Are T-LGL leukemia and NK-chronic lymphoproliferative disorder really two distinct diseases?

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

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

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

Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae

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

STAT3 mutation impacts biological and clinical features of T-LGL leukemia

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\ub1) 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\ub1 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

Longevity mutation in SCH9 prevents recombination errors and premature genomic instability in a Werner/Bloom model system

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 sgs1Δ 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

HS1, a Lyn Kinase Substrate, Is Abnormally Expressed in B-Chronic Lymphocytic Leukemia and Correlates with Response to Fludarabine-Based Regimen

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

Oncogene homologue Sch9 promotes age-dependent mutations by a superoxide and Rev1/Polζ-dependent mechanism

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 sch9Δ 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 Polζ, 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

Role of inflammation in the development of lung fibrosis and in the pathogenesis of pulmunary hypertension

ABSTRACT Background: The bleomycin is a glycopeptide with antitumor and antiviral activity utilized in clinical for chemotherapeutic treatment of several neoplasms. Unfortunately the 10% of treated patients develops interstitial pneumonia that progresses to fibrosis. The fibrogenesis is linked to develop of a Th2 cellular response with proinflammatory chemokines release. Glycogen Synthase Kinase 3 (GSK-3) is a pivotal element for the control of immune response because modulates the inflammatory cytokines production. Since lung injury caused by bleomycin is characterized by an inflammatory response followed by a fibrotic degeneration, we postulated that blocking GSK-3 kinase activity with a specific inhibitor could affect the bleomycin-induced pulmonary inflammatory and pro-fibrotic cytokine network. Moreover it has been demonstrated that the develop of pathologies which elicit a Th2 inflammatory response could be mitigated by agents able to induce a Th1 immune response. Indeed administration of Neutrophil-Activating Protein of Helicobacter pylori (HP-NAP) was found to be able to down modulate the Th2 immune response in a asthma ovalbumin-induced model mouse by eliciting a Th1 immune response and resulted effective in preventing allergic asthma in the model mouse. Purpose of the study: We investigated the effects of the specific and selective ATP-competitive GSK-3 inhibitor, SB216763, and the effects of HP-NAP administration on the onset and development of inflammation and fibrosis in a bleomycin-induced lung fibrosis mouse model. Methods: We differently randomised cohorts of C57BL6 mice to receive intratracheal instillation of inhibitor SB216763, HP-NAP, bleomycin, bleomycin plus SB216763 or bleomycin plus HP-NAP and followed their health status for 28 days. Bronchoalveolar lavage (BAL) was performed and mice were sacrificed at different interval times (2, 7, 14 and 28 days). Histopathological analysis of the lungs, flow-cytometry studies and Cell Sorting of BAL pulmonary monocytes were then performed. Finally we examined cytokines gene expression levels in lung monocytes trough Real Time PCR analysis. Results: SB216763 and HP-NAP administration prevented lung inflammation and the subsequent fibrosis when co-administrated with bleomycin. BALF analysis of mice revealed a significant reduction in bleomycin-induced alveolitis. SB216763 treatment was associated with a significantly lower production of inflammatory cytokines (TNF-α and CCL12) by macrophages. Moreover we observed that HP-NAP administration downmodulated the Th2 inflammatory response bleomycin-induced by eliciting a Th1 phenotype (downmodulating IL-4 chemokine and upregulating the IFN-γ). Conclusions: These findings suggest that GSK-3 inhibition induces a protective effect on lung fibrosis triggered by bleomycin and candidate GSK-3 as a potential therapeutic target for preventing pulmonary fibrosis. Furthermore our results suggest that also HP-NAP, preventing lung fibrosis develop, might be a possible new tool for therapeutic strategies aimed to redirect Th2 into less aggressive Th1 response.RIASSUNTO Background: La bleomicina è un glicopeptite con attività antitumorale e antivirale utilizzata in clinica come chemio-terapico per il trattamento di diversi tumori. Sfortunatamente il 10% dei pazienti trattati sviluppa polmonite interstiziale che progredisce in fibrosi. La fibrogenesi è collegata allo sviluppo di una risposta immunitaria di tipo Th2 accompagnata dal rilascio di chemiochine pro-infiammatorie. La Glicogeno Sintetasi Chinasi 3 (GSK-3, Glycogen Synthase Kinase 3) è un fattore centrale per il controllo della risposta immunitaria poiché modula la produzione di citochine infiammatorie. Visto che il danno polmonare è caratterizzato da una risposta infiammatoria seguita da una degenerazione fibrotica, noi abbiamo ipotizzato che l’inibizione dell’attività di GSK-3, tramite uno specifico inibitore, potrebbe influenzare l’infiammazione del polmone ed il network di citochine pro-infiammatorie indotti dalla bleomicina. Recentemente è anche stato visto che lo sviluppo di patologie sostenute da un’infiammazione di tipo Th2 possono essere mitigate da agenti in grado di indurre una risposta infiammatoria di tipo Th1. In accordo con questi risultati la proteina attivante i neutrofili, prodotta dal batterio Helicobacter pylori (HP-NAP), stimolando una risposta immunitaria di tipo Th1, si è dimostrata capace di modulare la risposta immunitaria di tipo Th2 indotta dall’ ovalbumina in un modello murino di asma, risultando quindi efficace nel prevenire lo sviluppo dell’asma allergica. Scopo dello studio: In questa ricerca abbiamo investigato gli effetti della somministrazione dell’inibitore SB216763, specifico per GSK-3, e della somministrazione della proteina HP-NAP nello sviluppo dell’infiammazione e della fibrosi nel modello murino di fibrosi polmonare indotta da bleomicina. Metodi: Differenti coorti di topi C57BL6 sono state randomizzate per ricevere instillazione di SB216763, HP-NAP, bleomicina, bleomicina più SB216763 o bleomicina più HP-NAP, ed il loro stato di salute è stato monitorato per 28 giorni. I topi sono stati sacrificati ed i lavaggi broncho alveolari (BALs) eseguiti a diversi intervalli di tempo (a 2, 7, 14 e 28 giorni). Quindi sono stati eseguiti l’analisi istopatologia dei polmoni e valutazioni citoflurimetriche del BAL. Infine sono stati esaminati, tramite Real Time PCR, i livelli di espressione genica delle citochine nei monociti di polmone purificati dai BALs tramite sorting. Risultati: La somministrazione dell’inibitore SB216763 e della proteina HP-NAP, quando co-somministrati con la bleomicina, sono stati in grado di prevenire l’instaurarsi dell’infiammazione e il conseguente sviluppo di fibrosi nel polmone. L’analisi dei BALs ha infatti rivelato una significativa riduzione dell’alveolite indotta dalla bleomicina. Il trattamento con l’SB216763 si è associato con una ridotta produzione, da parte di macrofagi, di citochine infiammatorie (TNF-α and CCL12), mentre la somministrazione di HP-NAP ha down-modulato la risposta infiammatoria di tipo Th2 indotta dalla bleomicina, stimolando il tipo Th1(diminuendo il livello di espressione delle chemiochina IL-4 ed aumentando invece quello di IFN-γ). .Conclusioni: I risultati ottenuti in questo progetto di ricerca suggeriscono che l’inibizione di GSK-3 ha un effetto protettivo nella fibrosi polmonare indotta dalla bleomicina e candidano GSK-3 come potenziale target terapeutico per prevenire la fibrosi polmonare; inoltre suggeriscono che la proteina HP-NAP, prevenendo lo sviluppo della fibrosi polmonare tramite l’induzione di una risposta di tipo Th1, potrebbe essere una nuova strategia terapeutica per ridirezionare la risposta infiammatoria dal fenotipo Th2 verso quello meno aggressivo Th1

A simple model system for age-dependent DNA damage and cancer

International audienceAging is the major risk factor for many human cancers. However, the mechanisms responsible for the effect of aging on tumor incidence are poorly understood, in part because few model systems are available to study age-dependent genomic instability. Furthermore, the role of DNA mutations in "normal aging" and "life span extension" is unclear. Our laboratory has developed a novel method to study aging in yeast based on the survival of non-dividing populations (chronological life span). Two major pathways have been identified that control chronological aging: the Ras/PKA/Msn2/4 and the Sch9 pathways. The downregulation of either of them promotes life span extension. Importantly, similar pathways (insulin/IGF1-like), regulate longevity in higher eukaryotes suggesting a common evolutionary origin for the life span-regulatory mechanisms. Moreover, both Ras and Sch9 are functional homologs of two major mammalian oncogenes (Ras and Akt), which underlines the close link between cancer and aging. By combining chronological life span with simple assays for the detection of DNA mutations and dedifferentiation we have developed a powerful system to identify genes that regulate genomic instability and understand the fundamental mechanisms that may be responsible for age-dependent DNA mutations and cancer in mammals. Here we describe the use of this system to monitor the age-dependent accumulation of different types of DNA mutations including base substitutions, frame-shift mutations, and gross chromosomal rearrangements (GCRs)