Wilms' tumor suppressor gene (WT1) loss in T-cell acute lymphoblastic leukemia promotes cell survival and resistance to DNA damage

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor, resulting from the transformation of T-cell progenitors. Thanks to advances in molecular techniques, many alterations have been identified in T-ALL cells opening new opportunities for targeted therapy. WT1 gene deletions and mutations have been reported in 10-12% of T-ALL patients, but the mechanisms downstream of WT1 alterations in T-ALL have not been elucidated. The WT1 gene encodes a zinc-finger transcription factor which is characterized by multiple alternative isoforms. The isoforms that lack the three amino acids lysine-threonine-serine (KTS') between zinc finger 3 and 4, are conserved throughout vertebrate evolution and have high DNA-binding affinity and transcriptional activity. Most of WT1 mutations found in T-ALL are heterozygous frameshifts in exon 7 predicted to produce a truncated protein which lacks the DNA binding domain. Our main hypothesis is that WT1 acts as a haplo-insufficient tumor suppressor gene in T-ALL and that WT1 loss in T-ALL leads to de-regulation of pathway in T-ALL. In this study, we first analyzed the effects of full-length and mutant WT1 isoform over-expression on the survival and proliferation of T-ALL cells. We observed that only the (KTS') isoforms negatively affected growth of T-ALL and impaired colony formation in soft-agar. Importantly, the truncated WT1 proteins, derived from a characteristic frameshift mutation in exon 7 (E384Stop), had no effects. In parallel, we also analyzed the effects of WT1 loss in T-ALL cells. We found that WT1 knockdown in MOLT4 cells significantly increased the number of colonies in clonogenic assays in comparison with control cells. Overall these results indicated that WT1 most probably works as an haplo-insufficient tumor suppressor gene in T-ALL. In order to evaluate if mutations in WT1 locus are most likely responsible for an impaired transcriptional program we mainly focused on the analysis of WT1 deregulated targets following WT1 loss in T-ALL cells. To define the structure of the transcriptional network activated by loss of function of WT1, we performed ChIP-chip and gene expression analysis in MOLT4 T-ALL cells. ChIP-chip analysis showed that WT1 direct targets were enriched in pathways responsible for cellular response to stress, such as p53, nucleotide excision repair and Mitogen-Activated Protein Kinases (MAPK) signalling pathways. Integration of ChIP-chip data with gene expression analysis performed under WT1 loss of function conditions in MOLT4 cells provided an enrichment in the MAPK pathway. Stemming from these results, we finally evaluated if the loss of WT1 conferred increased survival after DNA damage, such as ionizing radiation or chemotherapeutic drugs, in MOLT4 T-ALL cells and primary T-ALL xenografts. Analysis of cell viability and apoptosis showed that WT1 alterations induced increased survival following DNA-damaging conditions, mainly affecting directly the transcription of important mediators of p53 apoptotic response. A master regulator of these effects was BBC3/PUMA, whose induction was augmented in the presence of both WT1 and p53 proteins. In conclusion, analyzing WT1 loss in T-ALL cells we determined a deregulation of several genes involved in the pathogenesis of T-ALL, in particular genes responsible for cellular response to stress, strongly suggesting WT1 acts as tumor suppressor gene in T-ALL cells

WT1 loss attenuates the TP53-induced DNA damage response in T-cell acute lymphoblastic leukemia

open12siLoss of function mutations and deletions in Wilms tumor 1 (WT1) gene are present in about 10% of T-cell acute lymphoblastic leukemia. Clinically, (WT1) mutations are enriched in relapsed series and are associated to inferior relapse-free survival in thymic T-cell acute lymphoblastic leukemia cases. Here, we demonstrate that WT1 plays a critical role in DNA damage response in T-cell leukemia. (WT1) loss conferred resistance to DNA damaging agents and attenuated the transcriptional activation of important apoptotic regulators downstream of TP53 in TP53-competent MOLT4 T-leukemia cells but not in TP53-mutant T-cell acute lymphoblastic leukemia cell lines. Notably, (WT1) loss positively affected the expression of the X-linked inhibitor of apoptosis protein (XIAP), and genetic or chemical inhibition with Embelin, a XIAP inhibitor, significantly restored sensitivity to γ-radiation in both T-cell acute lymphoblastic leukemia cell lines and patient xenografts. These results unveil an important role of (WT1) tumor suppressor gene in DNA damage response, and support a role for anti-XIAP targeted therapies in the treatment of (WT1)-mutant T-cell leukemia.openBordin, Fulvio; Piovan, Erich; Masiero, Elena; Ambesi-Impiombato, Alberto; Minuzzo, Sonia; Bertorelle, Roberta; Sacchetto, Valeria; Pilotto, Giorgia; Basso, Giuseppe; Zanovello, Paola; Amadori, Alberto; Tosello, ValeriaBordin, Fulvio; Piovan, Erich; Masiero, Elena; Ambesi-Impiombato, Alberto; Minuzzo, Sonia; Bertorelle, Roberta; Sacchetto, Valeria; Pilotto, Giorgia; Basso, Giuseppe; Zanovello, Paola; Amadori, Alberto; Tosello, Valeri

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3 epsilon

RSL3, a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating GPx4. Here we report the purification of the protein indispensable for GPx4 inactivation by RSL3. MS analysis reveals 14-3-3 isoforms as candidates and recombinant human 14-3-3epsilon confirms the identification. The function of 14-3-3\uf065 is redox-regulated. Moreover, overexpression and silencing of the gene coding for 14-3-3\uf065 consistently control the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein, operating in cell signalling, further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives

Non-invasive diagnostic tests for Helicobacter pylori infection

BACKGROUND: Helicobacter pylori (H pylori) infection has been implicated in a number of malignancies and non-malignant conditions including peptic ulcers, non-ulcer dyspepsia, recurrent peptic ulcer bleeding, unexplained iron deficiency anaemia, idiopathic thrombocytopaenia purpura, and colorectal adenomas. The confirmatory diagnosis of H pylori is by endoscopic biopsy, followed by histopathological examination using haemotoxylin and eosin (H & E) stain or special stains such as Giemsa stain and Warthin-Starry stain. Special stains are more accurate than H & E stain. There is significant uncertainty about the diagnostic accuracy of non-invasive tests for diagnosis of H pylori. OBJECTIVES: To compare the diagnostic accuracy of urea breath test, serology, and stool antigen test, used alone or in combination, for diagnosis of H pylori infection in symptomatic and asymptomatic people, so that eradication therapy for H pylori can be started. SEARCH METHODS: We searched MEDLINE, Embase, the Science Citation Index and the National Institute for Health Research Health Technology Assessment Database on 4 March 2016. We screened references in the included studies to identify additional studies. We also conducted citation searches of relevant studies, most recently on 4 December 2016. We did not restrict studies by language or publication status, or whether data were collected prospectively or retrospectively. SELECTION CRITERIA: We included diagnostic accuracy studies that evaluated at least one of the index tests (urea breath test using isotopes such as13C or14C, serology and stool antigen test) against the reference standard (histopathological examination using H & E stain, special stains or immunohistochemical stain) in people suspected of having H pylori infection. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the references to identify relevant studies and independently extracted data. We assessed the methodological quality of studies using the QUADAS-2 tool. We performed meta-analysis by using the hierarchical summary receiver operating characteristic (HSROC) model to estimate and compare SROC curves. Where appropriate, we used bivariate or univariate logistic regression models to estimate summary sensitivities and specificities. MAIN RESULTS: We included 101 studies involving 11,003 participants, of which 5839 participants (53.1%) had H pylori infection. The prevalence of H pylori infection in the studies ranged from 15.2% to 94.7%, with a median prevalence of 53.7% (interquartile range 42.0% to 66.5%). Most of the studies (57%) included participants with dyspepsia and 53 studies excluded participants who recently had proton pump inhibitors or antibiotics.There was at least an unclear risk of bias or unclear applicability concern for each study.Of the 101 studies, 15 compared the accuracy of two index tests and two studies compared the accuracy of three index tests. Thirty-four studies (4242 participants) evaluated serology; 29 studies (2988 participants) evaluated stool antigen test; 34 studies (3139 participants) evaluated urea breath test-13C; 21 studies (1810 participants) evaluated urea breath test-14C; and two studies (127 participants) evaluated urea breath test but did not report the isotope used. The thresholds used to define test positivity and the staining techniques used for histopathological examination (reference standard) varied between studies. Due to sparse data for each threshold reported, it was not possible to identify the best threshold for each test.Using data from 99 studies in an indirect test comparison, there was statistical evidence of a difference in diagnostic accuracy between urea breath test-13C, urea breath test-14C, serology and stool antigen test (P = 0.024). The diagnostic odds ratios for urea breath test-13C, urea breath test-14C, serology, and stool antigen test were 153 (95% confidence interval (CI) 73.7 to 316), 105 (95% CI 74.0 to 150), 47.4 (95% CI 25.5 to 88.1) and 45.1 (95% CI 24.2 to 84.1). The sensitivity (95% CI) estimated at a fixed specificity of 0.90 (median from studies across the four tests), was 0.94 (95% CI 0.89 to 0.97) for urea breath test-13C, 0.92 (95% CI 0.89 to 0.94) for urea breath test-14C, 0.84 (95% CI 0.74 to 0.91) for serology, and 0.83 (95% CI 0.73 to 0.90) for stool antigen test. This implies that on average, given a specificity of 0.90 and prevalence of 53.7% (median specificity and prevalence in the studies), out of 1000 people tested for H pylori infection, there will be 46 false positives (people without H pylori infection who will be diagnosed as having H pylori infection). In this hypothetical cohort, urea breath test-13C, urea breath test-14C, serology, and stool antigen test will give 30 (95% CI 15 to 58), 42 (95% CI 30 to 58), 86 (95% CI 50 to 140), and 89 (95% CI 52 to 146) false negatives respectively (people with H pylori infection for whom the diagnosis of H pylori will be missed).Direct comparisons were based on few head-to-head studies. The ratios of diagnostic odds ratios (DORs) were 0.68 (95% CI 0.12 to 3.70; P = 0.56) for urea breath test-13C versus serology (seven studies), and 0.88 (95% CI 0.14 to 5.56; P = 0.84) for urea breath test-13C versus stool antigen test (seven studies). The 95% CIs of these estimates overlap with those of the ratios of DORs from the indirect comparison. Data were limited or unavailable for meta-analysis of other direct comparisons. AUTHORS' CONCLUSIONS: In people without a history of gastrectomy and those who have not recently had antibiotics or proton ,pump inhibitors, urea breath tests had high diagnostic accuracy while serology and stool antigen tests were less accurate for diagnosis of Helicobacter pylori infection.This is based on an indirect test comparison (with potential for bias due to confounding), as evidence from direct comparisons was limited or unavailable. The thresholds used for these tests were highly variable and we were unable to identify specific thresholds that might be useful in clinical practice.We need further comparative studies of high methodological quality to obtain more reliable evidence of relative accuracy between the tests. Such studies should be conducted prospectively in a representative spectrum of participants and clearly reported to ensure low risk of bias. Most importantly, studies should prespecify and clearly report thresholds used, and should avoid inappropriate exclusions

Proteine coinvolte nella riparazione di danni al DNA indotti da radiazioni gamma: analisi tramite immunofluorescenza

In questa tesi è stato presentato uno studio degli effetti indotti da radiazioni ionizzanti y su fibroblasti umani, allo scopo di verificare, attraverso analisi con immunofluorescenza diretta, la presenza della proteina 53BP1 nel pathway di segnalazione e riparazione del danno al DNA

Wilms' tumor suppressor gene (WT1) loss in T-cell acute lymphoblastic leukemia promotes cell survival and resistance to DNA damage

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor, resulting from the transformation of T-cell progenitors. Thanks to advances in molecular techniques, many alterations have been identified in T-ALL cells opening new opportunities for targeted therapy. WT1 gene deletions and mutations have been reported in 10-12% of T-ALL patients, but the mechanisms downstream of WT1 alterations in T-ALL have not been elucidated. The WT1 gene encodes a zinc-finger transcription factor which is characterized by multiple alternative isoforms. The isoforms that lack the three amino acids lysine-threonine-serine (KTS') between zinc finger 3 and 4, are conserved throughout vertebrate evolution and have high DNA-binding affinity and transcriptional activity. Most of WT1 mutations found in T-ALL are heterozygous frameshifts in exon 7 predicted to produce a truncated protein which lacks the DNA binding domain. Our main hypothesis is that WT1 acts as a haplo-insufficient tumor suppressor gene in T-ALL and that WT1 loss in T-ALL leads to de-regulation of pathway in T-ALL. In this study, we first analyzed the effects of full-length and mutant WT1 isoform over-expression on the survival and proliferation of T-ALL cells. We observed that only the (KTS') isoforms negatively affected growth of T-ALL and impaired colony formation in soft-agar. Importantly, the truncated WT1 proteins, derived from a characteristic frameshift mutation in exon 7 (E384Stop), had no effects. In parallel, we also analyzed the effects of WT1 loss in T-ALL cells. We found that WT1 knockdown in MOLT4 cells significantly increased the number of colonies in clonogenic assays in comparison with control cells. Overall these results indicated that WT1 most probably works as an haplo-insufficient tumor suppressor gene in T-ALL. In order to evaluate if mutations in WT1 locus are most likely responsible for an impaired transcriptional program we mainly focused on the analysis of WT1 deregulated targets following WT1 loss in T-ALL cells. To define the structure of the transcriptional network activated by loss of function of WT1, we performed ChIP-chip and gene expression analysis in MOLT4 T-ALL cells. ChIP-chip analysis showed that WT1 direct targets were enriched in pathways responsible for cellular response to stress, such as p53, nucleotide excision repair and Mitogen-Activated Protein Kinases (MAPK) signalling pathways. Integration of ChIP-chip data with gene expression analysis performed under WT1 loss of function conditions in MOLT4 cells provided an enrichment in the MAPK pathway. Stemming from these results, we finally evaluated if the loss of WT1 conferred increased survival after DNA damage, such as ionizing radiation or chemotherapeutic drugs, in MOLT4 T-ALL cells and primary T-ALL xenografts. Analysis of cell viability and apoptosis showed that WT1 alterations induced increased survival following DNA-damaging conditions, mainly affecting directly the transcription of important mediators of p53 apoptotic response. A master regulator of these effects was BBC3/PUMA, whose induction was augmented in the presence of both WT1 and p53 proteins. In conclusion, analyzing WT1 loss in T-ALL cells we determined a deregulation of several genes involved in the pathogenesis of T-ALL, in particular genes responsible for cellular response to stress, strongly suggesting WT1 acts as tumor suppressor gene in T-ALL cells.La leucemia linfoblastica acuta a cellule T (LLA-T) è un tumore ematologico derivante dalla trasformazione neoplastica dei progenitori dei linfociti T. Grazie ai numerosi progressi effettuati nelle tecniche di biologia molecolare, numerose alterazioni genetiche sono state identificate nei pazienti affetti da LLA-T, generando nuove opportunità per la messa a punto di una terapia più mirata. Delezioni e mutazioni a carico del gene WT1 sono state identificate nel 10-12% dei pazienti affetti da LLA-T, ma tuttora gli effetti derivanti da tali alterazioni non sono stati ancora ben chiariti. Il gene WT1 codifica per un fattore di trascrizione caratterizzato da diverse isoforme derivante da splicing alternativo. Le isoforme sono conservate in tutti i vertebrati ma differiscono tra loro per la presenza o assenza dell'esone 5 e di un tripeptide composto dagli amminoacidi lisina-trenonina-serina (KTS), a cavallo tra il terzo e quarto dominio zinc finger. Le isoforme prive del KTS (KTS') possiedono una forte affinità nel legare il DNA e quindi sono in grado di regolare la trascrizione dei geni bersaglio. La nostra ipotesi è che WT1 agisca come oncosopressore in condizioni di aplo-insufficienza nella LLA-T e che la sua assenza comporti un'alterata regolazione del suo assetto trascrizionale. In questo studio sono stati analizzati gli effetti della sovra-espressione di WT1, sia delle isoforme normali che di un caratteristico mutante trovato nella LLA-T, sulla sopravvivenza e proliferazione delle cellule leucemiche. Abbiamo osservato che esclusivamente le isoforme (KTS') erano in grado di influenzare negativamente la crescita delle cellule leucemiche e di diminuire la loro capacità di formare colonie in soft-agar. La sovra-epressione delle isoforme derivate da una caratteristica mutazione frameshift (E384Stop) nell'esone 7 del gene WT1, invece, non produceva alcun effetto sulla crescita delle cellule di LLA-T. Per mimare la deplezione di WT1 abbiamo indotto il silenziamento del gene nella linea cellulare di leucemia MOLT4 e abbiamo valutato l'effetto sulla crescita delle cellule mediante saggio clonogenico. Abbiamo riscontrato che le cellule leucemiche con bassi livelli di espressione di WT1 presentavano un significativo incremento di colonie rispetto alle cellule di controllo. Questi risultati suggeriscono che le mutazioni di WT1 possano determinare l'aplo-insufficenza del gene nelle cellule di LLA-T, favorendo la crescita e lo sviluppo tumorale. Allo scopo di valutare se le mutazioni a carico di WT1 potessero indurre un'alterazione del suo assetto trascrizionale in cellule di LLA-T, ci siamo focalizzati nellâanalisi dei suoi bersagli dopo deplezione di WT1 in una linea cellulare di LLA-T. Mediante lâanalisi di immunoprecipitazione della cromatina su tecnologia microarray (Chromatin Immunoprecipitation on chip; ChIP-chip), effettuata nella linea cellulare MOLT4, abbiamo ottenuto circa 800 geni regolati direttamente da WT1 nella LLA-T. Analisi bioinformatiche hanno dimostrato un arricchimento di questi geni in alcune vie di segnale implicate nella risposta allo stress cellulare: la via di attivazione di p53, le vie coinvolte nella riparazione del DNA dopo danno cellulare e la via di segnalazione della Mitogen-Activated Protein (MAP) chinasi. Integrando lâanalisi ChIP-chip con l'analisi del profilo di espressione genica, ottenuta dopo silenziamento di WT1 nelle cellule MOLT4, abbiamo ulteriormente definito i bersagli diretti di WT1 che risultano anche de-regolati nella LLA-T. Questi risultano arricchiti nella via di segnalazione delle MAP chinasi. Prendendo spunto da questi risultati, abbiamo infine verificato se la ridotta espressione di WT1 nelle cellule di LLA-T, sia in linee cellulari che in campioni primari derivanti da pazienti affetti da LLA-T, ne favorisca la sopravvivenza in seguito a danno al DNA, per esempio dopo trattamento con radiazioni ionizzanti o farmaci chemioterapici. Le analisi della vitalità cellulare e dell'apoptosi di queste cellule hanno chiaramente mostrato come le alterazioni di WT1 inducano una maggior resistenza a condizioni di stress, in quanto vanno ad interferire con la trascrizione di importanti geni apoptotici, soprattutto quelli a valle di p53. In particolare l'induzione del gene BBC3/PUMA, un fattore chiave nella risposta apoptotica, è significativamente più elevata nelle cellule in cui WT1 e p53 risultano funzionali. In conclusione, analizzando l'effetto della perdita dellâespressione di WT1 nella LLA-T, abbiamo riscontrato un'alterata regolazione di numerosi geni coinvolti nella patogenesi della LLA-T, in particolare quelli responsabili della risposta cellulare in seguito al danno al DNA, suggerendo ulteriormente un ruolo di WT1 come gene oncosopressore in questa neoplasia

Calcineurin complex isolated from T-cell acute lymphoblastic leukemia (T-ALL) cells identifies new signaling pathways including mTOR/AKT/S6K whose inhibition synergize with Calcineurin inhibition to promote T-ALL cell death

Calcineurin (Cn) is a calcium activated protein phosphatase involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined. In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL) we used tandem affinity chromatography, followed by mass spectrometry to purify novel Cn-interacting partners. We found the Cn-interacting proteins to be part of numerous cellular signaling pathways including eIF2 signaling and mTOR signaling. Coherently, modulation of Cn activity in T-ALL cells determined alterations in the phosphorylation status of key molecules implicated in protein translation such as eIF-2\u3b1 and ribosomal protein S6. Joint targeting of PI3K-mTOR, eIF-2\u3b1 and 14-3-3 signaling pathways with Cn unveiled novel synergistic pro-apoptotic drug combinations. Further analysis disclosed that the synergistic interaction between PI3K-mTOR and Cn inhibitors was prevalently due to AKT inhibition. Finally, we showed that the synergistic pro-apoptotic response determined by jointly targeting AKT and Cn pathways was linked to down-modulation of key anti-apoptotic proteins including Mcl-1, Claspin and XIAP. In conclusion, we identify AKT inhibition as a novel promising drug combination to potentiate the pro-apoptotic effects of Cn inhibitors

Calcineurin and GSK-3 inhibition sensitizes T-cell acute lymphoblastic leukemia cells to apoptosis through X-linked inhibitor of apoptosis protein degradation

The calcineurin (Cn)-nuclear factor of activated T cells signaling pathway is critically involved in many aspects of normal T-cell physiology; however, its direct implication in leukemogenesis is still ill-defined. Glycogen synthase kinase-3 beta (GSK-3 beta) has recently been reported to interact with Cn in neuronal cells and is implicated in MLL leukemia. Our biochemical studies clearly demonstrated that Cn was able to interact with GSK-3 beta in T-cell acute lymphoblastic leukemia (T-ALL) cells, and that this interaction was direct, leading to an increased catalytic activity of GSK-3 beta, possibly through autophosphorylation of Y216. Sensitivity to GSK-3 inhibitor treatment correlated with altered GSK-3 beta phosphorylation and was more prominent in T-ALL with Pre/Pro immunophenotype. In addition, dual Cn and GSK-3 inhibitor treatment in T-ALL cells promoted sensitization to apoptosis through proteasomal degradation of X-linked inhibitor of apoptosis protein (XIAP). Consistently, resistance to drug treatments in primary samples was strongly associated with higher XIAP protein levels. Finally, we showed that dual Cn and GSK-3 inhibitor treatment in vitro and in vivo is effective against available models of T-ALL, indicating an insofar untapped therapeutic opportunity