Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML 1‐ ETO mouse model
2013; Springer Nature; Volume: 5; Issue: 12 Linguagem: Inglês
10.1002/emmm.201302661
ISSN1757-4684
AutoresNina Cabezas‐Wallscheid, Victoria Eichwald, Jos de Graaf, Martin Löwer, Hans‐Anton Lehr, Andreas Kreft, Leonid Eshkind, Andreas Hildebrandt, Yasmin Abassi, Rosario Heck, Anna Katharina Dehof, Svetlana Ohngemach, Rolf Sprengel, Simone Wörtge, Steffen Schmitt, Johannes Lotz, Claudius U. Meyer, Thomas Kindler, Dong‐Er Zhang, Bernd Kaina, John C. Castle, Andreas Trumpp, Uğur Şahin, Ernesto Bockamp,
Tópico(s)Cancer Genomics and Diagnostics
ResumoResearch Article4 October 2013Open Access Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model Nina Cabezas-Wallscheid Nina Cabezas-Wallscheid Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany German Cancer Research Center, Department of Stem Cells and Cancer, Heidelberg, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Victoria Eichwald Victoria Eichwald German Cancer Research Center, Division of Molecular Immunology, Heidelberg, Germany Search for more papers by this author Jos de Graaf Jos de Graaf TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Martin Löwer Martin Löwer TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Hans-Anton Lehr Hans-Anton Lehr University of Lausanne, Institut Universitaire de Pathologie, CHUV, Lausanne, Switzerland Search for more papers by this author Andreas Kreft Andreas Kreft Medical Center of the Johannes Gutenberg-University Mainz, Department of Pathology, Mainz, Germany Search for more papers by this author Leonid Eshkind Leonid Eshkind Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Andreas Hildebrandt Andreas Hildebrandt Johannes Gutenberg-University Mainz, Institute for Informatics, Mainz, Germany Search for more papers by this author Yasmin Abassi Yasmin Abassi Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Rosario Heck Rosario Heck Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Anna Katharina Dehof Anna Katharina Dehof Saarland University, Center for Bioinformatics, Saarbrücken, Germany Search for more papers by this author Svetlana Ohngemach Svetlana Ohngemach Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Rolf Sprengel Rolf Sprengel Max-Planck-Institute for Medical Research, Heidelberg, Germany Search for more papers by this author Simone Wörtge Simone Wörtge Max-Planck-Institute for Medical Research, Heidelberg, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany Search for more papers by this author Steffen Schmitt Steffen Schmitt German Cancer Research Center, FACS Core Facility, Heidelberg, Germany Search for more papers by this author Johannes Lotz Johannes Lotz Medical Center of the Johannes Gutenberg-University Mainz, Institute of Clinical Chemistry and Laboratory Medicine, Mainz, Germany Search for more papers by this author Claudius Meyer Claudius Meyer Medical Center of the Johannes Gutenberg-University Mainz Children's Hospital, Mainz, Germany Search for more papers by this author Thomas Kindler Thomas Kindler Me, dical Center of the Johannes Gutenberg-University Mainz, Division of Haematology, Oncology and Pneumology, III. Medical Department, Mainz, Germany Search for more papers by this author Dong-Er Zhang Dong-Er Zhang Department of Pathology, University of California San Diego, Division of Biological Sciences and Moores UCSD Cancer Center, San Diego, CA, USA Search for more papers by this author Bernd Kaina Bernd Kaina Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author John C. Castle John C. Castle TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Andreas Trumpp Andreas Trumpp German Cancer Research Center, Department of Stem Cells and Cancer, Heidelberg, Germany Search for more papers by this author Ugur Sahin Ugur Sahin TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Ernesto Bockamp Corresponding Author Ernesto Bockamp Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Nina Cabezas-Wallscheid Nina Cabezas-Wallscheid Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany German Cancer Research Center, Department of Stem Cells and Cancer, Heidelberg, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Victoria Eichwald Victoria Eichwald German Cancer Research Center, Division of Molecular Immunology, Heidelberg, Germany Search for more papers by this author Jos de Graaf Jos de Graaf TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Martin Löwer Martin Löwer TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Hans-Anton Lehr Hans-Anton Lehr University of Lausanne, Institut Universitaire de Pathologie, CHUV, Lausanne, Switzerland Search for more papers by this author Andreas Kreft Andreas Kreft Medical Center of the Johannes Gutenberg-University Mainz, Department of Pathology, Mainz, Germany Search for more papers by this author Leonid Eshkind Leonid Eshkind Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Andreas Hildebrandt Andreas Hildebrandt Johannes Gutenberg-University Mainz, Institute for Informatics, Mainz, Germany Search for more papers by this author Yasmin Abassi Yasmin Abassi Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Rosario Heck Rosario Heck Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Anna Katharina Dehof Anna Katharina Dehof Saarland University, Center for Bioinformatics, Saarbrücken, Germany Search for more papers by this author Svetlana Ohngemach Svetlana Ohngemach Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Rolf Sprengel Rolf Sprengel Max-Planck-Institute for Medical Research, Heidelberg, Germany Search for more papers by this author Simone Wörtge Simone Wörtge Max-Planck-Institute for Medical Research, Heidelberg, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany Search for more papers by this author Steffen Schmitt Steffen Schmitt German Cancer Research Center, FACS Core Facility, Heidelberg, Germany Search for more papers by this author Johannes Lotz Johannes Lotz Medical Center of the Johannes Gutenberg-University Mainz, Institute of Clinical Chemistry and Laboratory Medicine, Mainz, Germany Search for more papers by this author Claudius Meyer Claudius Meyer Medical Center of the Johannes Gutenberg-University Mainz Children's Hospital, Mainz, Germany Search for more papers by this author Thomas Kindler Thomas Kindler Me, dical Center of the Johannes Gutenberg-University Mainz, Division of Haematology, Oncology and Pneumology, III. Medical Department, Mainz, Germany Search for more papers by this author Dong-Er Zhang Dong-Er Zhang Department of Pathology, University of California San Diego, Division of Biological Sciences and Moores UCSD Cancer Center, San Diego, CA, USA Search for more papers by this author Bernd Kaina Bernd Kaina Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author John C. Castle John C. Castle TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Andreas Trumpp Andreas Trumpp German Cancer Research Center, Department of Stem Cells and Cancer, Heidelberg, Germany Search for more papers by this author Ugur Sahin Ugur Sahin TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Ernesto Bockamp Corresponding Author Ernesto Bockamp Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany Search for more papers by this author Author Information Nina Cabezas-Wallscheid1,2,3, Victoria Eichwald4, Jos de Graaf5, Martin Löwer5, Hans-Anton Lehr6, Andreas Kreft7, Leonid Eshkind1,3, Andreas Hildebrandt8, Yasmin Abassi1,3, Rosario Heck1,3, Anna Katharina Dehof9, Svetlana Ohngemach1,3, Rolf Sprengel10, Simone Wörtge10,11, Steffen Schmitt12, Johannes Lotz13, Claudius Meyer14, Thomas Kindler15, Dong-Er Zhang16, Bernd Kaina3, John C. Castle5, Andreas Trumpp2, Ugur Sahin5 and Ernesto Bockamp 1,3 1Medical Center of the Johannes Gutenberg-University Mainz, Department of Internal Medicine III, Division of Translational and Experimental Oncology, Mainz, Germany 2German Cancer Research Center, Department of Stem Cells and Cancer, Heidelberg, Germany 3Medical Center of the Johannes Gutenberg-University Mainz, Institute for Toxicology, Mainz, Germany 4German Cancer Research Center, Division of Molecular Immunology, Heidelberg, Germany 5TRON – Translational Oncology at the Johannes Gutenberg-University Mainz, Mainz, Germany 6University of Lausanne, Institut Universitaire de Pathologie, CHUV, Lausanne, Switzerland 7Medical Center of the Johannes Gutenberg-University Mainz, Department of Pathology, Mainz, Germany 8Johannes Gutenberg-University Mainz, Institute for Informatics, Mainz, Germany 9Saarland University, Center for Bioinformatics, Saarbrücken, Germany 10Max-Planck-Institute for Medical Research, Heidelberg, Germany 11Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany 12German Cancer Research Center, FACS Core Facility, Heidelberg, Germany 13Medical Center of the Johannes Gutenberg-University Mainz, Institute of Clinical Chemistry and Laboratory Medicine, Mainz, Germany 14Medical Center of the Johannes Gutenberg-University Mainz Children's Hospital, Mainz, Germany 15Me, dical Center of the Johannes Gutenberg-University Mainz, Division of Haematology, Oncology and Pneumology, III. Medical Department, Mainz, Germany 16Department of Pathology, University of California San Diego, Division of Biological Sciences and Moores UCSD Cancer Center, San Diego, CA, USA *Corresponding author: Tel: +49 6131 17 9784; Fax: +49 6131 230506;E-mail: [email protected] EMBO Mol Med (2013)5:1804-1820https://doi.org/10.1002/emmm.201302661 →See accompanying article 10.1002/emmm.201303483 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMPs) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative disease (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option. INTRODUCTION Acute myeloid leukaemia (AML) is a heterogeneous group of severe haematological diseases characterized by a block in myeloid differentiation and the unrestrained proliferation of immature myeloid cells (Estey & Dohner, 2006). One of the most frequent genetic alterations found in human AML is the t(8;21)(q22;q22) AML1-ETO chromosomal translocation that is commonly associated with core binding factor (CBF) AML (Arber et al, 2008). Despite the great clinical improvements that have been made in the treatment of AML, t(8;21)-associated CBF AML remains a significant clinical problem, with 30% of patients relapsing and long-term survival rates ranging between 61 and 31% (Appelbaum et al, 2006; Döhner et al, 2010; Grimwade et al, 2010; Lin et al, 2008; Marcucci et al, 2005; Narimatsu et al, 2008). In case of the t(8;21) translocation, a fusion between the DNA-binding Runt homology domain of the haematopoietic master regulator AML1 (RUNX1, CBFα2 and PEBPαB) and the ETO gene (RUNX1T1 or MTG8) generates the 752 amino acid long chimeric AML1-ETO (AE) protein (Miyoshi et al, 1993). Although functionally similar to the AML1 transcription factor, the AE fusion protein has a different sub-cellular localization, distinct biochemical and molecular properties and altered transcriptional activity (Lam & Zhang, 2012; Reikvam et al, 2011). Important insights into the molecular consequences of aberrant AE expression have been gained from microarray and chromatin immunoprecipitation experiments. In these studies, transcriptional AE target genes and epigenetic modifications were identified that link AE function to cellular proliferation, self-renewal and differentiation (Alcalay et al, 2003; Balgobind et al, 2011; Kvinlaug et al, 2011; Ptasinska et al, 2012; Ross et al, 2004; Valk et al, 2004). However, because these experiments were based on the analysis of direct transcriptional modifications promoted by short-term AE expression or deletion and on biopsies from AML patients, the stepwise evolution of transcriptome-wide alterations downstream of the initial t(8;21) translocation are essentially unknown. In order to understand the cellular programs operating during the trajectory to leukaemia and to define novel therapeutic agents that can interfere with these pathways, it is critical to analyse preclinical mouse models that recapitulate the stepwise evolution and the initial mosaic expression of AE in blood cells characteristic of the human disease. In recent years leukaemic stem cells (LSCs) have attracted major attention as critical therapeutic targets as these cells have been proposed to drive leukaemia initiation, progression and maintenance (Baccelli & Trumpp, 2012; Dick, 2008). In addition, LSC are thought to be resistant to current chemotherapeutic regimes and thus might act as a reservoir for relapse (Ishikawa et al, 2007). For this reason, the identification and functional characterization of LSC has potentially profound clinical implications. Phenotypic, molecular and biochemical knowledge of LSC has been obtained for several AML subtypes. These studies demonstrated that AML LSC can be heterogeneous with respect to their cell surface phenotype and state of commitment [reviewed in (Horton & Huntly, 2012)]. However, the nature and molecular characteristics of t(8;21)-associated LSC still remain elusive. Finally, it is not known if ablation of AE function during manifest AML will provide a benefit to the patient. Indeed, specific inhibition of a single leukaemia-maintaining factor can be a highly effective therapy for chronic myeloid leukaemia (CML), as illustrated by targeted therapeutics like Imatinib (Druker et al, 2006). Since AE expression is a recurrent clinical feature in CBF AML, the concept of targeting AE has been proposed and first strategies that specifically inactivate AE function have been reported (Barton et al, 2009; Wang et al, 2011; Wichmann et al, 2010). In order to evaluate the potential therapeutic benefit of AE ablation and to decide if further research in this direction is warranted, in vivo proof-of-principle experiments are required. Using a novel experimental mouse model that recapitulates the slow disease evolution and mosaic expression of AE found in human AML, we report the first in vivo analysis of whole transcriptome alterations taking place immediately after the initial activation of AE and during the trajectory to leukaemic disease. We also show that the ability to initiate and maintain leukaemia is not only restricted to those cells that phenotypically resemble HSC but also resides in the granulocyte macrophage progenitor (GMP) population of committed myeloid cells. Finally, we demonstrate that long-term expression of AE consistently induces a phenotype of indolent myeloproliferative disease (MDP)-like myeloid leukaemia with complete penetrance and report that inactivation of AE function is a potential novel therapeutic option. RESULTS Generation of a blood cell-specific conditional AE mouse model We initiated this study with the aim to analyse global signalling pathways and cellular mechanisms that operate during the progressive disease evolution downstream of the t(8;21) translocation. In the past several mouse models for t(8;21)-associated leukaemia have been developed [reviewed in (Lam & Zhang, 2012; Reikvam et al, 2011)]. However, with these models the initial mosaic expression pattern of AE and the delayed appearance of secondary mutations, characteristic for human CBF AML, were not recapitulated. To be able to address the role of AE during the dynamic evolution towards manifest leukaemia and to dissect the molecular and cellular programs operating during this process, we wanted to establish a model that allows to conditionally and reversibly express AE in a fraction of all blood cell types. To completely restrict AE expression to haematopoietic cells, we transplanted whole bone marrow (BM) from compound ROSA26-iM2-tetO-GFP/TgPtet-AML1-ETO (R26/AE) mice into lethally irradiated recipients (Rhoades et al, 2000; Wortge et al, 2010). As illustrated in Fig 1A, in reconstituted mice the expression of AE and the GFP co-reporter gene can be regulated by administering doxycycline (DOX) [for review see (Bockamp et al, 2002; Bockamp et al, 2008)]. We have previously shown that the R26-iM2 effector mouse strain directed mosaic transgene expression to lineage−/c-Kit+/Sca1+(L−K+S+) haematopoietic progenitor cells and to different adult blood lineages (Wortge et al, 2010). In line with this mosaic expression pattern, we found conditional GFP activation in a percentage of long-term repopulating haematopoietic stem cells (LT-HSC, L−K+S+CD150+CD48− Flt3−CD34−), short-term repopulating haematopoietic stem cells (ST-HSC, L−K+S+CD150+CD48−Flt3−CD34+), common myeloid progenitors (CMPs, L−K+S−IL7Rα−CD34+FcγRII/IIIlow), granulocyte macrophage progenitors (GMPs, L−K+S−IL7Rα−CD34+FcγRII/III+), megakaryocyte erythrocyte progenitors (MEPs, L−K+S−IL7Rα−CD34−FcγRII/IIIlow) and common lymphoid progenitors (CLPs, LKintSintIL7Rα+) of R26/AE reconstituted recipients (Fig 1B, Supporting Information Fig S1A). Furthermore, analysis of median fluorescence revealed comparable GFP fluorescence in LT-, ST-HSC, CMP, GMP and MEP indicating that the R26-iM2 effector mouse model directed similar transgene activation levels to these populations (Supporting Information Fig S1B). To determine if AE induction was strictly dependent on the presence of DOX and to confirm AE transcription in different haematopoietic cell types, we subjected whole BM, immature and mature haematopoietic cells to quantitative rtPCR analysis. As shown in Fig 1C, in the absence of DOX AE mRNA was not detectable in BM cells of R26/AE animals (BM −DOX). By contrast, following DOX induction AE-specific transcripts were present in BM (BM +DOX), L−K+S+ cells, lineage-committed haematopoietic progenitors and adult blood cells (Fig 1C). Figure 1. Conditional AE expression alters normal blood cell development. DOX-dependent AE and GFP expression. Conditional GFP expression in blood stem and progenitor cells. Green bars represent the percentage of GFP-expressing cells ± SD in each lineage. Shown are mean values for four R26/AE BM-reconstituted recipients that had been DOX exposed during 10 days. LT-/ST-HSC, long-term/short-term repopulating haematopoietic stem cells; CMP, common myeloid progenitor; GMP, granulocyte macrophage progenitor; MEP, megakaryocyte erythroid progenitor; CLP, common lymphoid progenitor. Blue bars show mean AE mRNA levels ± SD from three compound R26/AE mice in the absence (−DOX) or after 3 days of DOX exposure (+DOX). AE mRNA was determined in BM, L−K+S+ cells, CMP, GMP, MEP, B220+ B-lymphoid cells, CD3+ T-lymphoid cells, Ter119+ erythrocytes, CD41+ megakaryocytes, CD11b+ monocytes and macrophages and Gr1+ myeloid cells. AE levels are represented relative to AE mRNA transcription in the Kasumi-1 cell line (white bar). Peripheral blood films from a representative control and an AE-expressing mouse. Arrowheads indicate abnormal red blood cells. Above the bar diagram a spleen (SPL), thymus (THY) and lymph node (LN) of a non-induced (left) and an AE-expressing mouse (right) are shown. Each bar represents the mean weight ± SD for five individual mice. White bars represent non-induced and black bars AE-expressing mice. Two-tailed unpaired t-test analysis demonstrated significant differences in spleen (p = 0.0331), thymus (p = 0.0021) and lymph node (p = 0.0093) weight between control and AE-induced mice. Flow cytometric analysis of erythroid, granulocytic, magakaryocytic, B- and T-cell maturation profiles from controls (white bars) and mice that have been DOX-induced (black bars, +DOX nine months). For each cell type the absolute percentages ± SD of cells in the BM or the thymus are shown. Roman numerals designate different stages of erythrocyte maturation: I, proerythroblasts; II, basophilic erythroblasts; III, late basophilic erythroblasts and chromatophilic erythroblasts; IV, orthochromatophilic erythroblasts and reticulocytes. 7AAD, 7-amino-actinomycin D; DN, double negative and DP, double positive for CD4 and CD8 surface markers. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant. Download figure Download PowerPoint Taken together, the finding that conditional GFP activation was restricted to a minor percentage of LT-, ST-HSC and blood cell progenitors further extended previous results and demonstrated that the ROSA26 promoter activated transgene expression only in a limited number of cells within the stem and progenitor cell compartment. Our findings also provide direct evidence that the R26/AE transplantation model is suitable for conditionally activating AE expression in blood cells with no background. AE expression alters normal haematopoiesis A growing body of evidence suggests that the t(8;21) translocation is only leukaemia-initiating but not sufficient for the induction of AML (Kelly & Gilliland, 2002; Lam & Zhang, 2012; Reikvam et al, 2011). The constant association between the t(8;21) translocation and AML furthermore argues that aberrant AE rearrangement in HSC and thus also in their more differentiated progeny will change normal haematopoiesis in a way that specifically favours the later development of AML. To assess downstream effects promoted by AE expression and to evaluate the time needed to induce a phenotype, haematopoietic parameters were monitored. In agreement with previous studies (Buchholz et al, 2000; Higuchi et al, 2002; Rhoades et al, 2000; Yuan et al, 2001), AE expression for 6 months did not produce any obvious effects in blood cell parameters and organ morphology. Conversely, between 8 and 10 months, we noticed first phenotypic alterations. Analysis of differential blood cell parameters between AE-expressing and non-induced control mice revealed a significant drop in peripheral red blood cells that was accompanied by reduced levels of haemoglobin and an increase in eosinophils and large peroxidase-negative cells (Supporting Information Fig S2). Furthermore, inspection of peripheral blood morphology documented atypical red blood cells characterized by a polychromatic, aniso- and poikilocytotic phenotype (Fig 1D, arrowheads). In addition, we noticed that AE-expressing animals presented marked splenomegaly and a significant reduction of the thymus and the lymph nodes (Fig 1E). Histological analysis of thymus and lymph nodes revealed no additional abnormalities. By contrast, AE spleens showed clear signs of extramedullary haematopoiesis and in particular an increase in erythropoiesis within the red pulp (Supporting Information Fig S3 arrowheads). Flow cytometry directly confirmed the accumulation of erythrocytes in AE spleens and documented a significant increase in CD11b+/Gr1+ granulocytes but not in B lymphocytes and L−K+S+ cells (Supporting Information Fig S4). Consistent with the immature morphology of peripheral erythrocytes and revealing a defect in red blood cell maturation, BM pro-erythroblasts were increased and the relative percentage of more mature orthochromatophilic erythroblasts and reticulocytes was significantly reduced in AE expressing animals (I and IV, Fig 1F, Supporting Information Fig S5A). Moreover, immature and the relative percentage of mature granulocytes were augmented while megakaryocytes did not significantly change (Fig 1F, Supporting Information Fig S5B and C). With regard to lymphopoiesis, we found a decrease in immature and mature B cells and a reduction in DN3 and CD4+/CD8+ double positive (DP) T lymphoid cells (Fig 1F, Supporting Information Fig S6). Collectively, these data provide direct in vivo evidence that mosaic expression of AE in blood cells for more than 6 months altered normal haematopoietic lineage development and specifically increased the output of myeloid cells at the expense of erythro- and lymphopoiesis. AE expression specifically increases lineage-committed myeloid progenitors Adoptive transfer experiments using retrovirus-transduced BM cells suggested that enforced AE expression expanded the number of HSC (de Guzman et al, 2002). However, in this study HSC were defined as L−K+S+ cells, which represent a heterogeneous group of immature blood cells, and well defined HSC subgroups like LT-, ST-HSC and blood cell progenitors were not explicitly analysed. To establish the relevance of blood cell-specific AE induction for the HSC compartment, we analysed LT-, ST-HSC and haematopoietic progenitors. Interestingly, DOX exposure for 8–10 months did not significantly change LT- and ST-HSC (Fig 2A, Supporting Information Fig S7). Equally, CLP were not affected (Fig 2B, Supporting Information Fig S8A). However, in AE-expressing mice, GMP were significantly expanded and the relative percentages of MEP as well as CMP were reduced (Fig 2C, Supporting Information Fig S8B). In line with the increase in myelopoiesis found in vivo and also in agreement with previous reports (de Guzman et al, 2002; Higuchi et al, 2002; Okuda et al, 1996; Rhoades et al, 2000; Schwieger et al, 2002) we observed a significant increase in ex vivo granulocyte macrophage (CFU-GM) colony formation (Fig 2D). These results demonstrated that AE induction did not significantly increase LT-, ST-HSC and blood cell progenitors but specifically expanded the GMP population. Figure 2.AE expression specifically increases GMP. * p < 0.05; ** p < 0.01; ns, not significant. Flow cytometric analysis of LT- and ST-HS
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