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1
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Petazzi P, Gutierrez-Agüera F, Roca-Ho H, Castaño J, Bueno C, Alvarez N, Forrester LM, Sevilla A, Fidanza A, Menendez P. Generation of an inducible dCas9-SAM human PSC line for endogenous gene activation. Front Cell Dev Biol 2024; 12:1484955. [PMID: 39676795 PMCID: PMC11638181 DOI: 10.3389/fcell.2024.1484955] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2024] [Accepted: 11/11/2024] [Indexed: 12/17/2024] Open
Abstract
The CRISPR/Cas9 system has transformed genome editing by enabling precise modifications for diverse applications. Recent advancements, including base editing and prime editing, have expanded its utility beyond conventional gene knock-out and knock-in strategies. Additionally, several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains have been developed to modulate endogenous gene expression when directed to their regulatory regions by specific single-guide RNAs. Here, we report the development of the H9 human pluripotent stem cell (hPSC) line expressing an inducible dCas9-SAM activator (H9-iCas9.SAM), designed to activate transcription of endogenous genes. The H9-iCas9.SAM cells were generated through targeted integration of an inducible CRISPR/Cas9-based gene activator cassette into the AAVS1 "safe-harbour" locus. Molecular analyses confirmed precise and specific integration, ensuring minimal off-target effects. Functional characterization revealed that H9-iCas9.SAM cells retain pluripotency and display inducible endogenous gene activation upon doxycycline treatment. The versatility of H9-iCas9.SAM cells was demonstrated in directed in vitro differentiation assays, yielding neural stem cells (ectoderm), hematopoietic progenitor cells (mesoderm), and hepatocytes (endoderm). This underscores their potential in developmental biology studies and cell therapy applications. The engineered H9-iCas9.SAM line provides a robust platform for investigating gene function and advancing next-generation cell-based therapies.
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Affiliation(s)
- Paolo Petazzi
- Josep Carreras Leukemia Research Institute, Campus Clinic-UB, Casanova 143, Barcelona, Spain
| | | | - Heleia Roca-Ho
- Josep Carreras Leukemia Research Institute, Campus Clinic-UB, Casanova 143, Barcelona, Spain
| | - Julio Castaño
- Josep Carreras Leukemia Research Institute, Campus Clinic-UB, Casanova 143, Barcelona, Spain
| | - Clara Bueno
- Josep Carreras Leukemia Research Institute, Campus Clinic-UB, Casanova 143, Barcelona, Spain
- Spanish Network for Advanced Cell Therapies (TERAV), Carlos III Health Institute, Barcelona, Spain
- Spanish Cancer Network (CIBERONC), Carlos III Health Institute, Barcelona, Spain
| | - Niuska Alvarez
- Department of Cell Biology, Physiology, and Immunology, Faculty of Biology, Institute of Neuroscience, University of Barcelona, Barcelona, Spain
| | - Lesley M Forrester
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, Edinburgh Medical School, Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom
| | - Ana Sevilla
- Department of Cell Biology, Physiology, and Immunology, Faculty of Biology, Institute of Neuroscience, University of Barcelona, Barcelona, Spain
- Institute of Biomedicine, University of Barcelona (IBUB), Barcelona, Spain
| | - Antonella Fidanza
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, Edinburgh Medical School, Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom
- Edinburgh Medical School, Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom
| | - Pablo Menendez
- Josep Carreras Leukemia Research Institute, Campus Clinic-UB, Casanova 143, Barcelona, Spain
- Spanish Network for Advanced Cell Therapies (TERAV), Carlos III Health Institute, Barcelona, Spain
- Spanish Cancer Network (CIBERONC), Carlos III Health Institute, Barcelona, Spain
- Department of Biomedicine, School of Medicine, Casanova 143, University of Barcelona, Barcelona, Spain
- Institució Catalana de Recerca I Estudis Avançats (ICREA), Barcelona, Spain
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2
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Closa A, Reixachs-Solé M, Fuentes-Fayos AC, Hayer K, Melero J, Adriaanse FRS, Bos R, Torres-Diz M, Hunger S, Roberts K, Mullighan C, Stam R, Thomas-Tikhonenko A, Castaño J, Luque R, Eyras E. A convergent malignant phenotype in B-cell acute lymphoblastic leukemia involving the splicing factor SRRM1. NAR Cancer 2022; 4:zcac041. [PMID: 36518527 PMCID: PMC9732526 DOI: 10.1093/narcan/zcac041] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Revised: 10/09/2022] [Accepted: 11/25/2022] [Indexed: 11/07/2024] Open
Abstract
A significant proportion of infant B-cell acute lymphoblastic leukemia (B-ALL) patients remains with a dismal prognosis due to yet undetermined mechanisms. We performed a comprehensive multicohort analysis of gene expression, gene fusions, and RNA splicing alterations to uncover molecular signatures potentially linked to the observed poor outcome. We identified 87 fusions with significant allele frequency across patients and shared functional impacts, suggesting common mechanisms across fusions. We further identified a gene expression signature that predicts high risk independently of the gene fusion background and includes the upregulation of the splicing factor SRRM1. Experiments in B-ALL cell lines provided further evidence for the role of SRRM1 on cell survival, proliferation, and invasion. Supplementary analysis revealed that SRRM1 potentially modulates splicing events associated with poor outcomes through protein-protein interactions with other splicing factors. Our findings reveal a potential convergent mechanism of aberrant RNA processing that sustains a malignant phenotype independently of the underlying gene fusion and that could potentially complement current clinical strategies in infant B-ALL.
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Affiliation(s)
- Adria Closa
- The Shine-Dalgarno Centre for RNA Innovation, John Curtin School of Medical Research, Australian National University, Canberra, Australia
- Centre for Computational Biomedical Sciences, John Curtin School of Medical Research, Australian National University, Canberra, Australia
- EMBL Australia Partner Laboratory Network at the Australian National University, Canberra, Australia
| | - Marina Reixachs-Solé
- The Shine-Dalgarno Centre for RNA Innovation, John Curtin School of Medical Research, Australian National University, Canberra, Australia
- Centre for Computational Biomedical Sciences, John Curtin School of Medical Research, Australian National University, Canberra, Australia
- EMBL Australia Partner Laboratory Network at the Australian National University, Canberra, Australia
| | - Antonio C Fuentes-Fayos
- Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Cordoba, Spain
- University of Cordoba (UCO), Cordoba, Spain
- Reina Sofía University Hospital, Cordoba, Spain
| | - Katharina E Hayer
- Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, USA
| | - Juan L Melero
- The Shine-Dalgarno Centre for RNA Innovation, John Curtin School of Medical Research, Australian National University, Canberra, Australia
- Centre for Computational Biomedical Sciences, John Curtin School of Medical Research, Australian National University, Canberra, Australia
- EMBL Australia Partner Laboratory Network at the Australian National University, Canberra, Australia
| | | | - Romy S Bos
- Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands
| | - Manuel Torres-Diz
- Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, USA
| | - Stephen P Hunger
- Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, USA
| | - Kathryn G Roberts
- Department of Pathology, St. Jude Children's Research Hospital, Memphis, USA
| | - Charles G Mullighan
- Department of Pathology, St. Jude Children's Research Hospital, Memphis, USA
| | - Ronald W Stam
- Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands
| | - Andrei Thomas-Tikhonenko
- Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, USA
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA
| | - Justo P Castaño
- Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Cordoba, Spain
- University of Cordoba (UCO), Cordoba, Spain
- Reina Sofía University Hospital, Cordoba, Spain
- Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición, (CIBERobn), Cordoba, Spain
| | - Raúl M Luque
- Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Cordoba, Spain
- University of Cordoba (UCO), Cordoba, Spain
- Reina Sofía University Hospital, Cordoba, Spain
- Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición, (CIBERobn), Cordoba, Spain
| | - Eduardo Eyras
- The Shine-Dalgarno Centre for RNA Innovation, John Curtin School of Medical Research, Australian National University, Canberra, Australia
- Centre for Computational Biomedical Sciences, John Curtin School of Medical Research, Australian National University, Canberra, Australia
- EMBL Australia Partner Laboratory Network at the Australian National University, Canberra, Australia
- Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain
- Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain
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3
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Panobinostat (LBH589) increase survival in adult xenografic model of acute lymphoblastic leukemia with t(4;11) but promotes antagonistic effects in combination with MTX and 6MP. MEDICAL ONCOLOGY (NORTHWOOD, LONDON, ENGLAND) 2022; 39:216. [PMID: 36175721 DOI: 10.1007/s12032-022-01813-w] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/25/2022] [Accepted: 07/29/2022] [Indexed: 10/14/2022]
Abstract
Patients diagnosed with acute lymphoblastic leukemia (ALL) bearing t(4;11)/MLL-AF4 have aggressive clinical features, poor prognosis and there is an urgent need for new therapies to improve outcomes. Panobinostat (LBH589) has been identified as a potential therapeutic agent for ALL with t(4;11) and studies suggest that the antineoplastic effects are associated with reduced MLL-AF4 fusion protein and reduced expression of HOX genes. Here, we evaluated the in vitro effects of the combination of LBH589 with methotrexate (MTX) or 6-mercaptopurine (6MP) by cell proliferation assays and Calcusyn software in ALL cell line (RS4;11); the in vivo effects of LBH589 in xenotransplanted NOD-scid IL2Rgammanull mice measuring human lymphoblasts by flow cytometry; and the expression of HOX genes by qPCR after treatment in an adult model of ALL with t(4;11). LBH589 combination with MTX or 6MP did not promote synergistic effects in RS4;11 cell line. LBH589 treatment leads to increased overall survival and reduction of blasts in xenotransplanted mice but caused no significant changes in HOXA7, HOXA9, HOXA10, and MEIS1 expression. The LBH589, alone, showed promising antineoplastic effects in vivo and may represent a potential agent for chemotherapy in ALL patients with t(4;11).
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Chen Y, Zheng Y, Hong Y, Wen J, Li J, Huang Y, Chen Y, Zheng X, Yang T, Xu Y, Zheng J, Hu J. Genomic heterogeneity contributed to different prognosis between adult and pediatric acute lymphoblastic. J Leukoc Biol 2022; 112:513-522. [PMID: 35172382 DOI: 10.1002/jlb.5a0721-361r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
The prognosis of acute lymphoblastic leukemia (ALL) in adults is inferior to that in children. Hence, ALL remains challenging to cure in the adult population. Aberrant genetic alterations have been observed in ALL, although the patterns of differential gene alterations in adult and pediatric ALL have not been comprehensively determined on a genome-wide scale. We investigated the biologic differences in genomic profiles between adults (n = 64) and children (n = 54) with ALL and relationship between genomic heterogeneity and prognosis. The 2 populations showed similar common mutation types but an increased prevalence of genetic alterations in adult ALL. The median numbers of gene mutations were 17 (range: 1-53) and 4.5 (range: 1-19) per sample in adult and pediatric ALL, respectively (p < 0.001). An increased number of gene mutations and age were significantly correlated (R2 = 0.5853, p < 0.001). We identified 122 and 53 driver genes in adult and pediatric ALL samples, respectively. IKZF1, IDH1, and TTN mutations were significantly enriched in adult patients with ALL. KRAS, ARID1A, and CREBBP mutations were significantly enriched in pediatric patients with ALL (p < 0.05). The incidence of relapse was 40.0% and 9.6% in adult and pediatric patients with ALL, respectively (p = 0.003). The overall survival and relapse-free survival of adult patients with ALL were poorer than those of pediatric patients with ALL (p = 0.002 and p < 0.001, respectively). This genomic landscape enhances the understanding of the biologic differences in ALL between the 2 populations and provides insight for developing therapeutic approaches.
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Affiliation(s)
- Yanxin Chen
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Yongzhi Zheng
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Yunda Hong
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Jingjing Wen
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Jiazheng Li
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Yan Huang
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Yi Chen
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Xiaoyun Zheng
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Ting Yang
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Yangqi Xu
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Jing Zheng
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
| | - Jianda Hu
- Fujian Institute of Hematology, Fujian Provincial Key Laboratory of Hematology, Fujian Medical University Union Hospital, Fuzhou, Fujian, China
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5
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Duguid A, Mattiucci D, Ottersbach K. Infant leukaemia - faithful models, cell of origin and the niche. Dis Model Mech 2021; 14:dmm049189. [PMID: 34713888 PMCID: PMC8560498 DOI: 10.1242/dmm.049189] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
For patients and their families, the diagnosis of infant leukaemia is devastating. This disease has not seen the improvements in outcomes experienced with other paediatric leukaemias and it is becoming ever more apparent that infant leukaemia is a distinct biological entity. Insights into some of the distinguishing features of infant leukaemia, such as a single mutation - the MLL-gene rearrangement, the biology of disease aggressiveness and lineage plasticity, and the high incidence of central nervous system involvement, are likely to be gained from understanding the interactions between leukaemic cells and their environment or niche. The origins of infant leukaemia lie in the embryonic haematopoietic system, which is characterised by shifting locations and dynamic changes in the microenvironment. Understanding this foetal or embryonic context is integral to understanding infant leukaemia development. Owing to its rarity and prenatal origins, developing accurate modelling systems for further investigation of infant leukaemia is essential. In this Review, we discuss how available in vitro, ex vivo and in vivo infant leukaemia models contribute to our current understanding of the leukaemia niche in embryonic development, established disease and specialised non-haematopoietic niches. The mechanistic insights provided by accurate models will help identify viable novel therapeutic options.
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Affiliation(s)
| | | | - Katrin Ottersbach
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK
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6
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Tejedor JR, Bueno C, Vinyoles M, Petazzi P, Agraz-Doblas A, Cobo I, Torres-Ruiz R, Bayón GF, Pérez RF, López-Tamargo S, Gutierrez-Agüera F, Santamarina-Ojeda P, Ramírez-Orellana M, Bardini M, Cazzaniga G, Ballerini P, Schneider P, Stam RW, Varela I, Fraga MF, Fernández AF, Menéndez P. Integrative methylome-transcriptome analysis unravels cancer cell vulnerabilities in infant MLL-rearranged B cell acute lymphoblastic leukemia. J Clin Invest 2021; 131:138833. [PMID: 33983906 DOI: 10.1172/jci138833] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2020] [Accepted: 05/11/2021] [Indexed: 01/04/2023] Open
Abstract
B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. As predicted by its prenatal origin, infant B-ALL (iB-ALL) shows an exceptionally silent DNA mutational landscape, suggesting that alternative epigenetic mechanisms may substantially contribute to its leukemogenesis. Here, we have integrated genome-wide DNA methylome and transcriptome data from 69 patients with de novo MLL-rearranged leukemia (MLLr) and non-MLLr iB-ALL leukemia uniformly treated according to the Interfant-99/06 protocol. iB-ALL methylome signatures display a plethora of common and specific alterations associated with chromatin states related to enhancer and transcriptional control in normal hematopoietic cells. DNA methylation, gene expression, and gene coexpression network analyses segregated MLLr away from non-MLLr iB-ALL and identified a coordinated and enriched expression of the AP-1 complex members FOS and JUN and RUNX factors in MLLr iB-ALL, consistent with the significant enrichment of hypomethylated CpGs in these genes. Integrative methylome-transcriptome analysis identified consistent cancer cell vulnerabilities, revealed a robust iB-ALL-specific gene expression-correlating dmCpG signature, and confirmed an epigenetic control of AP-1 and RUNX members in reshaping the molecular network of MLLr iB-ALL. Finally, pharmacological inhibition or functional ablation of AP-1 dramatically impaired MLLr-leukemic growth in vitro and in vivo using MLLr-iB-ALL patient-derived xenografts, providing rationale for new therapeutic avenues in MLLr-iB-ALL.
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Affiliation(s)
- Juan Ramón Tejedor
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología de Asturias (IUOPA), Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Asturias, Spain.,Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Asturias, Spain
| | - Clara Bueno
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) and.,RICORS-TERAV Network, ISCIII, Madrid, Spain
| | - Meritxell Vinyoles
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) and
| | - Paolo Petazzi
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) and
| | - Antonio Agraz-Doblas
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain
| | - Isabel Cobo
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología de Asturias (IUOPA), Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Asturias, Spain.,Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Raúl Torres-Ruiz
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,RICORS-TERAV Network, ISCIII, Madrid, Spain.,Molecular Cytogenetics Group, Human Cancer Genetics Program, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Gustavo F Bayón
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología de Asturias (IUOPA), Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Asturias, Spain
| | - Raúl F Pérez
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología de Asturias (IUOPA), Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Asturias, Spain.,Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Asturias, Spain
| | - Sara López-Tamargo
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología de Asturias (IUOPA), Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Asturias, Spain
| | - Francisco Gutierrez-Agüera
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,RICORS-TERAV Network, ISCIII, Madrid, Spain
| | - Pablo Santamarina-Ojeda
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología de Asturias (IUOPA), Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Asturias, Spain
| | - Manuel Ramírez-Orellana
- RICORS-TERAV Network, ISCIII, Madrid, Spain.,Hematology Diagnostic Laboratory, Hospital Universitario Niño Jesús, Madrid, Spain
| | - Michela Bardini
- Centro Ricerca Tettamanti, Department of Paediatrics, University of Milano Bicocca, Fondazione MBBM, Monza, Italy
| | - Giovanni Cazzaniga
- Centro Ricerca Tettamanti, Department of Paediatrics, University of Milano Bicocca, Fondazione MBBM, Monza, Italy
| | - Paola Ballerini
- Pediatric Hematology, Armand Trousseau Hospital, Paris, France
| | - Pauline Schneider
- Princess Maxima Center for Paediatric Oncology, Utrecht, Netherlands
| | - Ronald W Stam
- Princess Maxima Center for Paediatric Oncology, Utrecht, Netherlands
| | - Ignacio Varela
- Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain
| | - Mario F Fraga
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología de Asturias (IUOPA), Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Asturias, Spain.,Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Asturias, Spain
| | - Agustín F Fernández
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Instituto Universitario de Oncología de Asturias (IUOPA), Hospital Universitario Central de Asturias (HUCA), Universidad de Oviedo, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Asturias, Spain.,Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Asturias, Spain
| | - Pablo Menéndez
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) and.,RICORS-TERAV Network, ISCIII, Madrid, Spain.,Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
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7
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Serrano-Garrido O, Peris-Torres C, Redondo-García S, Asenjo HG, Plaza-Calonge MDC, Fernandez-Luna JL, Rodríguez-Manzaneque JC. ADAMTS1 Supports Endothelial Plasticity of Glioblastoma Cells with Relevance for Glioma Progression. Biomolecules 2020; 11:biom11010044. [PMID: 33396280 PMCID: PMC7823850 DOI: 10.3390/biom11010044] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2020] [Revised: 12/08/2020] [Accepted: 12/19/2020] [Indexed: 12/20/2022] Open
Abstract
Gliomas in general and the more advanced glioblastomas (GBM) in particular are the most usual tumors of the central nervous system with poor prognosis. GBM patients develop resistance to distinct therapies, in part due to the existence of tumor cell subpopulations with stem-like properties that participate in trans-differentiation events. Within the complex tumor microenvironment, the involvement of extracellular proteases remains poorly understood. The extracellular protease ADAMTS1 has already been reported to contribute to the plasticity of cancer cells. Accordingly, this basic knowledge and the current availability of massive sequencing data from human gliomas, reinforced the development of this work. We first performed an in silico study of ADAMTS1 and endothelial markers in human gliomas, providing the basis to further assess these molecules in several primary glioblastoma-initiating cells and established GBM cells with the ability to acquire an endothelial-like phenotype. Using a co-culture approach of endothelial and GBM cells, we noticed a relevant function of ADAMTS1 in GBM cells leading the organization of endothelial-like networks and, even more significantly, we found a blockade of the formation of tumor-spheres and a deficient response to hypoxia in the absence of ADAMTS1. Our data support a chief role of this protease modulating the phenotypic plasticity of GBM.
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Affiliation(s)
- Orlando Serrano-Garrido
- GENYO, Centre for Genomics and Oncological Research: Pfizer/Universidad de Granada/Junta de Andalucía, Avenida de la Ilustración, 114, 18016 Granada, Spain; (O.S.-G.); (C.P.-T.); (S.R.-G.); (H.G.A.); (M.d.C.P.-C.)
- Faculty of Medicine, University of Panama, Ciudad Universitaria, Panamá 3366, Panama
| | - Carlos Peris-Torres
- GENYO, Centre for Genomics and Oncological Research: Pfizer/Universidad de Granada/Junta de Andalucía, Avenida de la Ilustración, 114, 18016 Granada, Spain; (O.S.-G.); (C.P.-T.); (S.R.-G.); (H.G.A.); (M.d.C.P.-C.)
| | - Silvia Redondo-García
- GENYO, Centre for Genomics and Oncological Research: Pfizer/Universidad de Granada/Junta de Andalucía, Avenida de la Ilustración, 114, 18016 Granada, Spain; (O.S.-G.); (C.P.-T.); (S.R.-G.); (H.G.A.); (M.d.C.P.-C.)
| | - Helena G. Asenjo
- GENYO, Centre for Genomics and Oncological Research: Pfizer/Universidad de Granada/Junta de Andalucía, Avenida de la Ilustración, 114, 18016 Granada, Spain; (O.S.-G.); (C.P.-T.); (S.R.-G.); (H.G.A.); (M.d.C.P.-C.)
| | - María del Carmen Plaza-Calonge
- GENYO, Centre for Genomics and Oncological Research: Pfizer/Universidad de Granada/Junta de Andalucía, Avenida de la Ilustración, 114, 18016 Granada, Spain; (O.S.-G.); (C.P.-T.); (S.R.-G.); (H.G.A.); (M.d.C.P.-C.)
| | - José Luis Fernandez-Luna
- Molecular Genetics Unit, Hospital Universitario Marqués de Valdecilla, Avenida Valdecilla, s/n, 39008 Santander, Spain;
| | - Juan Carlos Rodríguez-Manzaneque
- GENYO, Centre for Genomics and Oncological Research: Pfizer/Universidad de Granada/Junta de Andalucía, Avenida de la Ilustración, 114, 18016 Granada, Spain; (O.S.-G.); (C.P.-T.); (S.R.-G.); (H.G.A.); (M.d.C.P.-C.)
- Correspondence: ; Tel.: +34-958-715-500 (ext. 118)
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8
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Antunes ETB, Ottersbach K. The MLL/SET family and haematopoiesis. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2020; 1863:194579. [PMID: 32389825 PMCID: PMC7294230 DOI: 10.1016/j.bbagrm.2020.194579] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Revised: 04/08/2020] [Accepted: 04/30/2020] [Indexed: 12/11/2022]
Abstract
As demonstrated through early work in Drosophila, members of the MLL/SET family play essential roles during embryonic development through their participation in large protein complexes that are central to epigenetic regulation of gene expression. One of its members, MLL1, has additionally received a lot of attention as it is a potent oncogenic driver in different types of leukaemia when aberrantly fused to a large variety of partners as a result of chromosomal translocations. Its exclusive association with cancers of the haematopoietic system has prompted a large number of investigations into the role of MLL/SET proteins in haematopoiesis, a summary of which was attempted in this review. Interestingly, MLL-rearranged leukaemias are particularly prominent in infant and paediatric leukaemia, which commonly initiate in utero. This, together with the known function of MLL/SET proteins in embryonic development, has focussed research efforts in recent years on understanding the role of this protein family in developmental haematopoiesis and how this may be subverted by MLL oncofusions in infant leukaemia. A detailed understanding of these prenatal events is essential for the development of new treatments that improve the survival specifically of this very young patient group.
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Affiliation(s)
- Eric T B Antunes
- MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, Scotland, UK
| | - Katrin Ottersbach
- MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, Scotland, UK.
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9
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MLL-rearranged infant leukaemia: A 'thorn in the side' of a remarkable success story. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2020; 1863:194564. [PMID: 32376390 DOI: 10.1016/j.bbagrm.2020.194564] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Revised: 04/16/2020] [Accepted: 04/16/2020] [Indexed: 12/20/2022]
Abstract
Advances in treatment of childhood leukaemia has led to vastly improved survival rates, however some subtypes such as those characterised by MLL gene rearrangement (MLL-r), especially in infants, continue to have high relapse rates and poor survival. Natural history and molecular studies indicate that infant acute lymphoblastic leukaemia (ALL) originates in utero, is distinct from childhood ALL, and most cases are caused by MLL-r resulting in an oncogenic MLL fusion protein. Unlike childhood ALL, only a very small number of additional mutations are present in infant ALL, indicating that MLL-r alone may be sufficient to give rise to this rapid onset, aggressive leukaemia in an appropriate fetal cell context. Despite modifications in treatment approaches, the outcome of MLL-r infant ALL has remained dismal and a clear understanding of the underlying biology of the disease is required in order to develop appropriate disease models and more effective therapeutic strategies.
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10
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Extracellular Protease ADAMTS1 Is Required at Early Stages of Human Uveal Melanoma Development by Inducing Stemness and Endothelial-Like Features on Tumor Cells. Cancers (Basel) 2020; 12:cancers12040801. [PMID: 32230715 PMCID: PMC7226337 DOI: 10.3390/cancers12040801] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Revised: 03/21/2020] [Accepted: 03/24/2020] [Indexed: 12/16/2022] Open
Abstract
Extracellular matrix remodeling within the tumor microenvironment has been recognized as a relevant dynamic framework during tumor growth. However, research on proteases that trigger this remodeling keeps revealing a wide range of actions including both pro- and anti-tumorigenic. The extracellular protease ADAMTS1 exemplifies this dual role. In this work, we first confirmed a positive correlation of ADAMTS1 with endothelial-like phenotype of human melanoma cells together with the finding of associated signatures, including key genes such as endothelial CDH5. Using a CRISPR-Cas9 approach, we observed that the inhibition of ADAMTS1 in an aggressive uveal melanoma model compromised its endothelial-like properties, and more importantly, caused a robust blockade on the progression of tumor xenografts. Although vasculature emerged affected in ADAMTS1-deficient tumors, the most relevant action implied the downregulation of endothelial CDH5 in tumor cells, in association with stemness markers. Indeed, melanoma sphere assays also revealed a deficient commitment to form spheres in the absence of ADAMTS1, directly correlating with stemness markers and, remarkably, also with CDH5. Finally, taking advantage of advanced bioinformatics tools and available public data of uveal melanomas, we disclosed new prognosis factors, including endothelial elements and ADAMTS proteases. Our findings support the key role of ADAMTS proteases for uveal melanoma development since earlier stages, modulating the complex crosstalk between extracellular matrix and the induction of stemness and endothelial-like features. To our knowledge, this is the first report that supports the development of therapeutic targets on the extracellular matrix to overcome uveal melanoma.
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11
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Enhancing Hematopoiesis from Murine Embryonic Stem Cells through MLL1-Induced Activation of a Rac/Rho/Integrin Signaling Axis. Stem Cell Reports 2020; 14:285-299. [PMID: 31951812 PMCID: PMC7013201 DOI: 10.1016/j.stemcr.2019.12.009] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2019] [Revised: 12/12/2019] [Accepted: 12/17/2019] [Indexed: 12/14/2022] Open
Abstract
The Mixed Lineage Leukemia (MLL1, KMT2A) gene is critical for development and maintenance of hematopoietic stem cells (HSCs), however, whether this protein is limiting for HSC development is unknown due to lack of physiologic model systems. Here, we develop an MLL1-inducible embryonic stem cell (ESC) system and show that induction of wild-type MLL1 during ESC differentiation selectively increases hematopoietic potential from a transitional c-Kit+/Cd41+ population in the embryoid body and also at sites of hematopoiesis in embryos. Single-cell sequencing analysis illustrates inherent heterogeneity of the c-Kit+/Cd41+ population and demonstrates that MLL1 induction shifts its composition toward multilineage hematopoietic identities. Surprisingly, this does not occur through increasing Hox or other canonical MLL1 targets but through an enhanced Rac/Rho/integrin signaling state, which increases responsiveness to Vla4 ligands and enhances hematopoietic commitment. Together, our data implicate a Rac/Rho/integrin signaling axis in the endothelial to hematopoietic transition and demonstrate that MLL1 actives this axis.
Increasing MLL1 enhances hematopoietic potential in vitro and in vivo scRNA sequencing illustrates the heterogeneity of an EMP-like population from EBs MLL1 activates Rac/Rho/integrin signaling during hematopoietic specification MLL1-induced HSPCs are primed for hematopoiesis via integrin-mediated adhesion
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12
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Inhibition of DOT1L and PRMT5 promote synergistic anti-tumor activity in a human MLL leukemia model induced by CRISPR/Cas9. Oncogene 2019; 38:7181-7195. [PMID: 31417187 DOI: 10.1038/s41388-019-0937-9] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2018] [Revised: 04/30/2019] [Accepted: 05/29/2019] [Indexed: 02/07/2023]
Abstract
MLL rearrangements play a crucial role in leukemogenesis and comprise a poor prognosis. Therefore, new treatment strategies are urgently needed. We used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4 or -AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient cells faithfully mimicking the nature of the disease. Thus, it serves as a fundamental basis for pharmacological studies: inhibition of histone methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is one specific therapeutic approach currently tested in clinical trials. However, success was limited by restricted response warranting further investigation of drug combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines and in MLL mouse models. Here, we used DOT1L and PRMT5 inhibitors in our human MLL-rearranged model demonstrating dose-dependent reduced proliferation, impairment of cell cycle, increasing differentiation, apoptosis, downregulation of target genes and sensitization to chemotherapy. Strikingly, the combination of both compounds led to synergistic anti-tumoral effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias.
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13
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The Impact of PI3-kinase/RAS Pathway Cooperating Mutations in the Evolution of KMT2A-rearranged Leukemia. Hemasphere 2019; 3:e195. [PMID: 31723831 PMCID: PMC6746018 DOI: 10.1097/hs9.0000000000000195] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2018] [Revised: 02/21/2019] [Accepted: 02/22/2019] [Indexed: 12/11/2022] Open
Abstract
Leukemia is an evolutionary disease and evolves by the accrual of mutations within a clone. Those mutations that are systematically found in all the patients affected by a certain leukemia are called "drivers" as they are necessary to drive the development of leukemia. Those ones that accumulate over time but are different from patient to patient and, therefore, are not essential for leukemia development are called "passengers." The first studies highlighting a potential cooperating role of phosphatidylinositol 3-kinase (PI3K)/RAS pathway mutations in the phenotype of KMT2A-rearranged leukemia was published 20 years ago. The recent development in more sensitive sequencing technologies has contributed to clarify the contribution of these mutations to the evolution of KMT2A-rearranged leukemia and suggested that these mutations might confer clonal fitness and enhance the evolvability of KMT2A-leukemic cells. This is of particular interest since this pathway can be targeted offering potential novel therapeutic strategies to KMT2A-leukemic patients. This review summarizes the recent progress on our understanding of the role of PI3K/RAS pathway mutations in initiation, maintenance, and relapse of KMT2A-rearranged leukemia.
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14
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Bueno C, Calero-Nieto FJ, Wang X, Valdés-Mas R, Gutiérrez-Agüera F, Roca-Ho H, Ayllon V, Real PJ, Arambilet D, Espinosa L, Torres-Ruiz R, Agraz-Doblas A, Varela I, de Boer J, Bigas A, Gottgens B, Marschalek R, Menendez P. Enhanced hemato-endothelial specification during human embryonic differentiation through developmental cooperation between AF4-MLL and MLL-AF4 fusions. Haematologica 2019; 104:1189-1201. [PMID: 30679325 PMCID: PMC6545840 DOI: 10.3324/haematol.2018.202044] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Accepted: 01/21/2019] [Indexed: 12/18/2022] Open
Abstract
The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is expressed in only half of the patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors, both endothelial- and hemogenic-primed. Double fusion-expressing hemato-endothelial precursors specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-sequencing and H3K79me3 chromatin immunoprecipitation-sequencing, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing hemato-endothelial precursors. Importantly, chromatin immunoprecipitation-sequencing analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion-expressing differentiating hematopoietic cells. Overall, these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.
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Affiliation(s)
- Clara Bueno
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC), ISCIII, Barcelona, Spain
| | - Fernando J Calero-Nieto
- Department of Hematology, Cambridge Institute for Medical Research and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, UK
| | - Xiaonan Wang
- Department of Hematology, Cambridge Institute for Medical Research and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, UK
| | | | - Francisco Gutiérrez-Agüera
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Spain
| | - Heleia Roca-Ho
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Spain
| | - Veronica Ayllon
- GENyO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government and University of Granada, Department of Biochemistry and Molecular Biology, Granada, Spain
| | - Pedro J Real
- GENyO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government and University of Granada, Department of Biochemistry and Molecular Biology, Granada, Spain
| | - David Arambilet
- Programa de Cáncer, Instituto Hospital del Mar de Investigaciones Médicas. Barcelona. Spain
| | - Lluis Espinosa
- Programa de Cáncer, Instituto Hospital del Mar de Investigaciones Médicas. Barcelona. Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC), ISCIII, Barcelona, Spain
| | - Raul Torres-Ruiz
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Spain
| | - Antonio Agraz-Doblas
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Spain
- Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-Sodercan), Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain
| | - Ignacio Varela
- Instituto de Biomedicina y Biotecnología de Cantabria (CSIC-UC-Sodercan), Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain
| | - Jasper de Boer
- Cancer Section, UCL Great Ormond Street Institute of Child Health, London, UK
| | - Anna Bigas
- Programa de Cáncer, Instituto Hospital del Mar de Investigaciones Médicas. Barcelona. Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC), ISCIII, Barcelona, Spain
| | - Bertie Gottgens
- Department of Hematology, Cambridge Institute for Medical Research and Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, UK
| | - Rolf Marschalek
- Institute of Pharmaceutical Biology, Goethe-University, Frankfurt, Germany
| | - Pablo Menendez
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC), ISCIII, Barcelona, Spain
- Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
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15
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Milan T, Canaj H, Villeneuve C, Ghosh A, Barabé F, Cellot S, Wilhelm BT. Pediatric leukemia: Moving toward more accurate models. Exp Hematol 2019; 74:1-12. [PMID: 31154068 DOI: 10.1016/j.exphem.2019.05.003] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Revised: 05/19/2019] [Accepted: 05/22/2019] [Indexed: 02/07/2023]
Abstract
Leukemia is a complex genetic disease caused by errors in differentiation, growth, and apoptosis of hematopoietic cells in either lymphoid or myeloid lineages. Large-scale genomic characterization of thousands of leukemia patients has produced a tremendous amount of data that have enabled a better understanding of the differences between adult and pediatric patients. For instance, although phenotypically similar, pediatric and adult myeloid leukemia patients differ in their mutational profiles, typically involving either chromosomal translocations or recurrent single-base-pair mutations, respectively. To elucidate the molecular mechanisms underlying the biology of this cancer, continual efforts have been made to develop more contextually and biologically relevant experimental models. Leukemic cell lines, for example, provide an inexpensive and tractable model but often fail to recapitulate critical aspects of tumor biology. Likewise, murine leukemia models of leukemia have been highly informative but also do not entirely reproduce the human disease. More recent advances in the development of patient-derived xenografts (PDXs) or human models of leukemias are poised to provide a more comprehensive, and biologically relevant, approach to directly assess the impact of the in vivo environment on human samples. In this review, the advantages and limitations of the various current models used to functionally define the genetic requirements of leukemogenesis are discussed.
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MESH Headings
- Adolescent
- Animals
- Cell Differentiation
- Child
- Child, Preschool
- Female
- Heterografts
- Humans
- Infant
- Infant, Newborn
- Leukemia, Myeloid/genetics
- Leukemia, Myeloid/pathology
- Leukemia, Myeloid/therapy
- Male
- Mice
- Neoplasm Transplantation
- Neoplasms, Experimental/genetics
- Neoplasms, Experimental/metabolism
- Neoplasms, Experimental/pathology
- Neoplasms, Experimental/therapy
- Translocation, Genetic
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Affiliation(s)
- Thomas Milan
- Laboratory for High Throughput Biology, Institute for Research in Immunology and Cancer, Montréal, QC, Canada
| | - Hera Canaj
- Laboratory for High Throughput Biology, Institute for Research in Immunology and Cancer, Montréal, QC, Canada
| | - Chloe Villeneuve
- Laboratory for High Throughput Biology, Institute for Research in Immunology and Cancer, Montréal, QC, Canada
| | - Aditi Ghosh
- Laboratory for High Throughput Biology, Institute for Research in Immunology and Cancer, Montréal, QC, Canada
| | - Frédéric Barabé
- Centre de recherche en infectiologie du CHUL, Centre de recherche du CHU de Québec, Quebec City, QC, Canada; CHU de Québec Hôpital Enfant-Jésus, Quebec City, QC, Canada; Department of Medicine, Université Laval, Quebec City, QC, Canada
| | - Sonia Cellot
- Division of Hematology, Department of Pediatrics, Ste-Justine Hospital, Montréal, Université de Montréal, Montréal, QC, Canada
| | - Brian T Wilhelm
- Laboratory for High Throughput Biology, Institute for Research in Immunology and Cancer, Montréal, QC, Canada; Department of Medicine, Université de Montréal, Montréal, QC, Canada.
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16
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Agraz-Doblas A, Bueno C, Bashford-Rogers R, Roy A, Schneider P, Bardini M, Ballerini P, Cazzaniga G, Moreno T, Revilla C, Gut M, Valsecchi MG, Roberts I, Pieters R, De Lorenzo P, Varela I, Menendez P, Stam RW. Unraveling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis. Haematologica 2019; 104:1176-1188. [PMID: 30679323 PMCID: PMC6545849 DOI: 10.3324/haematol.2018.206375] [Citation(s) in RCA: 74] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Accepted: 12/20/2018] [Indexed: 02/06/2023] Open
Abstract
B-cell acute lymphoblastic leukemia is the commonest childhood cancer. In infants, B-cell acute lymphoblastic leukemia remains fatal, especially in patients with t(4;11), present in ~80% of cases. The pathogenesis of t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemia remains difficult to model, and the pathogenic contribution in cancer of the reciprocal fusions resulting from derivative translocated-chromosomes remains obscure. Here, “multi-layered” genome-wide analyses and validation were performed on a total of 124 de novo cases of infant B-cell acute lymphoblastic leukemia uniformly diagnosed and treated according to the Interfant 99/06 protocol. These patients showed the most silent mutational landscape reported so far for any sequenced pediatric cancer. Recurrent mutations were exclusively found in K-RAS and N-RAS, were subclonal and were frequently lost at relapse, despite a larger number of non-recurrent/non-silent mutations. Unlike non-MLL-rearranged B-cell acute lymphoblastic leukemias, B-cell receptor repertoire analysis revealed minor, non-expanded B-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cell acute lymphoblastic leukemias and the most immature human fetal liver hematopoietic stem and progenitor cells, confirming a “pre-VDJ” fetal cellular origin for both t(4;11) and RASmut. The reciprocal fusion AF4-MLL was expressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA cluster genes are exclusively expressed in AF4-MLL-expressing patients. Importantly, AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs. 11.7%, P=0.001), and overall survival (73.7 vs. 25.2%, P=0.016). AF4-MLL expression retained its prognostic significance when analyzed in a Cox model adjusting for risk stratification according to the Interfant-06 protocol based on age at diagnosis, white blood cell count and response to prednisone. This study has clinical implications for disease outcome and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lymphoblastic leukemia.
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Affiliation(s)
- Antonio Agraz-Doblas
- Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain.,Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Spain
| | - Clara Bueno
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Spain
| | | | - Anindita Roy
- Department of Paediatrics, University of Oxford, UK
| | - Pauline Schneider
- Princess Maxima Center for Pediatric Oncology, Utrecht, the Netherlands
| | - Michela Bardini
- Centro Ricerca Tettamanti, Department of Pediatrics, University of Milano Bicocca, Fondazione MBBM, Monza, Italy
| | | | - Gianni Cazzaniga
- Centro Ricerca Tettamanti, Department of Pediatrics, University of Milano Bicocca, Fondazione MBBM, Monza, Italy
| | - Thaidy Moreno
- Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain
| | - Carlos Revilla
- Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain
| | - Marta Gut
- CNAG-CRG, Center for Genomic Regulation, Barcelona, Spain.,Universitat Pompeu Fabra, Barcelona, Spain
| | - Maria G Valsecchi
- Interfant Trial Data Center, University of Milano-Bicocca, Monza, Italy
| | - Irene Roberts
- Department of Paediatrics, University of Oxford, UK.,MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, UK
| | - Rob Pieters
- Princess Maxima Center for Pediatric Oncology, Utrecht, the Netherlands
| | - Paola De Lorenzo
- Interfant Trial Data Center, University of Milano-Bicocca, Monza, Italy
| | - Ignacio Varela
- Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain
| | - Pablo Menendez
- Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Spain .,Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.,Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), ISCIII, Barcelona, Spain
| | - Ronald W Stam
- Princess Maxima Center for Pediatric Oncology, Utrecht, the Netherlands
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17
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Alsayegh K, Cortés-Medina LV, Ramos-Mandujano G, Badraiq H, Li M. Hematopoietic Differentiation of Human Pluripotent Stem Cells: HOX and GATA Transcription Factors as Master Regulators. Curr Genomics 2019; 20:438-452. [PMID: 32194342 PMCID: PMC7062042 DOI: 10.2174/1389202920666191017163837] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Revised: 09/04/2019] [Accepted: 09/27/2019] [Indexed: 02/07/2023] Open
Abstract
Numerous human disorders of the blood system would directly or indirectly benefit from therapeutic approaches that reconstitute the hematopoietic system. Hematopoietic stem cells (HSCs), either from matched donors or ex vivo manipulated autologous tissues, are the most used cellular source of cell therapy for a wide range of disorders. Due to the scarcity of matched donors and the difficulty of ex vivo expansion of HSCs, there is a growing interest in harnessing the potential of pluripotent stem cells (PSCs) as a de novo source of HSCs. PSCs make an ideal source of cells for regenerative medicine in general and for treating blood disorders in particular because they could expand indefinitely in culture and differentiate to any cell type in the body. However, advancement in deriving functional HSCs from PSCs has been slow. This is partly due to an incomplete understanding of the molecular mechanisms underlying normal hematopoiesis. In this review, we discuss the latest efforts to generate human PSC (hPSC)-derived HSCs capable of long-term engraftment. We review the regulation of the key transcription factors (TFs) in hematopoiesis and hematopoietic differentiation, the Homeobox (HOX) and GATA genes, and the interplay between them and microRNAs. We also propose that precise control of these master regulators during the course of hematopoietic differentiation is key to achieving functional hPSC-derived HSCs.
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Affiliation(s)
- Khaled Alsayegh
- King Abdullah International Medical Research Centre, King Saud bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia.,Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
| | - Lorena V Cortés-Medina
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
| | - Gerardo Ramos-Mandujano
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
| | - Heba Badraiq
- King Abdullah International Medical Research Centre, King Saud bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia
| | - Mo Li
- Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
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18
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Lopez-Millan B, Diaz de la Guardia R, Roca-Ho H, Anguita E, Islam ABMMK, Romero-Moya D, Prieto C, Gutierrez-Agüera F, Bejarano-Garcia JA, Perez-Simon JA, Costales P, Rovira M, Marín P, Menendez S, Iglesias M, Fuster JL, Urbano-Ispizua A, Anjos-Afonso F, Bueno C, Menendez P. IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q- AML. Oncoimmunology 2018; 7:e1477460. [PMID: 30228947 PMCID: PMC6140592 DOI: 10.1080/2162402x.2018.1477460] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Revised: 05/10/2018] [Accepted: 05/12/2018] [Indexed: 12/25/2022] Open
Abstract
Treatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleiotropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syndromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited in vitro and in vivo preclinical AML models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard AML chemotherapy by interfering with the BM stroma-mediated chemoresistance. We report that IMiDs do not exert cytotoxic effects on either non-del5q/5q- AML cells nor BM-MSCs, but they enhance the immunomodulatory properties of BM-MSCs. When combined with AraC/Idarubicin, IMiDs fail to circumvent BM stroma-mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust extramedullary mobilization of AML cells. When administered as a single agent, lenalidomide specifically mobilizes non-del5q/5q- AML cells, but not healthy CD34+ cells, to peripheral blood (PB) through specific downregulation of CXCR4 in AML blasts. Global gene expression profiling supports a migratory/mobilization gene signature in lenalidomide-treated non-del5q/5q- AML blasts but not in CD34+ cells. Collectively, IMiDs mobilize non-del5q/5q- AML blasts to PB through CXCR4 downregulation, but fail to potentiate AraC/Idarubicin activity in preclinical models of non-del5q/5q- AML.
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Affiliation(s)
- Belen Lopez-Millan
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Rafael Diaz de la Guardia
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Heleia Roca-Ho
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Eduardo Anguita
- Hematology Department, Hospital Clínico San Carlos, IdISSC, Universidad Complutense de Madrid, Madrid, Spain
| | - Abul B M M K Islam
- Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka, Bangladesh
| | - Damia Romero-Moya
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Cristina Prieto
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Francisco Gutierrez-Agüera
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Jose Antonio Bejarano-Garcia
- Hematology department, Universidad de Sevilla, Instituto de Biomedicina de Sevilla (IBiS) Hospital Universitario Virgen del Rocío, CSIC, Seville, Spain.,Hematology Department, Hospital Universitario Virgen del Rocío, Seville, Spain
| | - Jose Antonio Perez-Simon
- Hematology department, Universidad de Sevilla, Instituto de Biomedicina de Sevilla (IBiS) Hospital Universitario Virgen del Rocío, CSIC, Seville, Spain
| | | | - Montse Rovira
- Hematology Department, Hospital Clínico de Barcelona, Barcelona, Spain
| | - Pedro Marín
- Hematology Department, Hospital Clínico de Barcelona, Barcelona, Spain
| | | | - Mar Iglesias
- Pathology Service, Hospital del Mar, Barcelona, Spain
| | - Jose Luis Fuster
- Oncohematology department, Sección de Oncohematología Pediátrica, Hospital Clínico Virgen de Arrixaca, Murcia, Spain
| | - Alvaro Urbano-Ispizua
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain.,Hematology Department, Hospital Clínico de Barcelona, Barcelona, Spain.,ISCIII, Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Barcelona, Spain
| | - Fernando Anjos-Afonso
- Cardiff School of Biosciences, European Cancer Stem Cell Research Institute, Cardiff, UK
| | - Clara Bueno
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Pablo Menendez
- Department of Biomedicine, Josep Carreras Leukemia Research Institute-Campus Clinic, School of Medicine, University of Barcelona, Barcelona, Spain.,ISCIII, Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Barcelona, Spain.,Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
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19
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Tejedor JR, Bueno C, Cobo I, Bayón GF, Prieto C, Mangas C, Pérez RF, Santamarina P, Urdinguio RG, Menéndez P, Fraga MF, Fernández AF. Epigenome-wide analysis reveals specific DNA hypermethylation of T cells during human hematopoietic differentiation. Epigenomics 2018; 10:903-923. [DOI: 10.2217/epi-2017-0163] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Aim: Epigenetic regulation plays an important role in cellular development and differentiation. A detailed map of the DNA methylation dynamics that occur during cell differentiation would contribute to decipher the molecular networks governing cell fate commitment. Methods: Illumina MethylationEPIC BeadChip platform was used to describe the genome-wide DNA methylation changes observed throughout hematopoietic maturation by analyzing multiple myeloid and lymphoid hematopoietic cell types. Results: We identified a plethora of DNA methylation changes that occur during human hematopoietic differentiation. We observed that T lymphocytes display substantial enhancement of de novo CpG hypermethylation as compared with other hematopoietic cell populations. T-cell-specific hypermethylated regions were strongly associated with open chromatin marks and enhancer elements, as well as binding sites of specific key transcription factors involved in hematopoietic differentiation, such as PU.1 and TAL1. Conclusion: These results provide novel insights into the role of DNA methylation at enhancer elements in T-cell development.
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Affiliation(s)
- J Ramón Tejedor
- Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Principado de Asturias, Spain
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Principado de Asturias, Spain
| | - Clara Bueno
- Department of Biomedicine, School of Medicine, Josep Carreras Leukemia Research Institute, University of Barcelona, Barcelona, Spain
| | - Isabel Cobo
- Department of Biomedicine, School of Medicine, Josep Carreras Leukemia Research Institute, University of Barcelona, Barcelona, Spain
| | - Gustavo F Bayón
- Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Principado de Asturias, Spain
| | - Cristina Prieto
- Department of Biomedicine, School of Medicine, Josep Carreras Leukemia Research Institute, University of Barcelona, Barcelona, Spain
| | - Cristina Mangas
- Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Principado de Asturias, Spain
| | - Raúl F Pérez
- Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Principado de Asturias, Spain
- Nanomaterials & Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Principado de Asturias, Spain
| | - Pablo Santamarina
- Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Principado de Asturias, Spain
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Principado de Asturias, Spain
| | - Rocío G Urdinguio
- Nanomaterials & Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Principado de Asturias, Spain
| | - Pablo Menéndez
- Department of Biomedicine, School of Medicine, Josep Carreras Leukemia Research Institute, University of Barcelona, Barcelona, Spain
- Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
- Josep Carreras Leukemia Research Institute-Campus ICO, Research Institut Germans Trias i Pujol (IGTP), Badalona, Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), ISCIII, Barcelona, Spain
| | - Mario F Fraga
- Nanomaterials & Nanotechnology Research Center (CINN-CSIC), Universidad de Oviedo, Principado de Asturias, Spain
| | - Agustín F Fernández
- Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Principado de Asturias, Spain
- Fundación para la Investigación Biosanitaria de Asturias (FINBA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Principado de Asturias, Spain
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20
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Zeisig BB, So CWE. MLL-AF4, a double-edged sword for iPSC respecification into HSPCs. Proc Natl Acad Sci U S A 2018; 115:1964-1966. [PMID: 29444865 PMCID: PMC5834742 DOI: 10.1073/pnas.1800622115] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Affiliation(s)
- Bernd B Zeisig
- Leukaemia and Stem Cell Biology Group, School of Cancer and Pharmaceutical Sciences, King's College London, London SE5 9NU, United Kingdom
| | - Chi Wai Eric So
- Leukaemia and Stem Cell Biology Group, School of Cancer and Pharmaceutical Sciences, King's College London, London SE5 9NU, United Kingdom
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21
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Malouf C, Ottersbach K. Molecular processes involved in B cell acute lymphoblastic leukaemia. Cell Mol Life Sci 2018; 75:417-446. [PMID: 28819864 PMCID: PMC5765206 DOI: 10.1007/s00018-017-2620-z] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2017] [Revised: 08/01/2017] [Accepted: 08/04/2017] [Indexed: 12/19/2022]
Abstract
B cell leukaemia is one of the most frequent malignancies in the paediatric population, but also affects a significant proportion of adults in developed countries. The majority of infant and paediatric cases initiate the process of leukaemogenesis during foetal development (in utero) through the formation of a chromosomal translocation or the acquisition/deletion of genetic material (hyperdiploidy or hypodiploidy, respectively). This first genetic insult is the major determinant for the prognosis and therapeutic outcome of patients. B cell leukaemia in adults displays similar molecular features as its paediatric counterpart. However, since this disease is highly represented in the infant and paediatric population, this review will focus on this demographic group and summarise the biological, clinical and epidemiological knowledge on B cell acute lymphoblastic leukaemia of four well characterised subtypes: t(4;11) MLL-AF4, t(12;21) ETV6-RUNX1, t(1;19) E2A-PBX1 and t(9;22) BCR-ABL1.
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Affiliation(s)
- Camille Malouf
- MRC Centre for Regenerative Medicine, The University of Edinburgh, 5 Little France Drive, Edinburgh, EH16 4UU, UK
| | - Katrin Ottersbach
- MRC Centre for Regenerative Medicine, The University of Edinburgh, 5 Little France Drive, Edinburgh, EH16 4UU, UK.
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22
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Prieto C, López-Millán B, Roca-Ho H, Stam RW, Romero-Moya D, Rodríguez-Baena FJ, Sanjuan-Pla A, Ayllón V, Ramírez M, Bardini M, De Lorenzo P, Valsecchi MG, Stanulla M, Iglesias M, Ballerini P, Carcaboso ÁM, Mora J, Locatelli F, Bertaina A, Padilla L, Rodríguez-Manzaneque JC, Bueno C, Menéndez P. NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL. Leukemia 2017; 32:633-644. [PMID: 28943635 PMCID: PMC5843903 DOI: 10.1038/leu.2017.294] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2017] [Revised: 08/24/2017] [Accepted: 08/29/2017] [Indexed: 12/11/2022]
Abstract
Mixed-lineage leukemia (MLL)-rearranged (MLLr) infant B-cell acute lymphoblastic leukemia (iMLLr-B-ALL) has a dismal prognosis and is associated with a pro-B/mixed phenotype, therapy refractoriness and frequent central nervous system (CNS) disease/relapse. Neuron-glial antigen 2 (NG2) is specifically expressed in MLLr leukemias and is used in leukemia immunophenotyping because of its predictive value for MLLr acute leukemias. NG2 is involved in melanoma metastasis and brain development; however, its role in MLL-mediated leukemogenesis remains elusive. Here we evaluated whether NG2 distinguishes leukemia-initiating/propagating cells (L-ICs) and/or CNS-infiltrating cells (CNS-ICs) in iMLLr-B-ALL. Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4+ leukemias indicated that NG2 is a malleable marker that does not enrich for L-IC or CNS-IC in iMLLr-B-All. However, NG2 expression was highly upregulated in blasts infiltrating extramedullar hematopoietic sites and CNS, and specific blockage of NG2 resulted in almost complete loss of engraftment. Indeed, gene expression profiling of primary blasts and primografts revealed a migratory signature of NG2+ blasts. This study provides new insights on the biology of NG2 in iMLLr-B-ALL and suggests NG2 as a potential therapeutic target to reduce the risk of CNS disease/relapse and to provide safer CNS-directed therapies for iMLLr-B-ALL.
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Affiliation(s)
- C Prieto
- Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - B López-Millán
- Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - H Roca-Ho
- Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - R W Stam
- Erasmus University Medical Center, Rotterdam, The Netherlands.,Princess Maxima Center for Paediatric Oncology, Utrecht, The Netherlands
| | - D Romero-Moya
- Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - F J Rodríguez-Baena
- GENYO, Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, Granada, Spain
| | - A Sanjuan-Pla
- Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - V Ayllón
- GENYO, Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, Granada, Spain
| | - M Ramírez
- Oncohematología, Hospital Universitario Niño Jesús, Madrid, Spain
| | - M Bardini
- Centro Ricerca Tettamanti, University of Milano-Bicocca, Ospedale San Gerardo Monza, Italy
| | - P De Lorenzo
- Interfant Trial Data Center, University of Milano-Bicocca, Monza, Italy
| | - M G Valsecchi
- Interfant Trial Data Center, University of Milano-Bicocca, Monza, Italy
| | - M Stanulla
- Department of Pediatric Hemato-Oncology, Hannover Medical School, Hannover, Germany
| | - M Iglesias
- Pathology Service, Hospital del Mar, Barcelona, Spain
| | - P Ballerini
- Pediatric Hematology, A. Trousseau Hospital, Paris, France
| | - Á M Carcaboso
- Developmental Tumor Biology Laboratory, Hospital Sant Joan de Deu, Barcelona, Spain
| | - J Mora
- Developmental Tumor Biology Laboratory, Hospital Sant Joan de Deu, Barcelona, Spain
| | - F Locatelli
- Department of Pediatric Hematology and Oncology, Ospedale Bambino Gesù, Rome, University of Pavia, Pavia, Italy
| | - A Bertaina
- Department of Pediatric Hematology and Oncology, Ospedale Bambino Gesù, Rome, University of Pavia, Pavia, Italy
| | - L Padilla
- Biomed Division, LEITAT Technological Centre, Barcelona, Spain
| | - Juan Carlos Rodríguez-Manzaneque
- GENYO, Centre for Genomics and Oncological Research: Pfizer/University of Granada/Andalusian Regional Government, Granada, Spain
| | - C Bueno
- Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,Centro de Investigacion Biomedica en Red-Oncología (CIBERONC), Barcelona, Spain
| | - P Menéndez
- Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.,Centro de Investigacion Biomedica en Red-Oncología (CIBERONC), Barcelona, Spain.,Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
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23
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Navarro-Montero O, Ayllon V, Lamolda M, López-Onieva L, Montes R, Bueno C, Ng E, Guerrero-Carreno X, Romero T, Romero-Moya D, Stanley E, Elefanty A, Ramos-Mejia V, Menendez P, Real PJ. RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling. Stem Cells 2017; 35:2253-2266. [PMID: 28869683 DOI: 10.1002/stem.2700] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2016] [Revised: 07/19/2017] [Accepted: 08/02/2017] [Indexed: 02/07/2023]
Abstract
Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266.
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Affiliation(s)
- Oscar Navarro-Montero
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain
| | - Veronica Ayllon
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain
| | - Mar Lamolda
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain.,Department of Biochemistry and Molecular Biology I, Faculty of Science, University of Granada, Granada, Spain
| | - Lourdes López-Onieva
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain
| | - Rosa Montes
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain
| | - Clara Bueno
- Josep Carreras Leukemia Research Institute and Biomedicine Department, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Elizabeth Ng
- Blood Cell Development and Disease Laboratory, Murdoch Childrens Research Institute. The Royal Children's Hospital, Parkville, Australia
| | - Xiomara Guerrero-Carreno
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain
| | - Tamara Romero
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain
| | - Damià Romero-Moya
- Josep Carreras Leukemia Research Institute and Biomedicine Department, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Ed Stanley
- Stem Cell Technology Laboratory, Murdoch Childrens Research Institute. The Royal Children's Hospital, Parkville, Australia
| | - Andrew Elefanty
- Blood Cell Development and Disease Laboratory, Murdoch Childrens Research Institute. The Royal Children's Hospital, Parkville, Australia
| | - Verónica Ramos-Mejia
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain
| | - Pablo Menendez
- Josep Carreras Leukemia Research Institute and Biomedicine Department, School of Medicine, University of Barcelona, Barcelona, Spain.,Instituciò Catalana de Reserca i EstudisAvançats (ICREA), Barcelona, Spain.,Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), ISCIII, Barcelona, Spain
| | - Pedro J Real
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO: Centre for Genomics and Oncological Research Pfizer-University of Granada-Junta de Andalucía, PTS Granada, Granada, Spain.,Department of Biochemistry and Molecular Biology I, Faculty of Science, University of Granada, Granada, Spain
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24
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Mouse models of MLL leukemia: recapitulating the human disease. Blood 2017; 129:2217-2223. [PMID: 28179274 DOI: 10.1182/blood-2016-10-691428] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Accepted: 02/03/2017] [Indexed: 12/13/2022] Open
Abstract
Chromosome translocations involving the mixed lineage leukemia (MLL) gene fuse it in frame with multiple partner genes creating novel fusion proteins (MLL-FPs) that cause aggressive acute leukemias in humans. Animal models of human disease are important for the exploration of underlying disease mechanisms as well as for testing novel therapeutic approaches. Patients carrying MLL-FPs have very few cooperating mutations, making MLL-FP driven leukemias ideal for animal modeling. The fact that the MLL-FP is the main driver mutation has allowed for a wide range of different experimental model systems designed to explore different aspects of MLL-FP leukemogenesis. In addition, MLL-FP driven acute myeloid leukemia (AML) in mice is often used as a general model for AML. This review provides an overview of different MLL-FP mouse model systems and discusses how well they have recapitulated aspects of the human disease as well as highlights the biological insights each model has provided into MLL-FP leukemogenesis. Many promising new drugs fail in the early stages of clinical trials. Lessons learned from past and present MLL-FP models may serve as a paradigm for designing more flexible and dynamic preclinical models for these as well as other acute leukemias.
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25
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Ayllón V, Vogel-González M, González-Pozas F, Domingo-Reinés J, Montes R, Morales-Cacho L, Ramos-Mejía V. New hPSC-based human models to study pediatric Acute Megakaryoblastic Leukemia harboring the fusion oncogene RBM15-MKL1. Stem Cell Res 2016; 19:1-5. [PMID: 28412998 DOI: 10.1016/j.scr.2016.12.019] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2016] [Accepted: 12/14/2016] [Indexed: 10/20/2022] Open
Abstract
Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL) is a rare disease with a dismal prognosis. Around 15% of patients carry the chromosomal translocation t(1;22) that originates the fusion oncogene RBM15-MKL1, which is linked to an earlier disease onset (median of 6months of age) and arises in utero. Here we report the generation of two hPSC cell lines constitutively expressing the oncogene RBM15-MKL1, resulting in an increased expression of known RBM15-MKL1 gene targets. These cell lines represent new disease models of pediatric AMKL to study the impact of the RBM15-MKL1 oncogene on human embryonic hematopoietic development.
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Affiliation(s)
- Verónica Ayllón
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO-Centre for Genomics and Oncological Research-Pfizer/University of Granada/Junta de Andalucía, PTS Granada, 18016 Granada, Spain.
| | - Marina Vogel-González
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO-Centre for Genomics and Oncological Research-Pfizer/University of Granada/Junta de Andalucía, PTS Granada, 18016 Granada, Spain
| | - Federico González-Pozas
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO-Centre for Genomics and Oncological Research-Pfizer/University of Granada/Junta de Andalucía, PTS Granada, 18016 Granada, Spain
| | - Joan Domingo-Reinés
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO-Centre for Genomics and Oncological Research-Pfizer/University of Granada/Junta de Andalucía, PTS Granada, 18016 Granada, Spain
| | - Rosa Montes
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO-Centre for Genomics and Oncological Research-Pfizer/University of Granada/Junta de Andalucía, PTS Granada, 18016 Granada, Spain
| | - Lucía Morales-Cacho
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO-Centre for Genomics and Oncological Research-Pfizer/University of Granada/Junta de Andalucía, PTS Granada, 18016 Granada, Spain
| | - Verónica Ramos-Mejía
- Gene Regulation, Stem Cells and Development Group, Department of Genomic Oncology, GENYO-Centre for Genomics and Oncological Research-Pfizer/University of Granada/Junta de Andalucía, PTS Granada, 18016 Granada, Spain.
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26
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Lin S, Luo RT, Ptasinska A, Kerry J, Assi SA, Wunderlich M, Imamura T, Kaberlein JJ, Rayes A, Althoff MJ, Anastasi J, O'Brien MM, Meetei AR, Milne TA, Bonifer C, Mulloy JC, Thirman MJ. Instructive Role of MLL-Fusion Proteins Revealed by a Model of t(4;11) Pro-B Acute Lymphoblastic Leukemia. Cancer Cell 2016; 30:737-749. [PMID: 27846391 DOI: 10.1016/j.ccell.2016.10.008] [Citation(s) in RCA: 85] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Revised: 04/21/2016] [Accepted: 10/12/2016] [Indexed: 01/11/2023]
Abstract
The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34+ cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.
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Affiliation(s)
- Shan Lin
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Roger T Luo
- Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA
| | - Anetta Ptasinska
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B15 2TT, UK
| | - Jon Kerry
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford OX3 9DS, UK
| | - Salam A Assi
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B15 2TT, UK
| | - Mark Wunderlich
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Toshihiko Imamura
- Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA
| | - Joseph J Kaberlein
- Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA
| | - Ahmad Rayes
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Mark J Althoff
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - John Anastasi
- Department of Pathology, University of Chicago, Chicago, IL 60637, USA
| | - Maureen M O'Brien
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Amom Ruhikanta Meetei
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Thomas A Milne
- MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford OX3 9DS, UK
| | - Constanze Bonifer
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham B15 2TT, UK
| | - James C Mulloy
- Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
| | - Michael J Thirman
- Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA.
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Mll-AF4 Confers Enhanced Self-Renewal and Lymphoid Potential during a Restricted Window in Development. Cell Rep 2016; 16:1039-1054. [PMID: 27396339 PMCID: PMC4967476 DOI: 10.1016/j.celrep.2016.06.046] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2016] [Revised: 04/27/2016] [Accepted: 06/09/2016] [Indexed: 01/15/2023] Open
Abstract
MLL-AF4+ infant B cell acute lymphoblastic leukemia is characterized by an early onset and dismal survival. It initiates before birth, and very little is known about the early stages of the disease’s development. Using a conditional Mll-AF4-expressing mouse model in which fusion expression is targeted to the earliest definitive hematopoietic cells generated in the mouse embryo, we demonstrate that Mll-AF4 imparts enhanced B lymphoid potential and increases repopulation and self-renewal capacity during a putative pre-leukemic state. This occurs between embryonic days 12 and 14 and manifests itself most strongly in the lymphoid-primed multipotent progenitor population, thus pointing to a window of opportunity and a potential cell of origin. However, this state alone is insufficient to generate disease, with the mice succumbing to B cell lymphomas only after a long latency. Future analysis of the molecular details of this pre-leukemic state will shed light on additional events required for progression to acute leukemia.
Mll-AF4 confers enhanced B cell potential and causes an expansion of pro-B cells Mll-AF4 increases self-renewal potential Mll-AF4 exerts its effects in a restricted developmental window The LMPP is a potential cell of origin for Mll-AF4-associated disease
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Prieto C, Stam RW, Agraz-Doblas A, Ballerini P, Camos M, Castaño J, Marschalek R, Bursen A, Varela I, Bueno C, Menendez P. Activated KRAS Cooperates with MLL-AF4 to Promote Extramedullary Engraftment and Migration of Cord Blood CD34+ HSPC But Is Insufficient to Initiate Leukemia. Cancer Res 2016; 76:2478-89. [PMID: 26837759 DOI: 10.1158/0008-5472.can-15-2769] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2015] [Accepted: 01/08/2016] [Indexed: 11/16/2022]
Abstract
The MLL-AF4 (MA4) fusion gene is the genetic hallmark of an aggressive infant pro-B-acute lymphoblastic leukemia (B-ALL). Our understanding of MA4-mediated transformation is very limited. Whole-genome sequencing studies revealed a silent mutational landscape, which contradicts the aggressive clinical outcome of this hematologic malignancy. Only RAS mutations were recurrently detected in patients and found to be associated with poorer outcome. The absence of MA4-driven B-ALL models further questions whether MA4 acts as a single oncogenic driver or requires cooperating mutations to manifest a malignant phenotype. We explored whether KRAS activation cooperates with MA4 to initiate leukemia in cord blood-derived CD34(+) hematopoietic stem/progenitor cells (HSPC). Clonogenic and differentiation/proliferation assays demonstrated that KRAS activation does not cooperate with MA4 to immortalize CD34(+) HSPCs. Intrabone marrow transplantation into immunodeficient mice further showed that MA4 and KRAS(G12V) alone or in combination enhanced hematopoietic repopulation without impairing myeloid-lymphoid differentiation, and that mutated KRAS did not cooperate with MA4 to initiate leukemia. However, KRAS activation enhanced extramedullary hematopoiesis of MA4-expressing cell lines and CD34(+) HSPCs that was associated with leukocytosis and central nervous system infiltration, both hallmarks of infant t(4;11)(+) B-ALL. Transcriptional profiling of MA4-expressing patients supported a cell migration gene signature underlying the mutant KRAS-mediated phenotype. Collectively, our findings demonstrate that KRAS affects the homeostasis of MA4-expressing HSPCs, suggesting that KRAS activation in MA4(+) B-ALL is important for tumor maintenance rather than initiation. Cancer Res; 76(8); 2478-89. ©2016 AACR.
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Affiliation(s)
- Cristina Prieto
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Ronald W Stam
- Pediatric Oncology/Hematology, Erasmus MC-Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Antonio Agraz-Doblas
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain. Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC-CSIC-UNIVERSIDAD DE CANTABRIA-SODERCAN), Santander, Spain
| | - Paola Ballerini
- Pediatric Hematology Department, A. Trousseau Hospital, Paris, France
| | - Mireia Camos
- Hematology Laboratory, Hospital Sant Joan de Deu, Barcelona, Spain
| | - Julio Castaño
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain
| | - Rolf Marschalek
- Institute Pharmaceutical Biology, Goethe-University, Frankfurt/Main, Germany
| | - Aldeheid Bursen
- Institute Pharmaceutical Biology, Goethe-University, Frankfurt/Main, Germany
| | - Ignacio Varela
- Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC-CSIC-UNIVERSIDAD DE CANTABRIA-SODERCAN), Santander, Spain
| | - Clara Bueno
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain.
| | - Pablo Menendez
- Josep Carreras Leukemia Research Institute and Department of Biomedicine, School of Medicine, University of Barcelona, Barcelona, Spain. Instituciò Catalana Recerca i Estudis Avançats (ICREA), Barcelona, Spain.
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Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα. Leukemia 2015; 30:674-82. [PMID: 26500142 DOI: 10.1038/leu.2015.294] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Revised: 09/03/2015] [Accepted: 09/17/2015] [Indexed: 12/21/2022]
Abstract
B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies.
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30
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Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia. Blood 2015; 126:2676-85. [PMID: 26463423 DOI: 10.1182/blood-2015-09-667378] [Citation(s) in RCA: 87] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2015] [Accepted: 09/30/2015] [Indexed: 02/06/2023] Open
Abstract
Infant B-cell acute lymphoblastic leukemia (B-ALL) accounts for 10% of childhood ALL. The genetic hallmark of most infant B-ALL is chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene. Despite improvement in the clinical management and survival (∼85-90%) of childhood B-ALL, the outcome of infants with MLL-rearranged (MLL-r) B-ALL remains dismal, with overall survival <35%. Among MLL-r infant B-ALL, t(4;11)+ patients harboring the fusion MLL-AF4 (MA4) display a particularly poor prognosis and a pro-B/mixed phenotype. Studies in monozygotic twins and archived blood spots have provided compelling evidence of a single cell of prenatal origin as the target for MA4 fusion, explaining the brief leukemia latency. Despite its aggressiveness and short latency, current progress on its etiology, pathogenesis, and cellular origin is limited as evidenced by the lack of mouse/human models recapitulating the disease phenotype/latency. We propose this is because infant cancer is from an etiologic and pathogenesis standpoint distinct from adult cancer and should be seen as a developmental disease. This is supported by whole-genome sequencing studies suggesting that opposite to the view of cancer as a "multiple-and-sequential-hit" model, t(4;11) alone might be sufficient to spawn leukemia. The stable genome of these patients suggests that, in infant developmental cancer, one "big-hit" might be sufficient for overt disease and supports a key contribution of epigenetics and a prenatal cell of origin during a critical developmental window of stem cell vulnerability in the leukemia pathogenesis. Here, we revisit the biology of t(4;11)+ infant B-ALL with an emphasis on its origin, genetics, and disease models.
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31
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Seifert A, Werheid DF, Knapp SM, Tobiasch E. Role of Hox genes in stem cell differentiation. World J Stem Cells 2015; 7:583-595. [PMID: 25914765 PMCID: PMC4404393 DOI: 10.4252/wjsc.v7.i3.583] [Citation(s) in RCA: 112] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Revised: 11/20/2014] [Accepted: 12/17/2014] [Indexed: 02/06/2023] Open
Abstract
Hox genes are an evolutionary highly conserved gene family. They determine the anterior-posterior body axis in bilateral organisms and influence the developmental fate of cells. Embryonic stem cells are usually devoid of any Hox gene expression, but these transcription factors are activated in varying spatial and temporal patterns defining the development of various body regions. In the adult body, Hox genes are among others responsible for driving the differentiation of tissue stem cells towards their respective lineages in order to repair and maintain the correct function of tissues and organs. Due to their involvement in the embryonic and adult body, they have been suggested to be useable for improving stem cell differentiations in vitro and in vivo. In many studies Hox genes have been found as driving factors in stem cell differentiation towards adipogenesis, in lineages involved in bone and joint formation, mainly chondrogenesis and osteogenesis, in cardiovascular lineages including endothelial and smooth muscle cell differentiations, and in neurogenesis. As life expectancy is rising, the demand for tissue reconstruction continues to increase. Stem cells have become an increasingly popular choice for creating therapies in regenerative medicine due to their self-renewal and differentiation potential. Especially mesenchymal stem cells are used more and more frequently due to their easy handling and accessibility, combined with a low tumorgenicity and little ethical concerns. This review therefore intends to summarize to date known correlations between natural Hox gene expression patterns in body tissues and during the differentiation of various stem cells towards their respective lineages with a major focus on mesenchymal stem cell differentiations. This overview shall help to understand the complex interactions of Hox genes and differentiation processes all over the body as well as in vitro for further improvement of stem cell treatments in future regenerative medicine approaches.
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Abstract
The molecular determinants regulating the specification of human embryonic stem cells (hESCs) into hematopoietic cells remain elusive. HOXA9 plays a relevant role in leukemogenesis and hematopoiesis. It is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and is downregulated upon differentiation. Hoxa9-deficient mice display impaired hematopoietic development, and deregulation of HOXA9 expression is frequently associated with acute leukemia. Analysis of the genes differentially expressed in cord blood HSPCs vs hESC-derived HSPCs identified HOXA9 as the most downregulated gene in hESC-derived HSPCs, suggesting that expression levels of HOXA9 may be crucial for hematopoietic differentiation of hESC. Here we show that during hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development, but is restricted to the hemogenic precursors (HEP) (CD31(+)CD34(+)CD45(-)), and diminishes as HEPs differentiate into blood cells (CD45(+)). Different gain-of-function and loss-of-function studies reveal that HOXA9 enhances hematopoietic differentiation of hESCs by specifically promoting the commitment of HEPs into primitive and total CD45(+) blood cells. Gene expression analysis suggests that nuclear factor-κB signaling could be collaborating with HOXA9 to increase hematopoietic commitment. However, HOXA9 on its own is not sufficient to confer in vivo long-term engraftment potential to hESC-hematopoietic derivatives, reinforcing the idea that additional molecular regulators are needed for the generation of definitive in vivo functional HSPCs from hESC.
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33
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Rodriguez R, Tornin J, Suarez C, Astudillo A, Rubio R, Yauk C, Williams A, Rosu-Myles M, Funes JM, Boshoff C, Menendez P. Expression of FUS-CHOP fusion protein in immortalized/transformed human mesenchymal stem cells drives mixoid liposarcoma formation. Stem Cells 2014; 31:2061-72. [PMID: 23836491 DOI: 10.1002/stem.1472] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2013] [Revised: 05/22/2013] [Accepted: 06/08/2013] [Indexed: 12/11/2022]
Abstract
Increasing evidence supports that mesenchymal stromal/stem cells (MSCs) may represent the target cell for sarcoma development. Although different sarcomas have been modeled in mice upon expression of fusion oncogenes in MSCs, sarcomagenesis has not been successfully modeled in human MSCs (hMSCs). We report that FUS-CHOP, a hallmark fusion gene in mixoid liposarcoma (MLS), has an instructive role in lineage commitment, and its expression in hMSC sequentially immortalized/transformed with up to five oncogenic hits (p53 and Rb deficiency, hTERT over-expression, c-myc stabilization, and H-RAS(v12) mutation) drives the formation of serially transplantable MLS. This is the first model of sarcoma based on the expression of a sarcoma-associated fusion protein in hMSC, and allowed us to unravel the differentiation processes and signaling pathways altered in the MLS-initiating cells. This study will contribute to test novel therapeutic approaches and constitutes a proof-of-concept to use hMSCs as target cell for modeling other fusion gene-associated human sarcomas.
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Affiliation(s)
- Rene Rodriguez
- Hospital Universitario Central de Asturias and Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain
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34
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Škorvaga M, Nikitina E, Kubeš M, Košík P, Gajdošechová B, Leitnerová M, Copáková L, Belyaev I. Incidence of common preleukemic gene fusions in umbilical cord blood in Slovak population. PLoS One 2014; 9:e91116. [PMID: 24621554 PMCID: PMC3951330 DOI: 10.1371/journal.pone.0091116] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2013] [Accepted: 02/06/2014] [Indexed: 12/11/2022] Open
Abstract
The first event in origination of many childhood leukemias is likely the presence of preleukemic clone (transformed hematopoietic stem/progenitor cells with preleukemic gene fusions (PGF)) in newborn. Thus, the screening of umbilical cord blood (UCB) for PGF may be of high importance for developing strategies for childhood leukemia prevention and treatment. However, the data on incidence of PGF in UCB are contradictive. We have compared multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (RT qPCR) in neonates from Slovak National Birth Cohort. According to multiplex PCR, all 135 screened samples were negative for the most frequent PGF of B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To explore the prevalence of prognostically important TEL-AML1, MLL-AF4 and BCR-ABL (p190), 200 UCB were screened using RT qPCR. The initial screening showed an unexpectedly high incidence of studied PGF. The validation of selected samples in two laboratories confirmed approximately ¼ of UCB positive, resulting in ∼4% incidence of TEL-AML1, ∼6.25% incidence of BCR-ABL1 p190, and ∼0.75% frequency of MLL-AF4. In most cases, the PGF presented at very low level, about 1–5 copies per 105 cells. We hypothesize that low PGF numbers reflect their relatively late origin and are likely to be eliminated in further development while higher number of PGF reflects earlier origination and may represent higher risk for leukemia.
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Affiliation(s)
- Milan Škorvaga
- Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic
| | - Ekaterina Nikitina
- Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic
- Laboratory of Oncovirology, Cancer Research Institute, Siberian Branch of the Russian Academy of Medical Sciences, Tomsk, Russian Federation
| | - Miroslav Kubeš
- Laboratory of R&D, Eurocord-Slovakia, Bratislava, Slovak Republic
| | - Pavol Košík
- Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic
| | - Beata Gajdošechová
- Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic
| | - Michaela Leitnerová
- Department of Clinical Oncology, National Cancer Institute, Bratislava, Slovak Republic
| | - Lucia Copáková
- Department of Clinical Oncology, National Cancer Institute, Bratislava, Slovak Republic
| | - Igor Belyaev
- Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic
- * E-mail:
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35
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Rodriguez RM, Suarez-Alvarez B, Salvanés R, Huidobro C, Toraño EG, Garcia-Perez JL, Lopez-Larrea C, Fernandez AF, Bueno C, Menendez P, Fraga MF. Role of BRD4 in hematopoietic differentiation of embryonic stem cells. Epigenetics 2014; 9:566-78. [PMID: 24445267 DOI: 10.4161/epi.27711] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
The bromodomain and extra terminal (BET) protein family member BRD4 is a transcriptional regulator, critical for cell cycle progression and cellular viability. Here, we show that BRD4 plays an important role in embryonic stem cell (ESC) regulation. During differentiation of ESCs, BRD4 expression is upregulated and its gene promoter becomes demethylated. Disruption of BRD4 expression in ESCs did not induce spontaneous differentiation but severely diminished hematoendothelial potential. Although BRD4 regulates c-Myc expression, our data show that the role of BRD4 in hematopoietic commitment is not exclusively mediated by c-Myc. Our results indicate that BRD4 is epigenetically regulated during hematopoietic differentiation ESCs in the context of a still unknown signaling pathway.
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Affiliation(s)
- Ramon M Rodriguez
- Cancer Epigenetics Laboratory; Instituto Universitario de Oncología del Principado de Asturias (IUOPA); HUCA; Universidad de Oviedo; Oviedo, Spain; Department of Immunology; Hospital Universitario Central de Asturias; Oviedo, Spain
| | | | - Ruben Salvanés
- Department of Immunology; Hospital Universitario Central de Asturias; Oviedo, Spain
| | - Covadonga Huidobro
- Cancer Epigenetics Laboratory; Instituto Universitario de Oncología del Principado de Asturias (IUOPA); HUCA; Universidad de Oviedo; Oviedo, Spain; MRC Human Genetics Unit; Institute of Genetics and Molecular Medicine; University of Edinburgh; Western General Hospital; Edinburgh, UK
| | - Estela G Toraño
- Cancer Epigenetics Laboratory; Instituto Universitario de Oncología del Principado de Asturias (IUOPA); HUCA; Universidad de Oviedo; Oviedo, Spain
| | - Jose L Garcia-Perez
- Department of Human DNA Variability; Pfizer-University of Granada and Andalusian Government Center for Genomics and Oncology (GENYO); Granada, Spain
| | - Carlos Lopez-Larrea
- Department of Immunology; Hospital Universitario Central de Asturias; Oviedo, Spain; Fundacion Renal "Íñigo Álvarez de Toledo"; Madrid, Spain
| | - Agustin F Fernandez
- Cancer Epigenetics Laboratory; Instituto Universitario de Oncología del Principado de Asturias (IUOPA); HUCA; Universidad de Oviedo; Oviedo, Spain
| | - Clara Bueno
- Josep Carreras Leukemia Research Institute; Barcelona, Spain; Centre for Genomics and Oncological Research (GENYO); Pfizer/University of Granada/Andalusian Government; Granada, Spain
| | - Pablo Menendez
- Josep Carreras Leukemia Research Institute; Barcelona, Spain; Instituciò Catalana de Reserca i Estudis Avançats (ICREA); Barcelona, Spain
| | - Mario F Fraga
- Cancer Epigenetics Laboratory; Instituto Universitario de Oncología del Principado de Asturias (IUOPA); HUCA; Universidad de Oviedo; Oviedo, Spain; Department of Immunology and Oncology; Centro Nacional de Biotecnología/CNB-CSIC; Cantoblanco; Madrid, Spain
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36
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Montes R, Ayllón V, Prieto C, Bursen A, Prelle C, Romero-Moya D, Real PJ, Navarro-Montero O, Chillón C, Marschalek R, Bueno C, Menendez P. Ligand-independent FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform cord blood CD34+ cells. Leukemia 2013; 28:666-74. [PMID: 24240202 DOI: 10.1038/leu.2013.346] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2013] [Revised: 10/18/2013] [Accepted: 11/08/2013] [Indexed: 01/11/2023]
Abstract
MLL-AF4 fusion is hallmark in high-risk infant pro-B-acute lymphoblastic leukemia (pro-B-ALL). Our limited understanding of MLL-AF4-mediated transformation reflects the absence of human models reproducing this leukemia. Hematopoietic stem/progenitor cells (HSPCs) constitute likely targets for transformation. We previously reported that MLL-AF4 enhanced hematopoietic engraftment and clonogenic potential in cord blood (CB)-derived CD34+ HSPCs but was not sufficient for leukemogenesis, suggesting that additional oncogenic lesions are required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL display enormous levels of FLT3, and occasionally FLT3-activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. We have explored whether FLT3.TKD (tyrosine kinase domain) mutation or increased expression of FLT3.WT (wild type) cooperates with MLL-AF4 to immortalize/transform CB-CD34+ HSPCs. In vivo, FLT3.TKD/FLT3.WT alone, or in combination with MLL-AF4, enhances hematopoietic repopulating function of CB-CD34+ HSPCs without impairing migration or hematopoietic differentiation. None of the animals transplanted with MLL-AF4+FLT3.TKD/WT-CD34+ HSPCs showed any sign of disease after 16 weeks. In vitro, enforced expression of FLT3.TKD/FLT3.WT conveys a transient overexpansion of MLL-AF4-expressing CD34+ HSPCs associated to higher proportion of cycling cells coupled to lower apoptotic levels, but does not augment clonogenic potential nor confer stable replating. Together, FLT3 activation does not suffice to immortalize/transform MLL-AF4-expressing CB-CD34+ HSPCs, suggesting the need of alternative (epi)-genetic cooperating oncogenic lesions.
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Affiliation(s)
- R Montes
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain
| | - V Ayllón
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain
| | - C Prieto
- 1] GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain [2] Faculty of Medicine, Department of Stem Cells, Development and Cancer, Cell Therapy Program of the University of Barcelona, Josep Carreras Leukemia Research Institute, Barcelona, Spain
| | - A Bursen
- Institute of Pharmaceutical Biology/ZAFES/DCAL, Goethe-University of Frankfurt, Biocenter, Frankfurt, Germany
| | - C Prelle
- Institute of Pharmaceutical Biology/ZAFES/DCAL, Goethe-University of Frankfurt, Biocenter, Frankfurt, Germany
| | - D Romero-Moya
- 1] GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain [2] Faculty of Medicine, Department of Stem Cells, Development and Cancer, Cell Therapy Program of the University of Barcelona, Josep Carreras Leukemia Research Institute, Barcelona, Spain
| | - P J Real
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain
| | - O Navarro-Montero
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain
| | - C Chillón
- Hospital Universitario de Salamanca, Servicio de Hematología, Salamanca, Spain
| | - R Marschalek
- Institute of Pharmaceutical Biology/ZAFES/DCAL, Goethe-University of Frankfurt, Biocenter, Frankfurt, Germany
| | - C Bueno
- 1] GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain [2] Faculty of Medicine, Department of Stem Cells, Development and Cancer, Cell Therapy Program of the University of Barcelona, Josep Carreras Leukemia Research Institute, Barcelona, Spain
| | - P Menendez
- 1] GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, Granada, Spain [2] Faculty of Medicine, Department of Stem Cells, Development and Cancer, Cell Therapy Program of the University of Barcelona, Josep Carreras Leukemia Research Institute, Barcelona, Spain [3] Instituciò Catalana de Reserca i Estudis Avançats (ICREA)
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Abstract
In this issue of Blood, Bueno and colleagues explore the developmental impact, as well as the transforming capacity, of the mixed-lineage leukemia (MLL)–AF4 fusion protein in combination with activation of FMS-like tyrosine receptor 3 (FLT3) in human embryonic stem cells (hESCs).1
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FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs. Blood 2013; 121:3867-78, S1-3. [PMID: 23479570 DOI: 10.1182/blood-2012-11-470146] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Mixed-lineage leukemia (MLL)-AF4 fusion arises prenatally in high-risk infant acute pro-B-lymphoblastic leukemia (pro-B-ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hematoendothelial specification but was insufficient for transformation, suggesting that additional oncogenic insults seem required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL expresses enormous levels of FLT3, occasionally because of activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. Here, we explored the developmental impact of FLT3 activation alone, or together with MLL-AF4, in the hematopoietic fate of hESCs. FLT3 activation does not affect specification of hemogenic precursors but significantly enhances the formation of CD45(+) blood cells, and CD45(+)CD34(+) blood progenitors with clonogenic potential. However, overexpression of FLT3 mutations or wild-type FLT3 (FLT3-WT) completely abrogates hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly affects hESC specification rather than proliferation or survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling of hESC-derived CD45(+) cells supports the FLT3-mediated inhibition of hematopoiesis in MLL-AF4-expressing hESCs, which is associated with large transcriptional changes and downregulation of genes involved in hematopoietic system development and function. Importantly, FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells, suggesting the need of alternative (epi)-genetic cooperating hits.
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Chillón MC, Gómez-Casares MT, López-Jorge CE, Rodriguez-Medina C, Molines A, Sarasquete ME, Alcoceba M, Miguel JDGS, Bueno C, Montes R, Ramos F, Rodríguez JN, Giraldo P, Ramírez M, García-Delgado R, Fuster JL, González-Díaz M, Menendez P. Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia. Leukemia 2012; 26:2360-6. [DOI: 10.1038/leu.2012.161] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
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Ramos-Mejía V, Montes R, Bueno C, Ayllón V, Real PJ, Rodríguez R, Menendez P. Residual expression of the reprogramming factors prevents differentiation of iPSC generated from human fibroblasts and cord blood CD34+ progenitors. PLoS One 2012; 7:e35824. [PMID: 22545141 PMCID: PMC3335819 DOI: 10.1371/journal.pone.0035824] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2012] [Accepted: 03/22/2012] [Indexed: 12/11/2022] Open
Abstract
Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation.
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Affiliation(s)
- Verónica Ramos-Mejía
- Centre Pfizer-Universidad de Granada-Junta de Andalucía for Genomics, Oncological Research (GENyO), Granada, Spain
- * E-mail: (VR); (PM)
| | - Rosa Montes
- Centre Pfizer-Universidad de Granada-Junta de Andalucía for Genomics, Oncological Research (GENyO), Granada, Spain
| | - Clara Bueno
- Centre Pfizer-Universidad de Granada-Junta de Andalucía for Genomics, Oncological Research (GENyO), Granada, Spain
| | - Verónica Ayllón
- Centre Pfizer-Universidad de Granada-Junta de Andalucía for Genomics, Oncological Research (GENyO), Granada, Spain
| | - Pedro J. Real
- Centre Pfizer-Universidad de Granada-Junta de Andalucía for Genomics, Oncological Research (GENyO), Granada, Spain
| | - René Rodríguez
- Centre Pfizer-Universidad de Granada-Junta de Andalucía for Genomics, Oncological Research (GENyO), Granada, Spain
| | - Pablo Menendez
- Centre Pfizer-Universidad de Granada-Junta de Andalucía for Genomics, Oncological Research (GENyO), Granada, Spain
- * E-mail: (VR); (PM)
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Real PJ, Ligero G, Ayllon V, Ramos-Mejia V, Bueno C, Gutierrez-Aranda I, Navarro-Montero O, Lako M, Menendez P. SCL/TAL1 regulates hematopoietic specification from human embryonic stem cells. Mol Ther 2012; 20:1443-53. [PMID: 22491213 DOI: 10.1038/mt.2012.49] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45(-)CD31(+)CD34(+)) and, to a lesser extent, on CD45(+) hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34(+)CD45(+)) and total (CD45(+)) blood cells with higher clonogenic potential. Short-hairpin RNA-based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.
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Affiliation(s)
- Pedro J Real
- Pfizer-Universidad de Granada-Junta de Andalucia Centre for Genomics and Oncological Research (GENyO), Granada, Spain.
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Zou C, Chou BK, Dowey SN, Tsang K, Huang X, Liu CF, Smith C, Yen J, Mali P, Zhang YA, Cheng L, Ye Z. Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines. Stem Cells Dev 2012; 21:2298-311. [PMID: 22225458 DOI: 10.1089/scd.2011.0688] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.
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Affiliation(s)
- Chunlin Zou
- Cell Therapy Center, Xuanwu Hospital, Capital Medical University, Beijing, China
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Affiliation(s)
- Ronald W Stam
- Department of Pediatric Oncology/Haematology, Erasmus MC-Sophia Children's Hospital, Dr. Molewaterplein 50, Room: Ee15-14a, 3015 GE Rotterdam, The Netherlands.
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