13 results on '"Antonio Niro"'
Search Results
2. Integrated Genomic, Functional, and Prognostic Characterization of Atypical Chronic Myeloid Leukemia
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Diletta Fontana, Daniele Ramazzotti, Andrea Aroldi, Sara Redaelli, Vera Magistroni, Alessandra Pirola, Antonio Niro, Luca Massimino, Cristina Mastini, Virginia Brambilla, Silvia Bombelli, Silvia Bungaro, Alessandro Morotti, Delphine Rea, Fabio Stagno, Bruno Martino, Leonardo Campiotti, Giovanni Caocci, Emilio Usala, Michele Merli, Francesco Onida, Marco Bregni, Elena Maria Elli, Monica Fumagalli, Fabio Ciceri, Roberto A. Perego, Fabio Pagni, Luca Mologni, Rocco Piazza, and Carlo Gambacorti-Passerini
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Abstract. Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.
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- 2020
- Full Text
- View/download PDF
3. Whole brain delivery of an instability-prone Mecp2 transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome
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Mirko Luoni, Serena Giannelli, Marzia Tina Indrigo, Antonio Niro, Luca Massimino, Angelo Iannielli, Laura Passeri, Fabio Russo, Giuseppe Morabito, Piera Calamita, Silvia Gregori, Benjamin Deverman, and Vania Broccoli
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intellectual disabilities ,AAV ,gene therapy ,neuronal disease ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of iMecp2 provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.
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- 2020
- Full Text
- View/download PDF
4. Whole brain delivery of an instability-prone Mecp2 transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome
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Angelo Iannielli, Laura Passeri, Benjamin E. Deverman, Mirko Luoni, Fabio Russo, Piera Calamita, Serena Giannelli, Silvia Gregori, Luca Massimino, Giuseppe Morabito, Antonio Niro, Vania Broccoli, and Marzia Indrigo
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0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,Mouse ,QH301-705.5 ,Methyl-CpG-Binding Protein 2 ,Science ,Transgene ,Genetic enhancement ,Mutant ,Gene Expression ,Rett syndrome ,Mice, Transgenic ,Biology ,neuronal disease ,General Biochemistry, Genetics and Molecular Biology ,MECP2 ,03 medical and health sciences ,0302 clinical medicine ,Neurodevelopmental disorder ,Gene duplication ,Gene expression ,mental disorders ,medicine ,Rett Syndrome ,Animals ,Transgenes ,Biology (General) ,General Immunology and Microbiology ,General Neuroscience ,Brain ,AAV ,General Medicine ,Genetic Therapy ,medicine.disease ,gene therapy ,nervous system diseases ,Disease Models, Animal ,030104 developmental biology ,Gene Expression Regulation ,Mutation ,Cancer research ,Medicine ,intellectual disabilities ,030217 neurology & neurosurgery ,Research Article ,Neuroscience - Abstract
Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of iMecp2 provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.
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- 2020
5. ETNK1 mutations in atypical chronic myeloid leukemia induce a mutator phenotype that can be reverted with phosphoethanolamine
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Clizia Chinello, Giovanni Zambrotta, Francesca Fanelli, Roberta Corti, Mi Jang, Steen Larsen, Carlo Gambacorti-Passerini, Rossella Renso, Rocco Piazza, Barbara Crescenzi, Mattia Docci, Lisa Marie Røst, Mayla Bertagna, Cristina Mecucci, Stefania Citterio, Deborah D'Aliberti, Francesco Mantegazza, Fulvio Magni, Ilaria Crespiatico, Guido Cavaletti, Antonio Niro, Diletta Fontana, Delphine Rea, Valeria Cassina, Mario Mauri, Domenico Salerno, Per Bruheim, M Bossi, Luca Nardo, Luca Massimino, Fontana, D, Mauri, M, Renso, R, Docci, M, Crespiatico, I, Rost, L, Jang, M, Niro, A, D'Aliberti, D, Massimino, L, Bertagna, M, Zambrotta, G, Bossi, M, Citterio, S, Crescenzi, B, Fanelli, F, Cassina, V, Corti, R, Salerno, D, Nardo, L, Chinello, C, Mantegazza, F, Mecucci, C, Magni, F, Cavaletti, G, Bruheim, P, Rea, D, Larsen, S, Piazza, R, and Gambacorti-Passerini, C
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business.industry ,Kinase ,Somatic cell ,Immunology ,Mutator phenotype ,Chronic myelomonocytic leukemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,medicine ,Atypical chronic myeloid leukemia ,Cancer research ,Eosinophilia ,Phosphorylation ,Systemic mastocytosis ,medicine.symptom ,business ,ETNK1, chronic myeloid leukemia - Abstract
ETNK1 kinase is responsible for the phosphorylation of ethanolamine to phosphoethanolamine (P-Et) (Kennedy, 1956, J Biol Chem). Recurrent somatic mutations occurring on ETNK1 were identified in about 13% of patients affected by atypical chronic myeloid leukemia (aCML), in 3-14% of chronic myelomonocytic leukemia (CMML), and in 20% of systemic mastocytosis (SM) patients with eosinophilia (Gambacorti-Passerini, 2015, Blood; Lasho, 2015, Blood Cancer J). ETNK1 mutations, encoding for H243Y, N244S/T/K, and G245V/A amino acid substitutions, cluster in a very narrow region of the ETNK1 catalytic domain and cause an impairment of ETNK1 enzymatic activity leading to a significant decrease in the intracellular concentration of P-Et (Gambacorti-Passerini, 2015, Blood). Despite this evidence, however, their oncogenic role remained largely unexplained. Here, we investigated the specific role of these mutations by using cellular CRISPR/Cas9 and ETNK1 overexpression models as well as aCML patients' samples. We showed that mutated ETNK1 causes a significant increase in mitochondrial activity (1.87 fold increase compared to WT; p=0.0002) and in ROS production (2.05 fold increase compared to WT; p Disclosures Rea: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.
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- 2020
6. ETNK1 mutations induce a mutator phenotype that can be reverted with phosphoethanolamine
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Francesca Fanelli, Steen Larsen, Stefania Citterio, Lisa Marie Røst, Roberta Corti, Barbara Crescenzi, Mattia Docci, Deborah D'Aliberti, Mi Jang, Delphine Rea, Rocco Piazza, Clizia Chinello, Mayla Bertagna, Guido Cavaletti, Fulvio Magni, Antonio Niro, M Bossi, Ilaria Crespiatico, Rossella Renso, Carlo Gambacorti-Passerini, Giovanni Zambrotta, Francesco Mantegazza, Domenico Salerno, Luca Massimino, Mario Mauri, Per Bruheim, Luca Nardo, Valeria Cassina, Cristina Mecucci, Diletta Fontana, Fontana, D, Mauri, M, Renso, R, Docci, M, Crespiatico, I, Rost, L, Jang, M, Niro, A, D'Aliberti, D, Massimino, L, Bertagna, M, Zambrotta, G, Bossi, M, Citterio, S, Crescenzi, B, Fanelli, F, Cassina, V, Corti, R, Salerno, D, Nardo, L, Chinello, C, Mantegazza, F, Mecucci, C, Magni, F, Cavaletti, G, Bruheim, P, Rea, D, Larsen, S, Gambacorti-Passerini, C, and Piazza, R
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0301 basic medicine ,Myeloid ,Cancer therapy ,Somatic cell ,Succinic Acid ,General Physics and Astronomy ,Cell Line ,Cell Respiration ,DNA Breaks ,Electron Transport Complex II ,Ethanolamines ,Humans ,Leukemia, Myeloid ,Mitochondria ,Mutation ,Phenotype ,Phosphotransferases (Alcohol Group Acceptor) ,Reactive Oxygen Species ,Tigecycline ,Mitochondrion ,medicine.disease_cause ,MITOCHONDRIAL ,0302 clinical medicine ,PHOSPHATIDYLETHANOLAMINE ,Ethanolamine ,PHOSPHOMETABOLOME ,Multidisciplinary ,Leukemia ,Hyperactivation ,Molecular medicine ,Chemistry ,DNA Break ,Cell biology ,030220 oncology & carcinogenesis ,Phosphorylation ,Reactive Oxygen Specie ,Intracellular ,Human ,DNA damage ,Science ,HIGH-THROUGHPUT ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,medicine ,TANDEM MASS-SPECTROMETRY ,PATHWAYS ,LEUKEMIA ,LIPIDOME ,REPAIR ,Haematological cancer ,General Chemistry ,030104 developmental biology - Abstract
Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype., ETNK1 mutations are recurrent in leukemia but how they contribute to oncogenesis is still unclear. Here, the authors show that ETNK1 mutations increase mitochondrial activity, ROS and H2AX levels and that these effects can be rescued upon phosphoethanolamine supplementation.
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- 2020
7. Author response: Whole brain delivery of an instability-prone Mecp2 transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome
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Mirko Luoni, Serena Giannelli, Marzia Tina Indrigo, Antonio Niro, Luca Massimino, Angelo Iannielli, Laura Passeri, Fabio Russo, Giuseppe Morabito, Piera Calamita, Silvia Gregori, Benjamin Deverman, and Vania Broccoli
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- 2019
8. Whole brain delivery of an instability-proneMecp2transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome
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Luca Massimino, Piera Calamita, Silvia Gregori, Benjamin E. Deverman, Angelo Iannielli, Serena Giannelli, Mirko Luoni, Antonio Niro, Marzia Indrigo, Vania Broccoli, Fabio Russo, Giuseppe Morabito, and Laura Passeri
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congenital, hereditary, and neonatal diseases and abnormalities ,business.industry ,Transgene ,Genetic enhancement ,Mutant ,Rett syndrome ,medicine.disease ,MECP2 ,nervous system diseases ,Neurodevelopmental disorder ,Gene expression ,Gene duplication ,mental disorders ,medicine ,Cancer research ,business - Abstract
Rett syndrome (RTT) is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles sinceMECP2is expressed throughout the brain and its duplication leads to severe neurological conditions as well. However, the recent introduction of AAV-PHP.eB, an engineered capsid with an unprecedented efficiency in crossing the blood-brain barrier upon intravenous injection, has provided an invaluable vehicle for gene transfer in the mouse nervous system. Herein, we use AAV-PHP.eB to deliver an instability-proneMecp2(iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viralMecp2transgene, limits supraphysiological Mecp2 protein levels in transduced neural tissues. Intravenous injections of the PHP.eB-iMecp2virus in symptomatic male and femaleMecp2mutant mice significantly ameliorated the disease progression with improved locomotor activity, coordination, lifespan and normalization of altered gene expression and mTOR signaling. Remarkably, PHP.eB-iMecp2administration did not result in severe toxicity effects either in femaleMecp2mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated maleMecp2mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of theiMecp2cassette provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain. This combination defines a novel viral system with significant therapeutic efficacy and increased safety which can contribute to overcome the hurdles that are delaying clinical applications of gene therapy for RTT.One Sentence SummaryGlobal brain transduction of the instability-proneMecp2transgene by systemic AAV-PHP.eB administration is both safe and effective in protecting male and femaleMecp2mutant mice from the RTT disease phenotype.
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- 2019
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9. Integrated Genomic, Functional and Prognostic Characterization of Atypical Chronic Myeloid Leukemia (aCML) in a Cohort of 43 Patients
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Elena Maria Elli, Bruno Martino, Diletta Fontana, Andrea Aroldi, Daniele Ramazzotti, Delphine Rea, Fabio Ciceri, Fabio Stagno, Leonardo Campiotti, Antonio Niro, Monica Fumagalli, Luca Massimino, Rocco Piazza, Carlo Gambacorti-Passerini, Giovanni Caocci, Fontana, D, Ramazzotti, D, Aroldi, A, Niro, A, Massimino, L, Rea, D, Stagno, F, Martino, B, Campiotti, L, Caocci, G, Elli, E, Fumagalli, M, Ciceri, F, Piazza, R, and GAMBACORTI PASSERINI, C
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Oncology ,medicine.medical_specialty ,business.industry ,Genetic heterogeneity ,Immunology ,Cell Biology ,Hematology ,Disease ,medicine.disease ,Biochemistry ,Somatic evolution in cancer ,Atypical chronic myeloid leukemia (aCML), philadelphia-negative myelogenous leukemia, sequence analysis, rna, cancer, gene expression profiling, leukemogenesis, myelodysplastic syndrome, myeloproliferative disease ,Uniparental disomy ,Gene expression profiling ,Log-rank test ,Internal medicine ,medicine ,Atypical chronic myeloid leukemia ,business ,Survival analysis - Abstract
Atypical chronic myeloid leukemia (aCML) is a rare BCR-ABL1 negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in several genes including SETBP1, ASXL1 and ETNK1, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. The clinical prognosis for aCML is poor, with a median overall survival of 18 months after diagnosis, and no established standards of care exist for its treatment. The dissection of the molecular processes underlying aCML leukemogenesis could therefore result decisive in ameliorating the prognosis for aCML. With the aim to provide a comprehensive genomic characterization of aCML and to link the detected alterations with the clinical course of the disease, we applied a high-throughput sequencing strategy to 43 aCML samples, including whole-exome sequencing and RNA sequencing. Our study confirms ASXL1 and SETBP1 as the most frequently mutated genes with a total of 43.2% and 30.2%, respectively; ETNK1 mutations are observed in 14% of patients. An average of 2 mutations per patient was observed [range: 0-5]. We characterized the clonal architecture in a subset of 8 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur very early in the clonal evolution history of aCML, while SETBP1 mutations represent a late event; interestingly, in the two cases where ASXL1 was mutated together with SETBP1, its mutations occupied an intermediate hierarchical position. CBL mutations, when present, showed a tendency toward reaching homozygosity through somatic uniparental disomy. Stratification based on RNA-sequencing gene expression data (Ramazzotti, Daniele, et al. Nature communications 9.1 (2018): 4453) identified two clearly different populations (26 and 17 patients) in terms of Overall Survival (OS), with 2 year OS of 69.23% [95% IC: 48.21%-86.67%] and 35.29% [95% IC: 14.21%-61.67%] respectively (logrank test for trend: p=0.004, Fig. 1A). In addition, the group with better prognosis showed a higher frequency of ETNK1 mutations (hypergeometric test: p=0.032). We next performed differential gene expression analysis to detect genes differentially expressed between the two patients' populations. This analysis revealed 38 significant genes (t-test p-value adjusted for false discovery rate p In conclusion, we present here the first description of a large aCML cohort, in which sequencing data, clonal hierarchy of mutations and gene expression profiles were integrated through bioinformatics analysis. RNA-sequencing data stratification characterizes two groups with different prognosis; a classifier based on the 4 top differently expressed genes accurately predicts patients' outcome. Figure 1. A) Overall Survival curve (Kaplan-Meier curve) at 24 months shows significant different outcomes (p=0.004). B) Random forest classifiers learn multiple decision trees in order to predict outcomes. In the figure, an example of decision tree where nodes are genes and leaves are outcomes (better/worse prognosis). C) Heatmap of expression fold change for the top four differentially expressed genes. Figure 1 Disclosures Rea: BMS: Honoraria; Incyte Biosciences: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Stagno:Pfizer: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Elli:Novartis: Membership on an entity's Board of Directors or advisory committees. Gambacorti-Passerini:Pfizer: Honoraria, Research Funding; Bristol-Meyers Squibb: Consultancy.
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- 2019
10. Integrated Genomic, Functional and Prognostic Characterization of Atypical Chronic Myeloid Leukemia (aCML) in a Cohort of 43 Patients
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Fontana, D, Ramazzotti, D, Aroldi, A, Niro, A, Massimino, L, Rea, D, Stagno, F, Martino, B, Campiotti, L, Caocci, G, Elli, E, Fumagalli, M, Ciceri, F, Piazza, R, GAMBACORTI PASSERINI, C, Diletta Fontana, Daniele Ramazzotti, Andrea Aroldi, Antonio Niro, Luca Massimino, Delphine Rea, Fabio Stagno, Bruno Martino, Leonardo Campiotti, Giovanni Caocci, Elena Maria Elli, Monica Fumagalli, Fabio Ciceri, Rocco Piazza, Carlo Gambacorti-Passerini, Fontana, D, Ramazzotti, D, Aroldi, A, Niro, A, Massimino, L, Rea, D, Stagno, F, Martino, B, Campiotti, L, Caocci, G, Elli, E, Fumagalli, M, Ciceri, F, Piazza, R, GAMBACORTI PASSERINI, C, Diletta Fontana, Daniele Ramazzotti, Andrea Aroldi, Antonio Niro, Luca Massimino, Delphine Rea, Fabio Stagno, Bruno Martino, Leonardo Campiotti, Giovanni Caocci, Elena Maria Elli, Monica Fumagalli, Fabio Ciceri, Rocco Piazza, and Carlo Gambacorti-Passerini
- Abstract
Atypical chronic myeloid leukemia (aCML) is a rare BCR-ABL1 negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in several genes including SETBP1, ASXL1 and ETNK1, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. The clinical prognosis for aCML is poor, with a median overall survival of 18 months after diagnosis, and no established standards of care exist for its treatment. The dissection of the molecular processes underlying aCML leukemogenesis could therefore result decisive in ameliorating the prognosis for aCML. With the aim to provide a comprehensive genomic characterization of aCML and to link the detected alterations with the clinical course of the disease, we applied a high-throughput sequencing strategy to 43 aCML samples, including whole-exome sequencing and RNA sequencing. Our study confirms ASXL1 and SETBP1 as the most frequently mutated genes with a total of 43.2% and 30.2%, respectively; ETNK1 mutations are observed in 14% of patients. An average of 2 mutations per patient was observed [range: 0-5]. We characterized the clonal architecture in a subset of 8 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur very early in the clonal evolution history of aCML, while SETBP1 mutations represent a late event; interestingly, in the two cases where ASXL1 was mutated together with SETBP1, its mutations occupied an intermediate hierarchical position. CBL mutations, when present, showed a tendency toward reaching homozygosity through somatic uniparental disomy. Stratification based on RNA-sequencing gene expression data (Ramazzotti, Daniele, et al. Nature communications 9.1 (2018): 4453) identified two clearly different populations (26 and 17 patients) in terms of Overall Survival (OS), with 2 year OS of 69.23% [95% IC: 48.21%-86.67%] and 35.29% [95% IC: 14.21%-61.67%] res
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- 2019
11. Integrated Genomic, Functional, and Prognostic Characterization of Atypical Chronic Myeloid Leukemia
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Emilio Usala, Giovanni Caocci, Daniele Ramazzotti, Alessandra Pirola, Leonardo Campiotti, Alessandro Morotti, Fabio Stagno, Roberto A. Perego, Silvia Bungaro, Cristina Mastini, Fabio Pagni, Silvia Bombelli, Fabio Ciceri, Vera Magistroni, Bruno Martino, Michele Merli, Antonio Niro, Sara Redaelli, Delphine Rea, Luca Mologni, Rocco Piazza, Diletta Fontana, Virginia Brambilla, Elena Maria Elli, Andrea Aroldi, Carlo Gambacorti-Passerini, Marco Bregni, Luca Massimino, Monica Fumagalli, Francesco Onida, Fontana, D, Ramazzotti, D, Aroldi, A, Redaelli, S, Magistroni, V, Pirola, A, Niro, A, Massimino, L, Mastini, C, Brambilla, V, Bombelli, S, Bungaro, S, Morotti, A, Rea, D, Stagno, F, Martino, B, Campiotti, L, Caocci, G, Usala, E, Merli, M, Onida, F, Bregni, M, Elli, E, Fumagalli, M, Ciceri, F, Perego, R, Pagni, F, Mologni, L, Piazza, R, and Gambacorti-Passerini, C
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Genetics ,lcsh:RC633-647.5 ,Somatic cell ,Genetic heterogeneity ,SETBP1 ,Whole exome sequencing ,lcsh:Diseases of the blood and blood-forming organs ,Hematology ,Disease ,ASXL1 ,Biology ,medicine.disease ,Somatic evolution in cancer ,MED/15 - MALATTIE DEL SANGUE ,In vivo ,Gene expression ,Atypical chronic myeloid leukemia ,medicine ,Original Article ,ETNK1 ,Gene ,Atypical Chronic Myeloid Leukemia - Abstract
Supplemental Digital Content is available for this article., Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.
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- 2020
12. Abstract 3385: ETNK1 mutations promote ROS production and DNA damage through increased mitochondrial activity
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Giovanni Signore, Rocco Piazza, Mayla Bertagna, Carlo Gambacorti-Passerini, Mario Mauri, Stefania Citterio, Guido Cavaletti, Luca Massimino, Antonio Niro, Giovanni Zambrotta, Diletta Fontana, Vincenzo Piazza, Barbara Crescenzi, M Bossi, Delphine Rea, and Cristina Mecucci
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chemistry.chemical_classification ,Cancer Research ,Mutation ,Mutation rate ,DNA damage ,Somatic cell ,Mitochondrion ,Biology ,medicine.disease_cause ,medicine.disease ,Molecular biology ,Enzyme ,Oncology ,chemistry ,medicine ,Atypical chronic myeloid leukemia ,Inner membrane - Abstract
Atypical chronic myeloid leukemia (aCML) is a clonal disorder belonging to the myelodysplastic/myeloproliferative syndromes. About 13% of aCML cases carry somatic mutations in ETNK1 gene, encoding for H243Y, N244S, and G245V substitutions. We previously showed that ETNK1 mutations cause a decreased catalytic activity of the enzyme. Despite this evidence however, their oncogenic role remained largely unexplained. Since ETNK1 activity is essential for the synthesis of phosphatidylethanolamine (PE) and given that PE is one of the most abundant phospholipids in the inner membrane of mitochondria, we focused our attention on mitochondrial activity. In order to characterize the oncogenic effect of ETNK1 variants we generated CRISPR/Cas9 clones carrying heterozygous N244S mutation and homozygous ETNK1 deletion (KO cells) on the 293 Flp-In™ cell-line. Both N244S and KO cells showed a significant increase in mitochondrial activity (1.78 and 2.13 fold increase, respectively; p= 0.0096 and p=0.0050) compared to WT, as assessed by MitoTracker™ Red. In line with this finding, electron microscopy revealed a significant modification in mitochondria morphology for N244S and KO cells, changing from an elongated, tubular shape to a round, swollen one. ATP (1.67 and 1.68 fold; p In line with these data, a higher mutation rate was detected in N244S and KO cells (8.09*10-7±9.6*10-8 and 8.20*10-7±1.28*10-7; p=0.0060 and p=0.0264) compared to WT (2.98*10-7±8.2*10-8) by 6-thioguanine assay. The reconstruction of the hierarchy of somatic mutations in ETNK1-mutated aCML patients revealed that ETNK1 variants invariably occur very early in the evolution history of the aCML patients. Taken together, our results show that impairment of ETNK1 function causes an increase in mitochondrial activity, which in turn leads to increased ROS production driving the accumulation of DNA mutations. Since the characterization of aCML subclonal architecture indicates ETNK1 mutations as a very early event in the history of the disease, we hypothesize that ETNK1 could contribute to the onset of aCML through the activation of a mutant phenotype, which in turn would accelerate the accumulation of further oncogenic mutations. Citation Format: Diletta Fontana, Mario Mauri, Antonio Niro, Luca Massimino, Mayla Bertagna, Giovanni Zambrotta, Mario Bossi, Stefania Citterio, Barbara Crescenzi, Giovanni Signore, Vincenzo Piazza, Cristina Mecucci, Guido Cavaletti, Delphine Rea, Carlo Gambacorti-Passerini, Rocco Piazza. ETNK1 mutations promote ROS production and DNA damage through increased mitochondrial activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3385.
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- 2018
13. ETNK1 is an early event and SETBP1 a late event in atypical Chronic Myeloid Leukemia
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Sara Redaelli, Rocco Piazza, Francesca Farina, Carlo Gambacorti-Passerini, Gabriele Merati, Antonio Niro, Diletta Fontana, Caterina Mezzatesta, Alessandra Pirola, Delphine Rea, Rossella Renso, Carla Donadoni, Niro, A, Piazza, R, Merati, G, Pirola, A, Donadoni, C, Fontana, D, Redaelli, S, Mezzatesta, C, Renso, R, Farina, F, Rea, D, and Gambacorti-Passerini, C
- Subjects
Genetics ,Neuroblastoma RAS viral oncogene homolog ,ETNK1 SETPB1 ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Philadelphia chromosome ,Biochemistry ,Molecular biology ,Somatic evolution in cancer ,Uniparental disomy ,medicine ,Atypical chronic myeloid leukemia ,Exome ,Exome sequencing - Abstract
Atypical Chronic Myeloid Leukemia (aCML) is a clonal disorder belonging to the myelodisplastic-myeloproliferative neoplasms, according to the WHO-2008 classification. From a clinical point of view it closely resembles the classical Chronic Myeloid Leukemia (CML), however it lacks the presence of the Philadelphia chromosome and of the BCR-ABL1 fusion gene. In recent works, we and others characterized the somatic lesions present in the aCML genome, mainly by using Next Generation Sequencing (NGS) technologies, demonstrating the presence of a large set of recurrent somatic mutations involving, among the others, SETBP1, ETNK1, ASXL1, EZH2, CBL, TET2, NRAS and U2AF1 genes. The identification of somatic variants occurring in a large number of genes clearly indicates that the genetic bases of aCML are very heterogeneous, in striking contrast with classical CML. This heterogeneity poses a great challenge to the dissection of the molecular steps required for aCML leukemogenesis. The hierarchical reconstruction of the different mutations occurring in a clonal disorder can have important biological, prognostic and therapeutic repercussions; therefore we started a project focused on the dissection of the aCML clonal evolution steps through the analysis of individual leukemic clones by methylcellulose assays in samples whose mutational status has been previously characterized by matched whole-exome sequencing. Patient CMLPh-019 was characterized by the presence of a complex mutational status, with somatic variants occurring in SETBP1, ETNK1, ASXL1 and CBL genes (Fig. 1a). Targeted resequencing analysis of individual clones revealed the presence of all the 4 variants in 44/60 (73.3%) clones; in 15/60 (25%) we detected the presence of mutated ETNK1, ASXL1 and CBL and wild-type (WT) SETBP1. Of these 15 clones, 33% carried heterozygous and 67% homozygous CBL mutations. In one clone (1.7%) we detected heterozygous ETNK1, homozygous CBL and WT sequences for ASXL1 and SETBP1, suggesting a strong selective pressure towards the acquisition of homozygous CBL mutations. Identification of homozygous CBL mutations in all the main clonal phases suggests that a significant positive selective pressure is associated with this event. Allelic imbalance analysis of CMLPh-019 exome using CEQer revealed that CBL homozygosity is caused by a somatic uniparental disomy event occurring in the telomeric region of the long arm of chromosome 11. Patient CMLPh-005 (Fig. 1b) was mutated in ASXL1, CBL and SETBP1. Targeted analysis done on 68 clones revealed a complex, branching evolution, with 63 clones carrying all the 3 variants. Of them, 47 (74.6%) had a heterozygous and 16 (25.4%) a homozygous CBL variant. Four clones (4.2%) carried ASXL1 and SETBP1 but not CBL mutations, while 1 clone was mutated in ASXL1 and CBL in absence of SETBP1 mutations, which suggests that CBL mutations occurred independently in two different subclones. Also in this case, allelic imbalance analysis of exome data revealed that CBL homozygosity was caused by a telomeric somatic uniparental disomy event. According to exome sequencing, patient CMLPh-003 carried SETBP1 mutation G870S and NRAS variant G12R. Clonal analysis confirmed the presence of SETBP1 G870S in all the clones analyzed, while heterozygous NRAS G12R mutation was detected in 67% (Fig. 1c). Notably in the remaining 33% another heterozygous NRAS variant, G12D, was detected. Retrospective reanalysis of exome data confirmed the presence of the newly identified variant, which had been previously filtered-out from exome data because of the low frequency. Patient CMLPh-013 was mutated in ASXL1, ETNK1, NRAS and SETBP1. Of the 39 clones analyzed, 34 (82.9%) showed the coexistence of ASXL1, ETNK1, NRAS and SETBP1, 4 were mutated in ASXL1, ETNK1 and NRAS and 1 in ETNK1 and NRAS, suggesting that ETNK1 and NRAS were early events, ASXL1 an intermediate one and SETBP1 a late variant (Fig. 1d). Taken globally, these data indicate that ETNK1 variants occur very early in the clonal evolution history of aCML, while ASXL1 represents an early/intermediate event and SETBP1 is often a late event. They also suggest that, in the context of aCML, there is a strong selective pressure towards the accumulation of homozygous CBL variants, as already shown in other leukemias. Figure 1. Clonal analysis of four aCML cases. The asterisks indicate hypothetical clones. Figure 1. Clonal analysis of four aCML cases. The asterisks indicate hypothetical clones. Disclosures Rea: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria.
- Published
- 2015
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