Lmo2 expression defines tumor cell identity during T‐cell leukemogenesis

Article7 June 2018Open Access Transparent process Lmo2 expression defines tumor cell identity during T-cell leukemogenesis Idoia García-Ramírez Idoia García-Ramírez Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Sanil Bhatia Sanil Bhatia Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany Search for more papers by this author Guillermo Rodríguez-Hernández Guillermo Rodríguez-Hernández Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Inés González-Herrero Inés González-Herrero Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Carolin Walter Carolin Walter Institute of Medical Informatics, University of Muenster, Muenster, Germany Search for more papers by this author Sara González de Tena-Dávila Sara González de Tena-Dávila Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Salma Parvin Salma Parvin Division of Hematology-Oncology, Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Department of Molecular and Cellular Pharmacology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Search for more papers by this author Oskar Haas Oskar Haas Children's Cancer Research Institute, St Anna Children's Hospital, Vienna, Austria Search for more papers by this author Wilhelm Woessmann Wilhelm Woessmann Department of Pediatric Hematology and Oncology, Justus-Liebig-University Giessen, Giessen, Germany Search for more papers by this author Martin Stanulla Martin Stanulla Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany Search for more papers by this author Martin Schrappe Martin Schrappe Department of Pediatrics, Christian-Albrechts-University of Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany Search for more papers by this author Martin Dugas Martin Dugas Institute of Medical Informatics, University of Muenster, Muenster, Germany Search for more papers by this author Yasodha Natkunam Yasodha Natkunam Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Alberto Orfao Alberto Orfao Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Servicio de Citometría and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Verónica Domínguez Verónica Domínguez Transgenesis Facility CNB-CBMSO, CSIC-UAM, Madrid, Spain Search for more papers by this author Belén Pintado Belén Pintado Transgenesis Facility CNB-CBMSO, CSIC-UAM, Madrid, Spain Search for more papers by this author Oscar Blanco Oscar Blanco Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Departamento de Anatomía Patológica, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Diego Alonso-López Diego Alonso-López Bioinformatics Unit, Cancer Research Center (CSIC-USAL), Salamanca, Spain Search for more papers by this author Javier De Las Rivas Javier De Las Rivas Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Bioinformatics and Functional Genomics Research Group, Cancer Research Center (CSIC-USAL), Salamanca, Spain Search for more papers by this author Alberto Martín-Lorenzo Alberto Martín-Lorenzo orcid.org/0000-0003-0558-890X Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Rafael Jiménez Rafael Jiménez Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Departamento de Fisiología y Farmacología, Edificio Departamental, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Francisco Javier García Criado Francisco Javier García Criado Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Departamento de Cirugía, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author María Begoña García Cenador María Begoña García Cenador Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Departamento de Cirugía, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Izidore S Lossos Izidore S Lossos Division of Hematology-Oncology, Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Department of Molecular and Cellular Pharmacology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Search for more papers by this author Carolina Vicente-Dueñas Corresponding Author Carolina Vicente-Dueñas [email protected] orcid.org/0000-0002-5401-8295 Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Arndt Borkhardt Corresponding Author Arndt Borkhardt [email protected] orcid.org/0000-0002-6121-4737 Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany Search for more papers by this author Julia Hauer Corresponding Author Julia Hauer [email protected] orcid.org/0000-0002-4058-3058 Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany Search for more papers by this author Isidro Sánchez-García Corresponding Author Isidro Sánchez-García [email protected] orcid.org/0000-0001-6989-9905 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Idoia García-Ramírez Idoia García-Ramírez Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Sanil Bhatia Sanil Bhatia Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany Search for more papers by this author Guillermo Rodríguez-Hernández Guillermo Rodríguez-Hernández Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Inés González-Herrero Inés González-Herrero Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Carolin Walter Carolin Walter Institute of Medical Informatics, University of Muenster, Muenster, Germany Search for more papers by this author Sara González de Tena-Dávila Sara González de Tena-Dávila Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Salma Parvin Salma Parvin Division of Hematology-Oncology, Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Department of Molecular and Cellular Pharmacology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Search for more papers by this author Oskar Haas Oskar Haas Children's Cancer Research Institute, St Anna Children's Hospital, Vienna, Austria Search for more papers by this author Wilhelm Woessmann Wilhelm Woessmann Department of Pediatric Hematology and Oncology, Justus-Liebig-University Giessen, Giessen, Germany Search for more papers by this author Martin Stanulla Martin Stanulla Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany Search for more papers by this author Martin Schrappe Martin Schrappe Department of Pediatrics, Christian-Albrechts-University of Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany Search for more papers by this author Martin Dugas Martin Dugas Institute of Medical Informatics, University of Muenster, Muenster, Germany Search for more papers by this author Yasodha Natkunam Yasodha Natkunam Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Alberto Orfao Alberto Orfao Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Servicio de Citometría and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Verónica Domínguez Verónica Domínguez Transgenesis Facility CNB-CBMSO, CSIC-UAM, Madrid, Spain Search for more papers by this author Belén Pintado Belén Pintado Transgenesis Facility CNB-CBMSO, CSIC-UAM, Madrid, Spain Search for more papers by this author Oscar Blanco Oscar Blanco Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Departamento de Anatomía Patológica, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Diego Alonso-López Diego Alonso-López Bioinformatics Unit, Cancer Research Center (CSIC-USAL), Salamanca, Spain Search for more papers by this author Javier De Las Rivas Javier De Las Rivas Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Bioinformatics and Functional Genomics Research Group, Cancer Research Center (CSIC-USAL), Salamanca, Spain Search for more papers by this author Alberto Martín-Lorenzo Alberto Martín-Lorenzo orcid.org/0000-0003-0558-890X Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Rafael Jiménez Rafael Jiménez Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Departamento de Fisiología y Farmacología, Edificio Departamental, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Francisco Javier García Criado Francisco Javier García Criado Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Departamento de Cirugía, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author María Begoña García Cenador María Begoña García Cenador Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Departamento de Cirugía, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Izidore S Lossos Izidore S Lossos Division of Hematology-Oncology, Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Department of Molecular and Cellular Pharmacology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Search for more papers by this author Carolina Vicente-Dueñas Corresponding Author Carolina Vicente-Dueñas [email protected] orcid.org/0000-0002-5401-8295 Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Arndt Borkhardt Corresponding Author Arndt Borkhardt [email protected] orcid.org/0000-0002-6121-4737 Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany Search for more papers by this author Julia Hauer Corresponding Author Julia Hauer [email protected] orcid.org/0000-0002-4058-3058 Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany Search for more papers by this author Isidro Sánchez-García Corresponding Author Isidro Sánchez-García [email protected] orcid.org/0000-0001-6989-9905 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain Search for more papers by this author Author Information Idoia García-Ramírez1,2,‡, Sanil Bhatia3,‡, Guillermo Rodríguez-Hernández1,2, Inés González-Herrero1,2, Carolin Walter4, Sara González de Tena-Dávila1,2, Salma Parvin5,6, Oskar Haas7, Wilhelm Woessmann8, Martin Stanulla9, Martin Schrappe10, Martin Dugas4, Yasodha Natkunam11, Alberto Orfao2,12, Verónica Domínguez13, Belén Pintado13, Oscar Blanco2,14, Diego Alonso-López15, Javier De Las Rivas2,16, Alberto Martín-Lorenzo1,2, Rafael Jiménez2,17, Francisco Javier García Criado2,18, María Begoña García Cenador2,18, Izidore S Lossos5,6, Carolina Vicente-Dueñas *,2,‡, Arndt Borkhardt *,3,‡, Julia Hauer *,3,‡ and Isidro Sánchez-García *,1,2,‡ 1Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-USAL, Salamanca, Spain 2Institute of Biomedical Research of Salamanca (IBSAL), Salamanca, Spain 3Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich-Heine University Dusseldorf, Dusseldorf, Germany 4Institute of Medical Informatics, University of Muenster, Muenster, Germany 5Division of Hematology-Oncology, Department of Medicine, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA 6Department of Molecular and Cellular Pharmacology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA 7Children's Cancer Research Institute, St Anna Children's Hospital, Vienna, Austria 8Department of Pediatric Hematology and Oncology, Justus-Liebig-University Giessen, Giessen, Germany 9Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany 10Department of Pediatrics, Christian-Albrechts-University of Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany 11Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA 12Servicio de Citometría and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain 13Transgenesis Facility CNB-CBMSO, CSIC-UAM, Madrid, Spain 14Departamento de Anatomía Patológica, Universidad de Salamanca, Salamanca, Spain 15Bioinformatics Unit, Cancer Research Center (CSIC-USAL), Salamanca, Spain 16Bioinformatics and Functional Genomics Research Group, Cancer Research Center (CSIC-USAL), Salamanca, Spain 17Departamento de Fisiología y Farmacología, Edificio Departamental, Universidad de Salamanca, Salamanca, Spain 18Departamento de Cirugía, Universidad de Salamanca, Salamanca, Spain ‡These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +34 923294813; E-mail: [email protected] *Corresponding author. Tel: +49 211 81 17680; E-mail: [email protected] *Corresponding author. Tel: +49 211 81 17680; E-mail: [email protected] *Corresponding author. Tel: +34 923294813; E-mail: [email protected] The EMBO Journal (2018)37:e98783https://doi.org/10.15252/embj.201798783 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 ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The impact of LMO2 expression on cell lineage decisions during T-cell leukemogenesis remains largely elusive. Using genetic lineage tracing, we have explored the potential of LMO2 in dictating a T-cell malignant phenotype. We first initiated LMO2 expression in hematopoietic stem/progenitor cells and maintained its expression in all hematopoietic cells. These mice develop exclusively aggressive human-like T-ALL. In order to uncover a potential exclusive reprogramming effect of LMO2 in murine hematopoietic stem/progenitor cells, we next showed that transient LMO2 expression is sufficient for oncogenic function and induction of T-ALL. The resulting T-ALLs lacked LMO2 and its target-gene expression, and histologically, transcriptionally, and genetically similar to human LMO2-driven T-ALL. We next found that during T-ALL development, secondary genomic alterations take place within the thymus. However, the permissiveness for development of T-ALL seems to be associated with wider windows of differentiation than previously appreciated. Restricted Cre-mediated activation of Lmo2 at different stages of B-cell development induces systematically and unexpectedly T-ALL that closely resembled those of their natural counterparts. Together, these results provide a novel paradigm for the generation of tumor T cells through reprogramming in vivo and could be relevant to improve the response of T-ALL to current therapies. Synopsis Genetic lineage tracing in cell type-specific mouse models of T-cell lymphoblastic leukemia (T-ALL) reveals that tumor cell identity is imposed by expression of the oncogene LMO2, rather than by the target cell phenotype. Maintained conditional expression of LMO2 in the hematopoietic lineage results in aggressive T-ALLin vivo. Restricted early LMO2 expression in hematopoietic stem/progenitor cells is sufficient to induce histological, genomic and transcriptional features of human T-ALL. Thymus deficiency impedes secondary genomic alterations required for T-ALL development. B-cell-specific LMO2 expression reprograms pro-B cells and germinal center B cells into T-ALL cells. Introduction The identification of the cell-of-origin from which acute lymphoblastic leukemia (ALL) initially arises is of great importance, both for our understanding of the basic biology of tumors and for the translation of this knowledge to the prevention, treatment, and precise prognosis of ALL (Visvader, 2011). Traditionally, the identity of the cell-of-origin was extrapolated from the immunophenotypic characterization of a leukemic cell. However, several transcriptome studies have shown that the molecular characteristics of leukemic cells do not correspond, in many cases, to what they seem to be according to their immunophenotype (Lim et al, 2009; Gilbertson, 2011). For this reason, extrapolating the identity of the cancer cell-of-origin from the ALL phenotype, without appropriate functional lineage tracing, can lead to the wrong conclusions (Molyneux et al, 2010). Lmo2 is one of the most frequent drivers of childhood T-ALL (Van Vlierberghe et al, 2006; Liu et al, 2017). LMO2 serves as a T-cell oncogene, recurrently translocated in T-ALL, and is implicated in leukemogenesis among X-linked severe combined immunodeficiency (SCID) patients, who received retroviral IL2Rγc gene therapy (Hacein-Bey-Abina et al, 2003, 2008; Pike-Overzet et al, 2007; Howe et al, 2008). Aberrant expression of LMO2 in hematopoietic stem/progenitor cells (HSC/PC) or in immature T cells (present in the thymus) leads to thymocyte self-renewal, early lymphoid precursor's accumulation, and transformation to T-ALL (McCormack et al, 2010; Treanor et al, 2011; Cleveland et al, 2013; Chambers & Rabbitts, 2015). Moreover, LMO2 was recently identified as one of the six transcription factors required for reprogramming committed murine blood cells into induced hematopoietic stem cells (Riddell et al, 2014). Notably, in addition to T-ALL, LMO2 is expressed in hematologic cancer of the B-cell lineage including DLBCL (Natkunam et al, 2007; Cubedo et al, 2012) and BCP-ALL (de Boer et al, 2011; Malumbres et al, 2011; Deucher et al, 2015). Induction of pluripotency in blood cells and LMO2 expression in B-cell malignancies suggest that LMO2 might exert leukemogenic potential in specific hematopoietic cell lineages other than the T-cell lineage. Besides that, a significant proportion of human T-ALL displays rearrangements of immunoglobulin heavy-chain genes, which additionally supports this hypothesis (Mizutani et al, 1986; Szczepanski et al, 1999; Meleshko et al, 2005). However, despite frequent alterations of Lmo2 in hematologic tumors, its impact on lineage organization during leukemogenesis and the importance of the cell-of-origin for heterogeneity and aggressiveness of Lmo2-driven tumors have remained unclear. By using in vivo genetic lineage tracing, we show that Lmo2 expression in HSC/PC as well as a precursor and mature B cells causes reprogramming and induction of T-ALL. Thereby the differentiation state of the tumor cell-of-origin influences the frequency and latency of T-ALL. These findings unveil a novel role of Lmo2 expression and demonstrate that Lmo2 promotes tumorigenesis in a manner contrasting that of other traditional oncogenes, which are persistently active in fully evolved tumor cells (Weinstein, 2002). Results Generation of a targeted mouse line conditionally expressing Lmo2 in HSCs Cell type-specific conditional activation of Lmo2 is a powerful tool for investigating the cell-of-origin of T-ALL. To achieve this aim, the Lmo2 cDNA was targeted to the ubiquitously expressed Rosa26 locus (Mao et al, 1999) where the green fluorescent protein (eGFP) was linked to the mouse Lmo2 cDNA via an internal ribosomal entry site (IRES). In the absence of Cre, neither Lmo2 nor eGFP is expressed (Appendix Fig S1A and B). Two sets of observations suggest a reprogramming effect of non-T-cell lineage cells by LMO2. First, LMO2 expression due to retroviral insertion and transactivation in CD34+ HSCs of X-SCID patients caused T-ALL but no other hematopoietic tumors (Hacein-Bey-Abina et al, 2008; Howe et al, 2008). And second, Lmo2 expression in murine blood cells negatively regulated erythroid differentiation (Visvader, 2011) and gives rise to induced pluripotent stem (iPS) cells (Batta et al, 2014; Riddell et al, 2014). We thus aimed to model the capability of Lmo2 to reprogram HSCs. Therefore, we initially crossed the Rosa26-Lmo2 mice with a Sca1-Cre mouse strain (Mainardi et al, 2014), in order to initiate Lmo2 expression in HSCs and maintain its expression in all hematopoietic cells (Appendix Fig S1C). Young Rosa26-Lmo2 + Sca1-Cre mice showed regular hematopoietic cell differentiation in the bone marrow, peripheral blood, spleen, and thymus (Appendix Figs S1C–E and S2A–D). Rosa26-Lmo2 + Sca1-Cre mice had a shorter lifespan than their wild-type (WT) littermates [Fig 1A; P < 0.0001; log-rank (Mantel–Cox) test] due to the development of T-ALL (96.7%; 30/31) that manifested as thymoma, splenomegaly, and disrupted thymic, liver, and splenic architectures (Fig 1B; Appendix Fig S3A and B). Fluorescent activating cell sorting (FACS) analysis of leukemic cells revealed an immature CD8+CD4+/− cell surface phenotype (Fig 1C; Appendix Fig S3C) with Lmo2 expression in the tumor T cells (Fig 1D) and clonal immature T-cell receptor (TCR) rearrangement (Fig 1E). We also performed whole-exome sequencing (WES) of these Lmo2+ T-ALLs (n = 9; Table 1), which were derived from thymuses of diseased Rosa26-Lmo2 + Sca1-Cre mice. We detected 23 somatic mutations, including six mutations in genes recorded in the cancer gene list (Table 1; Table EV1). Briefly, we identified recurrent Notch1 single-nucleotide variations (SNVs; 3/9) and Notch1 indels (4/9), Kras SNVs (3/9), and Nras SNVs (1/9; Table 1). This model corroborated previous findings, especially the observation from the SCID-X1 gene therapy trial, where integration of γC vector occurred close or in the LMO2 locus and Lmo2 expression was maintained throughout the progeny of the targeted cell (Hacein-Bey-Abina et al, 2003, 2008; Pike-Overzet et al, 2007; Howe et al, 2008). However, in our model Lmo2 expression was maintained constitutively, not only in HSC/PC but also in precursor and mature T cells (McCormack et al, 2010). Thus, a definite conclusion about an exclusive reprogramming effect of Lmo2 in murine HSC/PC in contrast to its expression in T-cell precursors and mature T cells was limited. Figure 1. T-ALL development in Rosa26-Lmo2 + Sca1-Cre mice Leukemia-specific survival of Rosa26-Lmo2 + Sca1-Cre mice (red line, n = 31), showing a significantly (log-rank ***P < 0.0001) shortened lifespan compared to control littermate WT mice (black line, n = 20) as a result of T-ALL development. An example of thymomas observed in the Rosa26-Lmo2 + Sca1-Cre mice studied. A thymus from a control littermate WT mouse is shown for reference. Hematoxylin and eosin staining showing infiltration of the thymus in Rosa26-Lmo2 + Sca1-Cre leukemic mice. Images are photographed at 400× magnification (scale bars: 200 μm). GFP expression in the pre-leukemic and leukemic cells from Rosa26-Lmo2 + Sca1-Cre mice, respectively. A control littermate WT mouse is shown for reference. Western blot analysis for Lmo2 and actin in T cells from the thymus of a wild-type mouse (1) and from the thymus of a Rosa26-Lmo2 + Sca1-Cre leukemic mouse (2). Tumoral cells of Rosa26-Lmo2 + Sca1-Cre T-ALL showed expression of the Lmo2 protein. TCR clonality in Rosa26-Lmo2 + Sca1-Cre mice. PCR analysis of TCR gene rearrangements in infiltrated thymuses of diseased Rosa26-Lmo2 + Sca1-Cre leukemic mice. Sorted DP T cells from the thymus of healthy mice served as a control for polyclonal TCR rearrangements. Leukemic thymus shows an increased clonality within their TCR repertoire (indicated by the code number of each Rosa26-Lmo2 + Sca1-Cre mouse analyzed). Download figure Download PowerPoint Table 1. Recurrent mutations in mouse models and human Lmo2+ T-ALL Lmo2 functions as a “hit-and-run” oncogene in T-ALL development We next addressed these limitations and modeled the scenario of HSC/PC restricted Lmo2 expression in vivo in a mouse strain where Lmo2 expression was initiated and maintained only in HSC/PC by placing Lmo2–TdTomato cDNA (Shaner et al, 2004) under the control of the stem-cell-specific Sca1 promoter (Sca1-Lmo2; Appendix Fig S4A). All T-cell subsets in the thymus contained a mosaic of Lmo2 expression (Appendix Fig S4B). Sca1-Lmo2 mice showed the regular distribution of hematopoietic populations in early post-gestational development, with TdTomato expression in all hematopoietic cell lineages (Appendix Fig S4C–G). By 3 months, a decrease in the double-positive (DP) T-cell population was accompanied by an increase in pre-leukemic double-negative (DN) T cells and CD8 T cells (Appendix Fig S4H). Lmo2 expression was detected by quantitative polymerase chain reaction (qPCR); enhanced expression of Cdkn2a was observed in the thymus in transgenic mice, consistent with the induction of Lmo2-dependent oncogenic stress (Appendix Fig S4I). The Sca1 promoter is active in subsets of T-cell precursors, and thus, both Lmo2-expressing and Lmo2-non-expressing precursor T cells coexisted in the thymus (Appendix Fig S4J). Studying whether the T-ALL cases are Lmo2-Tomato-positive or Lmo2-Tomato-negative has allowed identifying whether the Lmo2 expression is needed for the survival of T-ALL cells (Tomato+) or it serves as an earlier reprogramming event in leukemogenesis (Tomato−). Sca1-Lmo2 mice had a shorter lifespan than their wild-type (WT) littermates due to a highly disseminated form of T-ALL, consisting of a clonally immature CD8 or CD4 single-positive/DP-like population (Fig 2A–C; Appendix Fig S5A–E), as reported for human T-ALL (Van Vlierberghe et al, 2006) and Rosa26-Lmo2 + Sca1-Cre mice (Fig 1). Histological thymus sections were characterized by infiltrates of highly proliferative tumors and CD3 and TdT positivity (Fig 2B). Surprisingly, all Sca1-Lmo2 T-ALL cases studied (18 out of 21) were TdTomato−. Because there is evidence to suggest that the immunogenicity and cytotoxicity of the fluorescent marker potentially may confound the interpretation of in vivo experimental data (Ansari et al, 2016), we next formally excluded the possibility that the cells that were originally marked with the fluorescent marker cannot be accurately traced over time by showing that tumors had lost their Lmo2 expression by three different complementary approaches: immunohistochemistry (Fig 2C) and both real-time PCR and Western blot in sorted-purified leukemic Sca1-Lmo2 cells (Fig 2D and E). This observation indicates that an early expression of the Lmo2 oncogene in HSC/PC has the potential to induce aggressive T-ALL without any need for its perpetual expression to develop T-ALL. Figure 2. Reprogramming of HS/PCs cells to aggressive malignant mature T cells T-ALL-specific survival of Sca1-Lmo2 mice (red line, n = 21), showing a significantly (log-rank ***P < 0.0001) shortened lifespan compared to control littermate WT mice (blue line, n = 20) as a result of mature T-cell malignancies. An example of thymomas observed in 100% (21/21) of the Sca1-Lmo2 mice studied. A thymus from a control littermate WT mouse is shown for reference. Hema