatinib treatment in 25 chronic myelogenous leukemia patients. Fifteen of 25 patients (60 %) showed an increase in LGL. All 15 of these patients also showed an increase in NK cells, and 11 showed an increase in CD8 T cells. High frequencies of clonal rearrangements of TCR-β, -γ, and -δ genes were observed in LGL (+) patients, and at lower frequencies in LGL (−) patients as well. Clinical responses were favorable for all. With respect to their newly obtained complete molecular response after dasatinib treatment, LGL (+) patients showed higher response rates than did LGL (−) patients. In contrast, pleural effusions were more commonly observed in LGL (+) patients (60 %) than in LGL (−) patients (20 %). LGL counts significantly increased at 2 h after oral intake of dasatinib in all 25 patients. This was not observed in treatment with imatinib or nilotinib. Cytomegalovirus (CMV) C7-HRP tests were negative in all patients. Serum CMV-IgM antibodies were positive in only 2 of 25 patients without symptom of infection. Thus, LGL lymphocytosis during dasatinib treatment may be correlated with favorable molecular response, and with increased incidence of pleural effusions. In the clinical setting, CMV reactivation appears uncommon. [ABSTRACT FROM AUTHOR]

Large granular lymphocytic (LGL) leukemia is an uncommon disorder of mature T or natural killer (NK) cells. Most T-LGL proliferations are CD3(+)/CD8(+), although rare CD4(+) clonal T-LGL expansions have been reported. We report the clinicopathologic features of eight patients with aberrant CD4(+), cytotoxic T-cell lymphocytoses. Median follow-up was 29 months (range 8–100), during which all were alive without requirement for therapy. Four of eight patients had an additional malignancy; none had a history of rheumatoid arthritis, lymphadenopathy or hepatosplenomegaly. Morphologic expansions of granulated lymphocytes were evident in 6/8. All had immunophenotypically aberrant populations of CD4(+) T cells with uniform, moderate or bright CD56. Seven of eight expressed CD57, and four were CD8(partial dim +). Abnormal levels of expression of two or more T-cell antigens were seen in all cases. All tested cases were Tγ PCR positive. Our results support that CD4(+) T-LGL lymphocytosis is a clonal disorder with clinicopathologic characteristics distinct from the more common CD8(+) variant. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Simple Summary: TRBC1 expression analysis by flow cytometry (FCM) has been recently proved to be a useful, simple and fast approach to assessing Tαβ-cell clonality. The aim of this study was to validate the utility of this assay specifically for the diagnosis of T-cell clonality of T-large granular lymphocytic leukemias (T-LGLL), as more mature polyclonal Tαβ large granular lymphocytes (Tαβ-LGL) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. Our results showed that a TRBC1-FCM assay is also a fast and easy method for detecting T-cell clonality in T-LGLL based on altered (increased or decreased) percentages of TRBC1+ Tαβ cells of LGL expansions (i.e., with lymphocytosis) suspected of T-LGLL, whereas in the absence of lymphocytosis (or in TαβCD4-LGLL), the detection of increased absolute cell-counts of more precisely defined subpopulations of T-LGL expressing individual TCRVβ families is required. 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 (<0.9% or >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 <1.6% or >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. [ABSTRACT FROM AUTHOR]

The neoplastic tumour suppressors, Scribble, Dlg and Lgl, originally discovered in the vinegar fly Drosophila melanogaster, are currently being actively studied for their potential role in mammalian tumourigenesis. In Drosophila, these tumour suppressors function in a common genetic pathway to regulate apicobasal cell polarity and also play important roles in the control of cell proliferation, survival, differentiation and in cell migration/invasion. The precise mechanism by which Scribble, Dlg and Lgl function is not clear; however, they have been implicated in the regulation of signalling pathways, vesicle trafficking and in the Myosin II–actin cytoskeleton. We review the evidence for the involvement of Scribble, Dlg, and Lgl in cancer, and how the various functions ascribed to these tumour suppressors in Drosophila and mammalian systems may impact on the process of tumourigenesis.Oncogene (2008) 27, 6888–6907; doi:10.1038/onc.2008.341 [ABSTRACT FROM AUTHOR]

T cell large granular lymphocyte leukemia (T-LGL) is characterised by semiautonomous proliferation of monoclonal cytotoxic T lymphocytes, which can result in neutropenia, splenomegaly, and is associated with various autoimmune disorders, particularly rheumatoid arthritis. The coexistence of T-LGL leukemia with B cell abnormalities has previously been identified in case reports. However, no systematic analysis to determine the frequency of this co-association has been reported. Analysis of 63 T-LGL patients revealed a frequent association with humoral immune system abnormalities. We identified coexisting B cell dyscrasias in 17 T-LGL patients (27% of total), of whom 12 had monoclonal gammopathy of unknown significance (MGUS) (19%), and 5 had chronic lymphocytic leukemia (CLL) (8%). The presence of both MGUS and CLL was found in 2 patients (3%) and follicular lymphoma was identified with MGUS in another T-LGL patient (2%). Additionally, polyclonal hypergammaglobulinemia or hypogammaglobulinemia was found in 10 additional LGL leukemia patients bringing the total frequency of B cell abnormalities in T-LGL leukemia to 43% in our cohort. The co-association of B cell pathology with T-LGL suggests that either a common antigen drives clonal B and T cells, or that humoral malignancy could serve as the stimulus for lymphocyte expansion representing an overactive anti-tumour surveillance. [ABSTRACT FROM AUTHOR]

The intricately regulated differentiation of the somatic follicle cell lineages into distinct subpopulations with specific functions plays an essential role in Drosophila egg development. At early oogenesis, induction of the stalk cells generates the first anteroposterior (AP) asymmetry in the egg chamber by inducing the posterior localization of the oocyte. Later, the properly specified posterior follicle cells signal to polarize the oocyte along the AP and dorsoventral (DV) axes at mid-oogenesis. Here, we show that lethal(2)giant larvae (lgl), a Drosophila tumor suppressor gene, is required in the follicle cells for the differentiation of both stalk cells and posterior follicle cells. Loss-of-function mutations in lgl cause oocyte mispositioning in the younger one of the fused chambers, due to lack of the stalk. Removal of lgl function from the posterior follicle cells using the FLP/FRT system results in loss of the oocyte polarity that is elicited by the failure of those posterior cells to differentiate normally. Thus, we provide the first demonstration that lgl is implicated in the formation of the initial AP asymmetry and the patterning of the AP and DV axes in the oocyte by acting in the specification of a subset of somatic follicle cells.Cell Research (2008) 18:372–384. doi: 10.1038/cr.2008.25; published online 12 February 2008 [ABSTRACT FROM AUTHOR]

Abstract: The human lgl gene, Hugl-2 (llgl2, Lgl2), codes for a cytoskeletal protein involved in regulating cell polarity. Here, we report the identification and functional characterization of the promoter region (∼1.2kb) of the Hugl-2 gene. Luciferase expression assays show a high basal Hugl-2 promoter activity in different cell lines and primary human hepatocytes. Truncations of the promoter identified a GC-rich region important for this activity. Alignment of human and mouse genomic sequences demonstrate that this is an evolutionary conserved region fcontaining putative binding sites for several transcription factors including Elk-1 and Sp-1. Mithramycin A reduces Hugl-2 expression indicating Sp-1 transcription factors activate Hugl-2. Treatment of primary hepatocytes with epidermal growth factor (EGF) suppresses Hugl-2, suggesting regulation by the EGF-signaling pathway. Downregulation of Hugl-2 by EGF may contribute to loss of cell polarity and tumour progression, therefore supporting a tumour suppressor role for Hugl-2. [Copyright &y& Elsevier]

Summary: TCRαβ+/CD4+ T–large granular lymphocyte (LGL) lymphocytosis is a subgroup of monoclonal T-LGL lymphoproliferative disorders that are different from the CD8+ TCRαβT-LGL. An increasing evidence supports the involvement of a common antigen-driven mechanism in the etiology of TCRαβ+/CD4+ T-LGL. In this study, we tested several polymorphic markers associated with chronic viral infections and autoimmune diseases, including cytotoxic T-lymphocyte antigen–4 (CTLA-4), tumor necrosis factor–α (TNF-α), interleukin-10 (IL-10), interferon-γ (IFN-γ), RANTES, IL-1α, FAS, FAS-ligand (FASL), and NKG2D, to investigate the potential association of these immunogenetic factors with the development of T-LGL. Overall, 38 patients with CD4+ T-LGL were analyzed and compared with a group of both CD8+/TCRαβ+ T-LGL patients (n = 43) and a group of control subjects (n = 176). Our results did not show any clear association between the different single nucleotide polymorphisms (SNPs) analyzed and the development of CD4+/TCRαβ T-LGL. An increase in the frequency of −380 (AA/GA) TNF-α genotype associated with a greater production of this cytokine was found among CD8+ T LGL patients in comparison to the CD4+LGL patients and the control group. Our results suggest that the frequency of SNP of the genes coding for the studied immunoregulatory molecules are not associated with the development of CD4+/TCRαβ+ T-LGL. [Copyright &y& Elsevier]