Effect of aberrant ribosome biogenesis on wild-type and mutant p53 in cancer cells

2012/2013The tumor suppressor p53 plays a central role in the protection against DNA damage and other forms of stress, primarily by inducing cell cycle arrest or apoptosis. Missense mutation of p53, which is one of the most frequent genetic alterations detected in human cancers, inactivates these growth regulatory functions; in addition, mutant p53 often acquires tumor-promoting activities (gain-of-function). A complete and thorough understanding of the signaling circuitry that regulates wild-type and mutant p53 functions is therefore a primary objective for basic cancer research, since it may lead to development of important tools for diagnosis and therapy of tumors. One crucial component of such knowledge is the molecular mechanism leading to protein activation. Deregulated ribosome biogenesis is commonly observed in cancers as a result of increased biosynthetic demand due to uncontrolled cell proliferation. Cells actively monitor the fidelity of ribosome biogenesis, activating cellular checkpoints when this process is altered. Indeed, perturbations to many aspects of ribosome biogenesis generate a “nucleolar stress” that triggers a p53 response. Evidence in animal models indicates that the ribosomal-p53 checkpoint is indeed important for tumor suppression in vivo. In the recent past, we identified the nucleolar protein GTPBP4 as a novel p53 interactor, and established a functional link between the two proteins by demonstrating that GTPBP4 depletion promotes p53 accumulation and activation. In this Thesis, I demonstrate that GTPBP4 depletion affects 28S rRNA processing, generating a “nucleolar stress” responsible for p53 stabilization. The molecular mechanism responsible for such accumulation relies on the binding of ribosomal proteins to Mdm2. In parallel, I report that the same molecular mechanism leading to wild-type p53 stabilization upon ribosomal stress, can also promote mutant p53 accumulation in cancer cells. Thus, deregulated ribosome biogenesis may contribute to the stability of mutant p53 in tumors. These results collectively suggest that upon alterations in ribosome biogenesis, the TP53 status may be critical to drive the nucleolar surveillance pathway towards a tumor suppressive or oncogenic outcome.L’oncosoppressore p53 svolge un ruolo cruciale nella protezione della cellula da danni al DNA e da altre forme di stress, principalmente inducendo arresto del ciclo cellulare e apoptosi. Mutazioni missenso nel gene TP53 sono le alterazioni genetiche più frequenti trovate nei tumori umani ed inattivano queste funzioni regolative; oltre a questo, p53 mutata acquisisce nuove proprietà oncogeniche (gain-of-function). Capire i meccanismi regolativi che sono alla base delle funzioni di p53 wild-type e mutata è quindi un fondamentale obiettivo per la ricerca di base nel cancro, dal momento che può portare allo sviluppo di importanti strumenti sia diagnostici che terapeutici. Un aspetto fondamentale di tale ricerca è la comprensione dei meccanismi molecolari responsabili dell’attivazione della proteina. La sintesi dei ribosomi è frequentemente deregolata nei tumori, in quanto le cellule hanno un aumentato fabbisogno biosintetico a causa dell’incontrollata proliferazione. La cellula monitora attivamente la fedeltà della sintesi dei ribosomi, attivando meccanismi molecolari quando questo processo è compromesso. Infatti, alterazioni ad ogni livello nella sintesi dei ribosomi genera uno “stress nucleolare” che attiva la risposta di p53. Alcune evidenze in modelli animali infatti suggeriscono l’importanza delle funzioni oncosoppressive dell’asse molecolare costituito dalle proteine ribosomali e p53. In un lavoro pubblicato recentemente, abbiamo identificato la proteina nucleolare GTPBP4 come nuovo interattore di p53, inoltre abbiamo dimostrato un collegamento funzionale tra le due proteine, in quanto la deplezione di GTPBP4 promuove l’accumulo e l’attivazione di p53. In questa Tesi ho dimostrato che la deplezione di GTPBP4 altera il processamento del RNA ribosomale 28S, causando uno “stress nucleolare” responsabile per la stabilizzazione di p53. Il meccanismo molecolare necessario per la stabilizzazione di p53 dipende dal legame di proteine ribosomali a Mdm2. In parallelo, ho dimostrato che lo stesso meccanismo molecolare responsabile dell’accumulo di p53 wild-type in seguito allo stress ribosomiale, può indurre la stabilizzazione anche di p53 mutata. Quindi, un’alterata sintesi ribosomale può contribuire alla stabilità di mutant p53. Questi risultati suggeriscono che in seguito ad alterazioni nella sintesi dei ribosomi, lo stato di p53 può essere critico nel determinare se la risposta cellulare sia di natura oncosoppressiva oppure oncogenica.XXV Ciclo198

Cardiology of the future: xenotransplantation with porcine heart

The reduced availability of human donor hearts compared with the needs of patients with advanced heart failure refractory to medical therapy has promoted the search for therapeutic alternatives to cardiac allografts. Porcine heart xenotransplantation represents one of the most promising frontiers in this field today. From the first researches in the 1960s to today, the numerous advances achieved in the field of surgical techniques, genetic engineering and immunosuppression have made it possible at the beginning of 2022 to carry out the first swine-to-human heart transplant, attaining a survival of 2 months after surgery. The main intellectual and experimental stages that have marked the history of xenotransplantation, the latest acquisitions in terms of genetic editing, as well as the improvement of immunosuppressive therapy are discussed analytically in this article in order to illustrate the underlying complexity of this therapeutic model

Lymphocytic Myocarditis: A Genetically Predisposed Disease?

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Prognostic Prediction of Genotype vs Phenotype in Genetic Cardiomyopathies

Background: Diverse genetic backgrounds often lead to phenotypic heterogeneity in cardiomyopathies (CMPs). Previous genotype-phenotype studies have primarily focused on the analysis of a single phenotype, and the diagnostic and prognostic features of the CMP genotype across different phenotypic expressions remain poorly understood. Objectives: We sought to define differences in outcome prediction when stratifying patients based on phenotype at presentation compared with genotype in a large cohort of patients with CMPs and positive genetic testing. Methods: Dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy, left-dominant arrhythmogenic cardiomyopathy, and biventricular arrhythmogenic cardiomyopathy were examined in this study. A total of 281 patients (80% DCM) with pathogenic or likely pathogenic variants were included. The primary and secondary outcomes were: 1) all-cause mortality (D)/heart transplant (HT); 2) sudden cardiac death/major ventricular arrhythmias (SCD/MVA); and 3) heart failure-related death (DHF)/HT/left ventricular assist device implantation (LVAD). Results: Survival analysis revealed that SCD/MVA events occurred more frequently in patients without a DCM phenotype and in carriers of DSP, PKP2, LMNA, and FLNC variants. However, after adjustment for age and sex, genotype-based classification, but not phenotype-based classification, was predictive of SCD/MVA. LMNA showed the worst trends in terms of D/HT and DHF/HT/LVAD. Conclusions: Genotypes were associated with significant phenotypic heterogeneity in genetic cardiomyopathies. Nevertheless, in our study, genotypic-based classification showed higher precision in predicting the outcome of patients with CMP than phenotype-based classification. These findings add to our current understanding of inherited CMPs and contribute to the risk stratification of patients with positive genetic testing

Overview on electrical issues faced during the SPIDER experimental campaigns

SPIDER is the full-scale prototype of the ion source of the ITER Heating Neutral Beam Injector, where negative ions of Hydrogen or Deuterium are produced by a RF generated plasma and accelerated with a set of grids up to ~100 keV. The Power Supply System is composed of high voltage dc power supplies capable of handling frequent grid breakdowns, high current dc generators for the magnetic filter field and RF generators for the plasma generation. During the first 3 years of SPIDER operation different electrical issues were discovered, understood and addressed thanks to deep analyses of the experimental results supported by modelling activities. The paper gives an overview on the observed phenomena and relevant analyses to understand them, on the effectiveness of the short-term modifications provided to SPIDER to face the encountered issues and on the design principle of long-term solutions to be introduced during the currently ongoing long shutdown.Comment: 8 pages, 12 figures. Presented at SOFT 202

Changes in renal function after nephroureterectomy for upper urinary tract carcinoma: analysis of a large multicenter cohort (Radical Nephroureterectomy Outcomes (RaNeO) Research Consortium)

Purpose To investigate prevalence and predictors of renal function variation in a multicenter cohort treated with radical nephroureterectomy (RNU) for upper tract urothelial carcinoma (UTUC). Methods Patients from 17 tertiary centers were included. Renal function variation was evaluated at postoperative day (POD)-1, 6 and 12 months. Timepoints differences were Delta 1 = POD-1 eGFR - baseline eGFR; Delta 2 = 6 months eGFR - POD-1 eGFR; Delta 3 = 12 months eGFR - 6 months eGFR. We defined POD-1 acute kidney injury (AKI) as an increase in serum creatinine by >= 0.3 mg/dl or a 1.5 1.9-fold from baseline. Additionally, a cutoff of 60 ml/min in eGFR was considered to define renal function decline at 6 and 12 months. Logistic regression (LR) and linear mixed (LM) models were used to evaluate the association between clinical factors and eGFR decline and their interaction with follow-up. Results A total of 576 were included, of these 409(71.0%) and 403(70.0%) had an eGFR < 60 ml/min at 6 and 12 months, respectively, and 239(41.5%) developed POD-1 AKI. In multivariable LR analysis, age (Odds Ratio, OR 1.05, p < 0.001), male gender (OR 0.44, p = 0.003), POD-1 AKI (OR 2.88, p < 0.001) and preoperative eGFR < 60 ml/min (OR 7.58, p < 0.001) were predictors of renal function decline at 6 months. Age (OR 1.06, p < 0.001), coronary artery disease (OR 2.68, p = 0.007), POD-1 AKI (OR 1.83, p = 0.02), and preoperative eGFR < 60 ml/min (OR 7.80, p < 0.001) were predictors of renal function decline at 12 months. In LM models, age (p = 0.019), hydronephrosis (p < 0.001), POD-1 AKI (p < 0.001) and pT-stage (p = 0.001) influenced renal function variation (ss 9.2 +/- 0.7, p < 0.001) during follow-up. Conclusion Age, preoperative eGFR and POD-1 AKI are independent predictors of 6 and 12 months renal function decline after RNU for UTUC

Risks of Ventricular Arrhythmia and Heart Failure in Carriers of RBM20 Variants

BACKGROUND: Variants in RBM20 are reported in 2% to 6% of familial cases of dilated cardiomyopathy and may be associated with fatal ventricular arrhythmia and rapid heart failure progression. We sought to determine the risk of adverse events in RBM20 variant carriers and the impact of sex on outcomes. METHODS: Consecutive probands and relatives carrying RBM20 variants were retrospectively recruited from 12 cardiomyopathy units. The primary end point was a composite of malignant ventricular arrhythmia (MVA) and end-stage heart failure (ESHF). MVA and ESHF end points were also analyzed separately and men and women compared. Left ventricular ejection fraction (LVEF) contemporary to MVA was examined. RBM20 variant carriers with left ventricular systolic dysfunction (RBM20LVSD) were compared with variant-elusive patients with idiopathic left ventricular systolic dysfunction. RESULTS: Longitudinal follow-up data were available for 143 RBM20 variant carriers (71 men; median age, 35.5 years); 7 of 143 had an MVA event at baseline. Thirty of 136 without baseline MVA (22.0%) reached the primary end point, and 16 of 136 (11.8%) had new MVA with no significant difference between men and women (log-rank P=0.07 and P=0.98, respectively). Twenty of 143 (14.0%) developed ESHF (17 men and 3 women; log-rank P&lt;0.001). Four of 10 variant carriers with available LVEF contemporary to MVA had an LVEF &gt;35%. At 5 years, 15 of 67 (22.4%) RBM20LVSD versus 7 of 197 (3.6%) patients with idiopathic left ventricular systolic dysfunction had reached the primary end point (log-rank P&lt;0.001). RBM20 variant carriage conferred a 6.0-fold increase in risk of the primary end point. CONCLUSIONS: RBM20 variants are associated with a high risk of MVA and ESHF compared with idiopathic left ventricular systolic dysfunction. The risk of MVA in male and female RBM20 variant carriers is similar, but male sex is strongly associated with ESHF.</p

Risks of Ventricular Arrhythmia and Heart Failure in Carriers of RBM20 Variants

BACKGROUND: Variants in RBM20 are reported in 2% to 6% of familial cases of dilated cardiomyopathy and may be associated with fatal ventricular arrhythmia and rapid heart failure progression. We sought to determine the risk of adverse events in RBM20 variant carriers and the impact of sex on outcomes. METHODS: Consecutive probands and relatives carrying RBM20 variants were retrospectively recruited from 12 cardiomyopathy units. The primary end point was a composite of malignant ventricular arrhythmia (MVA) and end-stage heart failure (ESHF). MVA and ESHF end points were also analyzed separately and males and females compared. Left ventricular ejection fraction (LVEF) contemporary to MVA was examined. RBM20 variant carriers with left ventricular systolic dysfunction (RBM20LVSD) were compared with variant-elusive patients with idiopathic left ventricular systolic dysfunction. RESULTS: Longitudinal follow-up data were available for 143 RBM20 variant carriers (71 male; median age, 35.5 years); 7 of 143 had an MVA event at baseline. Thirty of 136 without baseline MVA (22.0%) reached the primary end point, and 16 of 136 (11.8%) had new MVA with no significant difference between males and females (log-rank P=0.07 and P=0.98, respectively). Twenty of 143 (14.0%) developed ESHF (17 males and 3 females; log-rank P35%. At 5 years, 15 of 67 (22.4%) RBM20LVSD versus 7 of 197 (3.6%) patients with idiopathic left ventricular systolic dysfunction had reached the primary end point (log-rank P<0.001). RBM20 variant carriage conferred a 6.0-fold increase in risk of the primary end point. CONCLUSIONS: RBM20 variants are associated with a high risk of MVA and ESHF compared with idiopathic left ventricular systolic dysfunction. The risk of MVA in male and female RBM20 variant carriers is similar, but male sex is strongly associated with ESHF

Truncating FLNC Mutations Are Associated With High-Risk Dilated and Arrhythmogenic Cardiomyopathies

BACKGROUND: Filamin C (encoded by the FLNC gene) is essential for sarcomere attachment to the plasmatic membrane. FLNC mutations have been associated with myofibrillar myopathies, and cardiac involvement has been reported in some carriers. Accordingly, since 2012, the authors have included FLNC in the genetic screening of patients with inherited cardiomyopathies and sudden death. OBJECTIVES: The aim of this study was to demonstrate the association between truncating mutations in FLNC and the development of high-risk dilated and arrhythmogenic cardiomyopathies. METHODS: FLNC was studied using next-generation sequencing in 2,877 patients with inherited cardiovascular diseases. A characteristic phenotype was identified in probands with truncating mutations in FLNC. Clinical and genetic evaluation of 28 affected families was performed. Localization of filamin C in cardiac tissue was analyzed in patients with truncating FLNC mutations using immunohistochemistry. RESULTS: Twenty-three truncating mutations were identified in 28 probands previously diagnosed with dilated, arrhythmogenic, or restrictive cardiomyopathies. Truncating FLNC mutations were absent in patients with other phenotypes, including 1,078 patients with hypertrophic cardiomyopathy. Fifty-four mutation carriers were identified among 121 screened relatives. The phenotype consisted of left ventricular dilation (68%), systolic dysfunction (46%), and myocardial fibrosis (67%); inferolateral negative T waves and low QRS voltages on electrocardiography (33%); ventricular arrhythmias (82%); and frequent sudden cardiac death (40 cases in 21 of 28 families). Clinical skeletal myopathy was not observed. Penetrance was >97% in carriers older than 40 years. Truncating mutations in FLNC cosegregated with this phenotype with a dominant inheritance pattern (combined logarithm of the odds score: 9.5). Immunohistochemical staining of myocardial tissue showed no abnormal filamin C aggregates in patients with truncating FLNC mutations. CONCLUSIONS: Truncating mutations in FLNC caused an overlapping phenotype of dilated and left-dominant arrhythmogenic cardiomyopathies complicated by frequent premature sudden death. Prompt implantation of a cardiac defibrillator should be considered in affected patients harboring truncating mutations in FLNC.Instituto de Salud Carlos III [PI11/0699, PI14/0967, PI14/01477, RD012/0042/0029, RD012/0042/0049, RD012/0042/0066, RD12/0042/0069]; Spanish Ministry of Economy and Competitiveness [SAF2015-71863-REDT]; Plan Nacional de I+D+I; Plan Estatalde I+D+I, European Regional Development Fund; Health in Code SLS

Importance of genotype for risk stratification in arrhythmogenic right ventricular cardiomyopathy using the 2019 ARVC risk calculator

none41siTo study the impact of genotype on the performance of the 2019 risk model for arrhythmogenic right ventricular cardiomyopathy (ARVC).Protonotarios, Alexandros; Bariani, Riccardo; Cappelletto, Chiara; Pavlou, Menelaos; García-García, Alba; Cipriani, Alberto; Protonotarios, Ioannis; Rivas, Adrian; Wittenberg, Regitze; Graziosi, Maddalena; Xylouri, Zafeirenia; Larrañaga-Moreira, José M; de Luca, Antonio; Celeghin, Rudy; Pilichou, Kalliopi; Bakalakos, Athanasios; Lopes, Luis Rocha; Savvatis, Konstantinos; Stolfo, Davide; Dal Ferro, Matteo; Merlo, Marco; Basso, Cristina; Freire, Javier Limeres; Rodriguez-Palomares, Jose F; Kubo, Toru; Ripoll-Vera, Tomas; Barriales-Villa, Roberto; Antoniades, Loizos; Mogensen, Jens; Garcia-Pavia, Pablo; Wahbi, Karim; Biagini, Elena; Anastasakis, Aris; Tsatsopoulou, Adalena; Zorio, Esther; Gimeno, Juan R; Garcia-Pinilla, Jose Manuel; Syrris, Petros; Sinagra, Gianfranco; Bauce, Barbara; Elliott, Perry MProtonotarios, Alexandros; Bariani, Riccardo; Cappelletto, Chiara; Pavlou, Menelaos; García-García, Alba; Cipriani, Alberto; Protonotarios, Ioannis; Rivas, Adrian; Wittenberg, Regitze; Graziosi, Maddalena; Xylouri, Zafeirenia; Larrañaga-Moreira, José M; de Luca, Antonio; Celeghin, Rudy; Pilichou, Kalliopi; Bakalakos, Athanasios; Lopes, Luis Rocha; Savvatis, Konstantinos; Stolfo, Davide; Dal Ferro, Matteo; Merlo, Marco; Basso, Cristina; Freire, Javier Limeres; Rodriguez-Palomares, Jose F; Kubo, Toru; Ripoll-Vera, Tomas; Barriales-Villa, Roberto; Antoniades, Loizos; Mogensen, Jens; Garcia-Pavia, Pablo; Wahbi, Karim; Biagini, Elena; Anastasakis, Aris; Tsatsopoulou, Adalena; Zorio, Esther; Gimeno, Juan R; Garcia-Pinilla, Jose Manuel; Syrris, Petros; Sinagra, Gianfranco; Bauce, Barbara; Elliott, Perry