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1. Developmental interplay between transcriptional alterations and a targetable cytokine signaling dependency in pediatric ETO2::GLIS2 leukemia

Author

Verónica Alonso-Pérez, Klaudia Galant, Fabien Boudia, Elie Robert, Zakia Aid, Laurent Renou, Vilma Barroca, Saryiami Devanand, Loélia Babin, Virginie Rouiller-Fabre, Delphine Moison, Didier Busso, Guillaume Piton, Christophe Metereau, Nassera Abermil, Paola Ballerini, Pierre Hirsch, Rima Haddad, Jelena Martinovic, Arnaud Petit, Hélène Lapillonne, Erika Brunet, Thomas Mercher, and Françoise Pflumio

Subjects
Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

Abstract Background Several fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia (AML) are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, associated with dismal prognosis. Methods We created novel ETO2::GLIS2 models of leukemogenesis through lentiviral transduction and CRISPR-Cas9 gene editing of human fetal and post-natal hematopoietic stem/progenitor cells (HSPCs), performed in-depth characterization of ETO2::GLIS2 transformed cells through multiple omics and compared them to patient samples. This led to a preclinical assay using patient-derived-xenograft models to test a combination of two clinically-relevant molecules. Results We showed that ETO2::GLIS2 expression in primary human fetal CD34+ hematopoietic cells led to more efficient in vivo leukemia development than expression in post-natal cells. Moreover, cord blood-derived leukemogenesis has a major dependency on the presence of human cytokines, including IL3 and SCF. Single cell transcriptomes revealed that this cytokine environment controlled two ETO2::GLIS2-transformed states that were also observed in primary patient cells. Importantly, this cytokine sensitivity may be therapeutically-exploited as combined MEK and BCL2 inhibition showed higher efficiency than individual molecules to reduce leukemia progression in vivo. Conclusions Our study uncovers an interplay between the cytokine milieu and transcriptional programs that extends a developmental window of permissiveness to transformation by the ETO2::GLIS2 AML fusion oncogene, controls the intratumoral cellular heterogeneity, and offers a ground-breaking therapeutical opportunity by a targeted combination strategy.

Published
2024
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2. The ETO2 transcriptional cofactor maintains acute leukemia by driving a MYB/EP300‐dependent stemness program

Author

Alexandre Fagnan, Zakia Aid, Marie Baille, Aneta Drakul, Elie Robert, Cécile K. Lopez, Cécile Thirant, Yann Lecluse, Julie Rivière, Cathy Ignacimouttou, Silvia Salmoiraghi, Eduardo Anguita, Audrey Naimo, Christophe Marzac, Françoise Pflumio, Sébastien Malinge, Christian Wichmann, Yun Huang, Camille Lobry, Julie Chaumeil, Eric Soler, Jean‐Pierre Bourquin, Claus Nerlov, Olivier A. Bernard, Juerg Schwaller, and Thomas Mercher

Subjects
Diseases of the blood and blood-forming organs, RC633-647.5
Abstract

Abstract Transcriptional cofactors of the ETO family are recurrent fusion partners in acute leukemia. We characterized the ETO2 regulome by integrating transcriptomic and chromatin binding analyses in human erythroleukemia xenografts and controlled ETO2 depletion models. We demonstrate that beyond its well‐established repressive activity, ETO2 directly activates transcription of MYB, among other genes. The ETO2‐activated signature is associated with a poorer prognosis in erythroleukemia but also in other acute myeloid and lymphoid leukemia subtypes. Mechanistically, ETO2 colocalizes with EP300 and MYB at enhancers supporting the existence of an ETO2/MYB feedforward transcription activation loop (e.g., on MYB itself). Both small‐molecule and PROTAC‐mediated inhibition of EP300 acetyltransferases strongly reduced ETO2 protein, chromatin binding, and ETO2‐activated transcripts. Taken together, our data show that ETO2 positively enforces a leukemia maintenance program that is mediated in part by the MYB transcription factor and that relies on acetyltransferase cofactors to stabilize ETO2 scaffolding activity.

Published
2024
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3. Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model

Author

Brahim Arkoun, Elie Robert, Fabien Boudia, Stefania Mazzi, Virginie Dufour, Aurélie Siret, Yasmine Mammasse, Zakia Aid, Matthieu Vieira, Aygun Imanci, Marine Aglave, Marie Cambot, Rachel Petermann, Sylvie Souquere, Philippe Rameau, Cyril Catelain, Romain Diot, Gérard Tachdjian, Olivier Hermine, Nathalie Droin, Najet Debili, Isabelle Plo, Sébastien Malinge, Eric Soler, Hana Raslova, Thomas Mercher, and William Vainchenker

Subjects
Hematology, Oncology, Medicine
Abstract

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/–, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell–based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

Published
2023
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4. De novo generation of the NPM-ALK fusion recapitulates the pleiotropic phenotypes of ALK+ ALCL pathogenesis and reveals the ROR2 receptor as target for tumor cells

Author

Loélia Babin, Alice Darchen, Elie Robert, Zakia Aid, Rosalie Borry, Claire Soudais, Marion Piganeau, Anne De Cian, Carine Giovannangeli, Olivia Bawa, Charlotte Rigaud, Jean-Yves Scoazec, Lucile Couronné, Layla Veleanu, Agata Cieslak, Vahid Asnafi, David Sibon, Laurence Lamant, Fabienne Meggetto, Thomas Mercher, and Erika Brunet

Subjects
ALK+ ALCL, Lymphoma, NPM-ALK fusion, CRISPR/Cas9 models, WNT, ROR2, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

Abstract Background Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. Methods We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. Results Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. Conclusions In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.

Published
2022
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5. The Pediatric Acute Leukemia Fusion Oncogene ETO2-GLIS2 Increases Self-Renewal and Alters Differentiation in a Human Induced Pluripotent Stem Cells-Derived Model

Author

Salvatore Nicola Bertuccio, Fabien Boudia, Marie Cambot, Cécile K. Lopez, Larissa Lordier, Alessandro Donada, Elie Robert, Cécile Thirant, Zakia Aid, Salvatore Serravalle, Annalisa Astolfi, Valentina Indio, Franco Locatelli, Andrea Pession, William Vainchenker, Riccardo Masetti, Hana Raslova, and Thomas Mercher

Subjects
Diseases of the blood and blood-forming organs, RC633-647.5
Published
2020
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6. Supplementary Figures and Legends from Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene–Driven Myeloid Leukemia

Author

Thomas Mercher, Juerg Schwaller, Françoise Pflumio, Arnaud Petit, Berthold Göttgens, Olivier A. Bernard, Isabelle Godin, Camille Lobry, Nathalie Droin, Claus Nerlov, Sébastien Malinge, Franco Locatelli, Riccardo Masetti, Muriel Gaudry, William Vainchenker, Antoine H.F.M. Peters, Eric Delabesse, Jean Luc Villeval, Loélia Babin, Erika Brunet, Yann Lecluse, Bastien Job, M'Boyba Diop, Sarah J. Kinston, Marie-Laure Arcangeli, Alexandre Fagnan, Cécile Thirant, Fabien Boudia, Hélène Lapillonne, Chrystèle Bilhou-Nabera, Paola Ballerini, Zakia Aid, Elie Robert, Vaia Stavropoulou, Esteve Noguera, and Cécile K. Lopez

Abstract

Supplementary Figures and Legends

Published
2023
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7. Data from Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene–Driven Myeloid Leukemia

Author

Thomas Mercher, Juerg Schwaller, Françoise Pflumio, Arnaud Petit, Berthold Göttgens, Olivier A. Bernard, Isabelle Godin, Camille Lobry, Nathalie Droin, Claus Nerlov, Sébastien Malinge, Franco Locatelli, Riccardo Masetti, Muriel Gaudry, William Vainchenker, Antoine H.F.M. Peters, Eric Delabesse, Jean Luc Villeval, Loélia Babin, Erika Brunet, Yann Lecluse, Bastien Job, M'Boyba Diop, Sarah J. Kinston, Marie-Laure Arcangeli, Alexandre Fagnan, Cécile Thirant, Fabien Boudia, Hélène Lapillonne, Chrystèle Bilhou-Nabera, Paola Ballerini, Zakia Aid, Elie Robert, Vaia Stavropoulou, Esteve Noguera, and Cécile K. Lopez

Abstract

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2–GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2–GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2–GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes.Significance:This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.See related commentary by Cruz Hernandez and Vyas, p. 1653.This article is highlighted in the In This Issue feature, p. 1631

Published
2023
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8. Supplementary Tables 1 to 11 from Ontogenic Changes in Hematopoietic Hierarchy Determine Pediatric Specificity and Disease Phenotype in Fusion Oncogene–Driven Myeloid Leukemia

Author

Thomas Mercher, Juerg Schwaller, Françoise Pflumio, Arnaud Petit, Berthold Göttgens, Olivier A. Bernard, Isabelle Godin, Camille Lobry, Nathalie Droin, Claus Nerlov, Sébastien Malinge, Franco Locatelli, Riccardo Masetti, Muriel Gaudry, William Vainchenker, Antoine H.F.M. Peters, Eric Delabesse, Jean Luc Villeval, Loélia Babin, Erika Brunet, Yann Lecluse, Bastien Job, M'Boyba Diop, Sarah J. Kinston, Marie-Laure Arcangeli, Alexandre Fagnan, Cécile Thirant, Fabien Boudia, Hélène Lapillonne, Chrystèle Bilhou-Nabera, Paola Ballerini, Zakia Aid, Elie Robert, Vaia Stavropoulou, Esteve Noguera, and Cécile K. Lopez

Abstract

Supplementary Tables 1 to 11

Published
2023
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9. Figure S7 from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

Efficacy of conventional chemotherapeutic agents alone or in combination with Trametinib in PDX B-ALL cells in vitro

Published
2023
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10. Supplementary Data from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

Material and Methods, Figure legends

Published
2023
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11. Supplementary Table 3 from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

List of the SNVs identified through RNA-sequencing

Published
2023
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12. Supplementary Table 7 from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

Differentially expressed genes in the human B-ALL cohort

Published
2023
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13. Supplementary Table 1 from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

Characteristics of the B-ALL cohort

Published
2023
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14. Supplementary Table 8 from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

Reagents and Resources

Published
2023
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15. Data from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

Purpose:Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL.Experimental Design:To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents.Results:Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRASG12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine.Conclusions:Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia.

Published
2023
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16. Supplementary Table 2 from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

List of the SNVs identified through whole exome sequencing

Published
2023
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17. Supplementary Table 4 from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

List of fusion transcripts identified through RNA-sequencing

Published
2023
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18. Supplementary Table 6 from Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Sebastien Malinge, Thomas Mercher, Eric Delabesse, Jean-Pierre Bourquin, Paola Ballerini, Birgit Geoerger, Rishi S. Kotecha, Carole Barin Bonnigal, Elizabeth Macintyre, Olivier A. Bernard, Muriel Gaudry, John D. Crispino, Laurence C. Cheung, Beat C. Bornhauser, Nathalie Droin, Yann Lecluse, Estelle Daudigeos, Gaelle Pierron, Damien Plassard, Stephanie Lagarde, Nais Prade, Zakia Aid, Damien Roos-Weil, Yi-Chien Tsai, Silvia Jenni, M'Boyba Diop, Kunjal Panchal, Cathy Ignacimouttou, Aurélie Siret, and Anouchka P. Laurent

Abstract

List of datasets enriched in murine B cell precursors (GSEA analyses)

Published
2023
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19. Stepwise GATA1 and SMC3 mutations alter megakaryocyte differentiation in a Down syndrome leukemia model

Author

Brahim Arkoun, Elie Robert, Fabien Boudia, Stefania Mazzi, Virginie Dufour, Aurélie Siret, Yasmine Mammasse, Zakia Aid, Matthieu Vieira, Imanci Aygun, Marine Aglave, Marie Cambot, Rachel Petermann, Sylvie Souquere, Philippe Rameau, Cyril Catelain, Romain Diot, Gérard Tachdjian, Olivier Hermine, Nathalie Droin, Najet Debili, Isabelle Plo, Sébastien Malinge, Eric Soler, Hana Raslova, Thomas Mercher, and William Vainchenker

Subjects
Chondroitin Sulfate Proteoglycans, Chromosomal Proteins, Non-Histone, Leukemia, Megakaryoblastic, Acute, Mutation, Humans, Cell Cycle Proteins, GATA1 Transcription Factor, Trisomy, General Medicine, Down Syndrome, Child, Megakaryocytes, Hematopoiesis
Abstract

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/-, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell-based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

Published
2022
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20. High caspase 3 and vulnerability to dual BCL2 family inhibition define ETO2::GLIS2 pediatric leukemia

Author

Zakia Aid, Elie Robert, Cécile K. Lopez, Maxence Bourgoin, Fabien Boudia, Melchior Le Mene, Julie Riviere, Marie Baille, Salima Benbarche, Laurent Renou, Alexandre Fagnan, Cécile Thirant, Laetitia Federici, Laure Touchard, Yann Lecluse, Anton Jetten, Birgit Geoerger, Hélène Lapillonne, Eric Solary, Muriel Gaudry, Soheil Meshinchi, Françoise Pflumio, Patrick Auberger, Camille Lobry, Arnaud Petit, Arnaud Jacquel, and Thomas Mercher

Subjects
Cancer Research, Oncology, Hematology
Abstract

Pediatric acute myeloid leukemia expressing the ETO2::GLIS2 fusion oncogene is associated with dismal prognosis. Previous studies have shown that ETO2::GLIS2 can efficiently induce leukemia development associated with strong transcriptional changes but those amenable to pharmacological targeting remained to be identified. By studying an inducible ETO2::GLIS2 cellular model, we uncovered that de novo ETO2::GLIS2 expression in human cells led to increased CASP3 transcription, CASP3 activation, and cell death. Patient-derived ETO2::GLIS2

Published
2022

21. Screening of ETO2-GLIS2-induced Super Enhancers identifies targetable cooperative dependencies in acute megakaryoblastic leukemia

Author

Salima Benbarche, Cécile K. Lopez, Eralda Salataj, Zakia Aid, Cécile Thirant, Marie-Charlotte Laiguillon, Séverine Lecourt, Yannis Belloucif, Camille Vaganay, Marion Antonini, Jiang Hu, Alexandra da Silva Babinet, Delphine Ndiaye-Lobry, Bryann Pardieu, Arnaud Petit, Alexandre Puissant, Julie Chaumeil, Thomas Mercher, and Camille Lobry

Subjects
Multidisciplinary
Abstract

Super Enhancers (SEs) are clusters of regulatory elements associated with cell identity and disease. However, whether these elements are induced by oncogenes and can regulate gene modules cooperating for cancer cell transformation or maintenance remains elusive. To address this question, we conducted a genome-wide CRISPRi-based screening of SEs in ETO2-GLIS2+acute megakaryoblastic leukemia. This approach revealed SEs essential for leukemic cell growth and survival that are induced by ETO2-GLIS2 expression. In particular, we identified a de novo SE specific of this leukemia subtype and regulating expression of tyrosine kinase–associated receptorsKITandPDGFRA. Combined expression of these two receptors was required for leukemic cell growth, and CRISPRi-mediated inhibition of this SE or treatment with tyrosine kinase inhibitors impaired progression of leukemia in vivo in patient-derived xenografts experiments. Our results show that fusion oncogenes, such as ETO2-GLIS2, can induce activation of SEs regulating essential gene modules synergizing for leukemia progression.

Published
2022

22. De novo generation of the NPM-ALK fusion recapitulates the pleiotropic phenotypes of ALK+ ALCL pathogenesis and reveals the ROR2 receptor as target for tumor cells

Author

Loélia Babin, Alice Darchen, Elie Robert, Zakia Aid, Rosalie Borry, Claire Soudais, Marion Piganeau, Anne De Cian, Carine Giovannangeli, Olivia Bawa, Charlotte Rigaud, Jean-Yves Scoazec, Lucile Couronné, Layla Veleanu, Agata Cieslak, Vahid Asnafi, David Sibon, Laurence Lamant, Fabienne Meggetto, Thomas Mercher, and Erika Brunet

Subjects
Cancer Research, Receptor Protein-Tyrosine Kinases, Protein-Tyrosine Kinases, Receptor Tyrosine Kinase-like Orphan Receptors, Translocation, Genetic, Mice, Phenotype, Oncology, hemic and lymphatic diseases, Molecular Medicine, Animals, Humans, Lymphoma, Large-Cell, Anaplastic, Anaplastic Lymphoma Kinase
Abstract

BackgroundAnaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion.MethodsWe performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes.ResultsBoth CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples.ConclusionsIn this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.

Published
2021

23. Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers

Author

Silvia Salmoiraghi, Daniel Birnbaum, Loïc Garçon, Frederik Otzen Bagger, Zakia Aid, Alexandre Fagnan, Benjamin Uzan, Stephane de Botton, Maria Riera Piqué-Borràs, Cécile K. Lopez, Christine Dierks, Connor Sweeney, Eric Delabesse, Juerg Schwaller, Elie Robert, Kazuya Shimoda, Zahra Kadri, Thomas Pabst, Jaroslaw P. Maciejewski, Betty Leite, Alexis Caulier, Sébastien Malinge, Samantha Tauchmann, Catherine Carmichael, Amina Kurtovic-Kozaric, Olivier A. Bernard, Virginie Deleuze, Ute M. Moll, Paresh Vyas, Martin Carroll, Veronique De Mas, Orietta Spinelli, Thomas Mercher, Hélène Lapillonne, Cathy Ignacimouttou, Charles G. Mullighan, Eric Soler, V. Gelsi-Boyer, Peter Valent, Cécile Thirant, Jean Baptiste Micol, Eduardo Anguita, Ilaria Iacobucci, Benjamin T. Kile, and Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, Myeloid, Erythroblasts, [SDV]Life Sciences [q-bio], Biochemistry, Transcriptome, Mice, 0302 clinical medicine, Mice, Inbred NOD, hemic and lymphatic diseases, Erythropoiesis, GATA1 Transcription Factor, Gene Knock-In Techniques, RNA-Seq, 0303 health sciences, education.field_of_study, Myeloid leukemia, GATA1, Hematology, Middle Aged, Neoplasm Proteins, 3. Good health, DNA-Binding Proteins, Leukemia, Haematopoiesis, Cell Transformation, Neoplastic, medicine.anatomical_structure, Radiation Chimera, 030220 oncology & carcinogenesis, Neoplastic Stem Cells, Female, Adult, Immunology, Population, Mice, Transgenic, Biology, Dioxygenases, Genetic Heterogeneity, Young Adult, 03 medical and health sciences, Transcriptional Regulator ERG, Proto-Oncogene Proteins, Exome Sequencing, medicine, Animals, Humans, Epigenetics, education, 030304 developmental biology, Cell Biology, Hematopoietic Stem Cells, medicine.disease, Mice, Inbred C57BL, Repressor Proteins, Mutation, Cancer research, Leukemia, Erythroblastic, Acute, Transcription Factors
Abstract

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

Published
2020
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24. Constitutive Activation of RAS/MAPK Pathway Cooperates with Trisomy 21 and Is Therapeutically Exploitable in Down Syndrome B-cell Leukemia

Author

Damien Plassard, Estelle Daudigeos, Elizabeth Macintyre, Kunjal Panchal, Yann Lécluse, Stéphanie Lagarde, Anouchka P. Laurent, Eric Delabesse, Carole Barin Bonnigal, Damien Roos-Weil, Olivier A. Bernard, Beat Bornhauser, M'Boyba Diop, Paola Ballerini, Sébastien Malinge, Aurelie Siret, Yi Chien Tsai, Cathy Ignacimouttou, Silvia Jenni, Zakia Aid, Naïs Prade, Jean-Pierre Bourquin, Thomas Mercher, Laurence C. Cheung, John D. Crispino, Rishi S. Kotecha, Birgit Geoerger, Gaëlle Pierron, Nathalie Droin, Muriel Gaudry, University of Zurich, Dynamique moléculaire de la transformation hématopoïétique (Dynamo), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Institut Gustave Roussy (IGR), The University of Western Australia (UWA), Immunologie des tumeurs et immunothérapie (UMR 1015), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), University Children’s Hospital Zurich, Centre de Recherches en Cancérologie de Toulouse (CRCT), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Curie [Paris], Curtin University [Perth], Planning and Transport Research Centre (PATREC), Northwestern University [Chicago, Ill. USA], Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Perth Children's Hospital [Nedlands, WA, Australia], CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Université Paris-Sorbonne (UP4), and univOAK, Archive ouverte

Subjects
0301 basic medicine, MAPK/ERK pathway, Cancer Research, Vincristine, Pyridones, Mice, Transgenic, 610 Medicine & health, [SDV.GEN] Life Sciences [q-bio]/Genetics, Pyrimidinones, Article, Immunophenotyping, 03 medical and health sciences, Mice, 0302 clinical medicine, Leukemia, B-Cell, Medicine, Animals, Humans, 1306 Cancer Research, Protein Kinase Inhibitors, Trametinib, [SDV.GEN]Life Sciences [q-bio]/Genetics, business.industry, MEK inhibitor, Gene Expression Profiling, Computational Biology, Oncogenes, medicine.disease, Leukemia, Disease Models, Animal, 030104 developmental biology, Oncology, 10036 Medical Clinic, 030220 oncology & carcinogenesis, B-cell leukemia, Cancer research, ras Proteins, 2730 Oncology, Disease Susceptibility, Down Syndrome, Mitogen-Activated Protein Kinases, business, Trisomy, Chromosome 21, medicine.drug, Signal Transduction
Abstract

Purpose: Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL. Experimental Design: To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents. Results: Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRASG12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine. Conclusions: Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia.

Published
2020
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25. The pediatric acute leukemia fusion oncogene ETO2-GLIS2 increases self-renewal and alters differentiation in a human induced pluripotent stem cells-derived model

Author

Franco Locatelli, Salvatore Serravalle, Cécile Thirant, Andrea Pession, Riccardo Masetti, Annalisa Astolfi, William Vainchenker, Valentina Indio, Alessandro Donada, Zakia Aid, Thomas Mercher, Salvatore Nicola Bertuccio, Elie Robert, Hana Raslova, Fabien Boudia, Larissa Lordier, Cécile K. Lopez, Marie Cambot, Bertuccio, SN, Boudia, F, Cambot, M, Lopez, CK, Lordier, L, Donada, A, Robert, E, Thirant, C, Aid, Z, Serravalle, S, Astolfi, A, Indio, V, Locatelli, F, Pession, A, Vainchenker, W, Masetti, R, Raslova, H, and Mercher, T

Subjects
Letter, Biology, Self renewal, NO, 03 medical and health sciences, 0302 clinical medicine, Text mining, GLIS2, Human Induced Pluripotent Stem Cells, 030304 developmental biology, 0303 health sciences, Acute leukemia, Oncogene, lcsh:RC633-647.5, business.industry, lcsh:Diseases of the blood and blood-forming organs, Hematology, 3. Good health, Settore MED/38 - PEDIATRIA GENERALE E SPECIALISTICA, 030220 oncology & carcinogenesis, ComputingMethodologies_DOCUMENTANDTEXTPROCESSING, ETO2-GLIS2, Cancer research, na, business
Abstract

Supplemental Digital Content is available in the text.

Published
2020

26. Nfkbie-deficiency leads to increased susceptibility to develop B-cell lymphoproliferative disorders in aged mice

Author

Veronique, Della-Valle, Damien, Roos-Weil, Laurianne, Scourzic, Enguerran, Mouly, Zakia, Aid, Walaa, Darwiche, Yann, Lecluse, Frederik, Damm, Sylvie, Mémet, Thomas, Mercher, Said, Aoufouchi, Florence, Nguyen-Khac, Olivier A, Bernard, Hussein, Ghamlouch, Hématopoïèse normale et pathologique (U1170 Inserm), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Gustave Roussy (IGR), Sorbonne Université (SU), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Université de Picardie Jules Verne (UPJV), CHU Amiens-Picardie, Plateforme imagerie et cytométrie (PFIC), Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse (AMMICa), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Humboldt University Of Berlin, Berlin Institute of Health (BIH), Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Intégrité du génome et cancers (IGC), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Service d'Hématologie clinique [CHU Pitié-Salpêtrière], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Humboldt-Universität zu Berlin, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Service d'Hématologie Biologique [CHU Pitié-Salpêtrière], and Gestionnaire, Hal Sorbonne Université

Subjects
Chronic lymphocytic leukaemia, B cells, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, B-cell lymphoma, [SDV]Life Sciences [q-bio], Oncogenes, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, Leukemia, Lymphocytic, Chronic, B-Cell, lcsh:RC254-282, Article, [SDV] Life Sciences [q-bio], Mice, Proto-Oncogene Proteins, Animals, I-kappa B Proteins, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Aberrant NF-κB activation is a hallmark of most B-cell malignancies. Recurrent inactivating somatic mutations in the NFKBIE gene, which encodes IκBε, an inhibitor of NF-κB-inducible activity, are reported in several B-cell malignancies with highest frequencies in chronic lymphocytic leukemia and primary mediastinal B-cell lymphoma, and account for a fraction of NF-κB pathway activation. The impact of NFKBIE deficiency on B-cell development and function remains, however, largely unknown. Here, we show that Nfkbie-deficient mice exhibit an amplification of marginal zone B cells and an expansion of B1 B-cell subsets. In germinal center (GC)-dependent immune response, Nfkbie deficiency triggers expansion of GC B-cells through increasing cell proliferation in a B-cell autonomous manner. We also show that Nfkbie deficiency results in hyperproliferation of a B1 B-cell subset and leads to increased NF-κB activation in these cells upon Toll-like receptor stimulation. Nfkbie deficiency cooperates with mutant MYD88 signaling and enhances B-cell proliferation in vitro. In aged mice, Nfkbie absence drives the development of an oligoclonal indolent B-cell lymphoproliferative disorders, resembling monoclonal B-cell lymphocytosis. Collectively, these findings shed light on an essential role of IκBε in finely tuning B-cell development and function.

Published
2020
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27. ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia

Author

Arnaud Petit, Françoise Pflumio, Cécile Thirant, Cécile K. Lopez, Sébastien Malinge, Hana Raslova, Aurelie Siret, Olivier A. Bernard, Clarisse Thiollier, Cathy Ignacimouttou, Mehdi Khaled, M'Boyba Diop, Jürg Schwaller, William Vainchenker, John D. Crispino, Paola Ballerini, Guy Leverger, Ce ' Line Lefebvre, Philippe Rameau, Benjamin T. Kile, Catherine Carmichael, Lou Le Mouel, Eric Soler, Phillipe Dessen, Thomas Mercher, Zakia Aid, Julie Riviere, Nathalie Droin, Christian Wichmann, Camille Lobry, Hématopoïèse normale et pathologique ( U1170 Inserm ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris Descartes - Faculté de Médecine ( UPD5 Médecine ), Université Paris Descartes - Paris 5 ( UPD5 ), Centre de génétique - Centre de référence des maladies rares, anomalies du développement et syndromes malformatifs (CHU de Dijon), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand ( CHU Dijon ), Lipides - Nutrition - Cancer [Dijon - U1231] ( LNC ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Plateforme imagerie et cytométrie ( PFIC ), Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse ( AMMICa ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Ondes et Imagerie ( O&I ), Laboratoire de Mécanique et d'Acoustique [Marseille] ( LMA ), Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Ecole Centrale de Marseille ( ECM ) -Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Ecole Centrale de Marseille ( ECM ), Hôpital Trousseau, CHRU Tours, CHU Trousseau [APHP], Centre de Recherche Saint-Antoine ( CR Saint-Antoine ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Institut Gustave Roussy ( IGR ), Centre de génétique et de physiologie moléculaire et cellulaire ( CGPhiMC ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Northwestern University Chicago, Université Nice Sophia Antipolis ( UNS ), Université Côte d'Azur ( UCA ), Plateforme de génomique, Laboratoire des Adaptations Physiologiques aux Activités Physiques ( LAPHAP ), Université de Poitiers, Aix Marseille Université ( AMU ) -Ecole Centrale de Marseille ( ECM ) -Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Ecole Centrale de Marseille ( ECM ) -Centre National de la Recherche Scientifique ( CNRS ), Centre National de la Recherche Scientifique ( CNRS ) -Université Claude Bernard Lyon 1 ( UCBL ), and Université de Lyon-Université de Lyon

Subjects
Transcriptional Activation, 0301 basic medicine, Cancer Research, Oncogene Proteins, Fusion, ChIP, Biology, [ SDV.CAN ] Life Sciences [q-bio]/Cancer, Mice, 03 medical and health sciences, Acute megakaryoblastic leukemia, Transcriptional Regulator ERG, GLIS2, Downregulation and upregulation, Leukemia, Megakaryoblastic, Acute, Transcription (biology), medicine, Animals, Humans, GATA1 Transcription Factor, Child, Enhancer, Gene, Transcription factor, transcription factor, CBFA2T3, AMKL, leukemia, Cell Differentiation, GLIS, medicine.disease, Leukemia, pediatric, Enhancer Elements, Genetic, 030104 developmental biology, Oncology, ERG, CRISPR, Cancer research, enhancer
Abstract

International audience; Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.

Published
2017
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28. Screening of clustered regulatory elements reveals functional cooperating dependencies in Leukemia

Author

Marion Antonini, Zakia Aid, Bryan Pardieu, Séverine Lecourt, Marie-Charlotte Laiguillon, Cécile Thirant, Arnaud Petit, Cécile K. Lopez, Eralda Salataj, Camille Lobry, Alexandre Puissant, Thomas Mercher, Julie Chaumeil, and Salima Benbarche

Subjects
BRD4, Massive parallel sequencing, Regulatory sequence, medicine, Cancer, Computational biology, Biology, medicine.disease, Genome, Gene, Fusion protein, Epigenomics
Abstract

In the recent years, massively parallel sequencing approaches identified hundreds of mutated genes in cancer(1) providing an unprecedented amount of information about mechanisms of cancer cell maintenance and progression. However, while (it is widely accepted that) transformation processes result from oncogenic cooperation between deregulated genes and pathways, the functional characterization of candidate key players is mostly performed at the single gene level which is generally inadequate to identify these oncogene circuitries. In addition, studies aimed at depicting oncogenic cooperation involve the generation of challenging mouse models or the deployment of tedious screening pipelines. Genome wide mapping of epigenomic modifications on histone tails or binding of factors such as MED1 and BRD4 allowed identification of clusters of regulatory elements, also termed Super-Enhancers (SE)(2). Functional annotation of these regions revealed their high relevance during normal tissue development and cancer ontogeny(3). An interesting paradigm of the tumorigenic function of these SE regions comes from ETO2-GLIS2-driven acute megakaryoblastic leukemia (AMKL) in which the fusion protein ETO2-GLIS2 is sufficient to promote an aberrant transcriptional network by the rewiring of SE regions(4). We thus hypothesized that important regulatory regions could control simultaneously expression of genes cooperating in functional modules to promote cancer development. In an effort to identify such modules, we deployed a genome-wide CRISPRi-based screening approach and nominated SE regions that are functionally linked to leukemia maintenance. In particular, we pinpointed a novel SE region regulating the expression of both tyrosine kinases KIT and PDGFRA. Whereas the inhibition of each kinase alone affected modestly cancer cell growth, combined inhibition of both receptors synergizes to impair leukemia cell growth and survival. Our results demonstrate that genome-wide screening of regulatory DNA elements can identify co-regulated genes collaborating to promote cancer and could open new avenues to the concept of combined gene inhibition upon single hit targeting.

Published
2019
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29. Ontogenic changes in hematopoietic hierarchy determine pediatric specificity and disease phenotype in fusion oncogene-driven myeloid leukemia

Author

Cécile K. Lopez, Marie Laure Arcangeli, Eric Delabesse, Sébastien Malinge, Alexandre Fagnan, Isabelle Godin, Franco Locatelli, Françoise Pflumio, Fabien Boudia, Cécile Thirant, Muriel Gaudry, Vaia Stavropoulou, Arnaud Petit, Claus Nerlov, Nathalie Droin, Riccardo Masetti, Paola Ballerini, Zakia Aid, Berthold Göttgens, Olivier Bernard, Sarah Kinston, Erika Brunet, Hélène Lapillonne, Loelia Babin, Juerg Schwaller, Antoine H.F.M. Peters, Elie Robert, Yann Lécluse, Bastien Job, Chrystele Bilhou-Nabera, Jean-Luc Villeval, Camille Lobry, William Vainchenker, Thomas Mercher, Esteve Noguera, M'Boyba Diop, Service d'hématologie-immunologie-oncologie pédiatrique [CHU Trousseau], Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre de Recherche Saint-Antoine (UMRS893), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Trousseau [APHP], Institut de psychiatrie et neurosciences (U894 / UMS 1266), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Stabilité génétique, Cellules Souches et Radiations (SCSR (U_967)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPC), Hématopoïèse normale et pathologique (U1170 Inserm), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme de Bioinformatique [Gustave Roussy], Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse (AMMICa), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Bases fondamentales et stratégies nouvelles en cancérologie (BFSNC - IFR54), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Régulation et dynamique des génomes, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherches en Cancérologie de Toulouse (CRCT), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Santa Lucia Foundation, IRCSS, Rome, University of Oxford [Oxford], Images et Modèles, Centre de Recherche en Acquisition et Traitement de l'Image pour la Santé (CREATIS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Cambridge [UK] (CAM), Service Procédés et Innovations Industriels (SPII), eRcane, Institut Cochin (UMR_S567 / UMR 8104), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Génétique des tumeurs (U985), Institut Gustave Roussy (IGR)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche et d'Etude en Droit et Science Politique (CREDESPO), Université de Bourgogne (UB), Université Jean Monnet [Saint-Étienne] (UJM)-Hospices Civils de Lyon (HCL)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Jean Monnet [Saint-Étienne] (UJM)-Hospices Civils de Lyon (HCL)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Lopez C.K., Noguera E., Stavropoulou V., Robert E., Aid Z., Ballerini P., Bilhou-Nabera C., Lapillonne H., Boudia F., Thirant C., Fagnan A., Arcangeli M.-L., Kinston S.J., Diop M., Job B., Lecluse Y., Brunet E., Babin L., Villeval J.L., Delabesse E., Peters A.H.F.M., Vainchenker W., Gaudry M., Masetti R., Locatelli F., Malinge S., Nerlov C., Droin N., Lobry C., Godin I., Bernard O.A., Gottgens B., Petit A., Pflumio F., Schwaller J., Mercher T., Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Trousseau [APHP], Centre de Recherche Saint-Antoine (CR Saint-Antoine), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire d'Hématologie [AP-HP Hôpital Armand Trousseau], Cellules Souches et Radiations (SCSR (U967 / UMR-E_008)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Brunet, Erika [0000-0002-1726-4673], Malinge, Sébastien [0000-0002-9533-7778], Droin, Nathalie [0000-0002-6099-5324], Godin, Isabelle [0000-0001-8577-8388], Göttgens, Berthold [0000-0001-6302-5705], Schwaller, Juerg [0000-0001-8616-0096], Mercher, Thomas [0000-0003-1552-087X], Apollo - University of Cambridge Repository, Centre de Psychiatrie et Neurosciences (U894), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Oxford, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Myeloid, Oncogene Proteins, Fusion, Oncogene Proteins, Fusion gene, Mice, 0302 clinical medicine, AML, CEBPA, GENOMIC ALTERATIONS, Tumor Cells, Cultured, TRANSCRIPTION FACTOR, RNA-SEQ, Child, ComputingMilieux_MISCELLANEOUS, GENE-EXPRESSION, Age Factors, Myeloid leukemia, GATA1, 3. Good health, Leukemia, Myeloid, Acute, Leukemia, Haematopoiesis, medicine.anatomical_structure, DIFFERENTIATION, Oncology, Settore MED/38 - PEDIATRIA GENERALE E SPECIALISTICA, Child, Preschool, 030220 oncology & carcinogenesis, Female, Adolescent, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, 03 medical and health sciences, children, ACUTE MEGAKARYOBLASTIC LEUKEMIA, transcription factors, medicine, Animals, Humans, FETAL, pediatric acute myeloid leukemia, LINEAGE COMMITMENT, Infant, medicine.disease, STEM-CELL, SELF-RENEWAL, 030104 developmental biology, Cancer research, Neoplasm Transplantation
Abstract

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2–GLIS2, are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2–GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2–GLIS2-induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. Significance: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state. See related commentary by Cruz Hernandez and Vyas, p. 1653. This article is highlighted in the In This Issue feature, p. 1631

Published
2019
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30. Modeling Acute Megakaryoblastic Leukemia of Down Syndrome Using Induced Pluripotent Stem Cells

Author

Isabelle Plo, Romain Diot, Marie Cambot, G Tachdjian, Eric Soler, Virginie Dufour, Najet Debili, Zakia Aid, Hana Raslova, Cyril Catelain, Rachel Petermann, Stefania Mazzi, Sébastien Malinge, Yasmine Mammasse, Thomas Mercher, Mathieu Vieira, Elie Robert, Sylvie Souquere, Aurelie Siret, Philippe Rameau, William Vainchenker, Fabien Boudia, and Brahim Arkoun

Subjects
Acute megakaryoblastic leukemia, Down syndrome, business.industry, Immunology, Cancer research, Medicine, Cell Biology, Hematology, business, medicine.disease, Induced pluripotent stem cell, Biochemistry
Abstract

Introduction Development of Acute megakaryoblastic leukemia in Down syndrome children (DS-AMKL) is a multi-step process. Acquired GATA1s mutation during fetal hematopoiesis is responsible of a transient myeloproliferative disorder (TMD) characterized by an accumulation of megakaryoblasts. Although most of TMD regress around birth, some TMD can progress from the initial GATA1s clone to AMKL through the acquisition of additional mutations, including in (i) the cohesin complex (i.e: SMC3), (ii) the JAK/STAT signaling pathway, such as MPL and (iii) the polycomb repressive complex 2 (EZH2). How these mutations cooperate to deregulate megakaryocyte (MK) differentiation and to induce a full-blown AMKL, along with the precise role of trisomy 21 (T21) during this transformation process remain unclear. Because modeling of DS-AMKL is particularly difficult in mice, we performed a step-wise introduction of GATA1s, a gain of function mutation of MPL (MPLW515K) and a heterozygous loss of function mutation in a cohesin (SMC3), separately or in combination, in T21 and isogenic disomic 21 (Dis21) human induced Pluripotent Stem Cells (iPSCs). Methods Trisomy 21 iPSCs were kindly provided by M. Weiss (Memphis, TN). CRISPR/Cas 9 genome editing of GATA1 or SMC3 allowed the generation of GATA1s T21, SMC3+/- T21 and GATA1s SMC3+/- T21 iPSC clones. CRISPR/Cas9-mediated knock-in of MPLW515K was performed in T21 GATA1s iPSCs. The subsequent T21 GATA1sMPLW515K/W515Kclones were selected as well as a revertant Dis21 GATA1sMPLW515K/W515Kclone. Finally, SMC3 insertion/deletion were obtained in isogenic T21 and Dis21 GATA1s MPLW515K/W515K SMC3+/-iPSCs clones. Hematopoietic differentiation was induced in 2D cultures in presence of a matrix and a cocktail of cytokines followed by a MK differentiation with SCF and TPO. MK differentiation was studied by clonogenic assays, flow cytometry, confocal microscopy and ultrastructural studies. Gene expression analyses were performed by RNA-seq on highly purified MK from all genotypes. Results GATA1s alone blocked MK maturation characterized by a persistent CD34 expression, an accumulation of abnormal large granules, a defect in the development of demarcation membranes (DMS), and a marked decrease in proplatelet formation. The typical GATA1s MK were large megakaryoblasts with numerous large granules and rare DMS. However, GATA1s alone had no effect on the clonogenic activity in CFU-MK assays and MK numbers. The introduction of the MPLW515K mutation did not modify this phenotype either in Dis21 or T21 GATA1s MK, but induced a complete TPO independence. SMC3+/- alone enhanced the MK maturation allowing the generation of a higher number of proplatelets-generating MK. Importantly, the combination of GATA1s and SMC3+/- mutations had a marked cooperative effect that worsened the MK maturation defect, led to the generation of abnormal megakaryoblasts with only a pre-DMS and resulted in enhanced proliferation and ploidization both in Dis21 and T21 iPSCs. Interestingly, the proliferation was markedly higher in T21 clones compared to Dis21 counterparts. RNA-seq and GSEA analyses showed that T21 GATA1s SMC3+/- mutant MK exhibited transcriptional signatures consistent with a dramatic decrease in the expression of maturation genes, including GATA1 target genes, while DNA replication gene markers were increased compared with GATA1s alone. T21 GATA1s MPLW515K/W515K SMC3+/- MK were enriched for AMKL signatures as compared to isogenic Dis21 GATA1sMPLW515K/W515K SMC3+/- MK. Ongoing ATAC-seq analyses will define the consequence of the different mutations on chromatin accessibility. Conclusion Using iPSC modeling, we analyzed in a human cell-context the consequences of the different combination of mutations associated with DS-AMKL that would be difficult to model using human primary cells. Our data demonstrate that GATA1s expression cooperates with SMC3+/- to enhance proliferation of megakaryoblasts from T21 iPSCs and isogenic Dis21 iPSCs hence reproducing the abnormalities observed in DS-AMKL. T21 is not directly involved in the MK differentiation defects but rather give a proliferative advantage supporting its role in leukemia development. Disclosures No relevant conflicts of interest to declare.

Published
2020
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31. S113 GENETICS AND MODELING OF HUMAN ACUTE ERYTHROID LEUKEMIA

Author

Loïc Garçon, Juerg Schwaller, S. DeBotton, A. Rambaldi, Cathy Ignacimouttou, Eric Delabesse, Alexandre Fagnan, Cécile K. Lopez, Hélène Lapillonne, Eduardo Anguita, V. Gelsi-Boyer, Jaroslaw P. Maciejewski, Benjamin T. Kile, M. Caroll, Catherine Carmichael, F. Otzen Bagger, Christine Dierks, Cécile Thirant, Peter Valent, Daniel Birnbaum, A. kurtovic, Zakia Aid, Thomas Pabst, Kazuya Shimoda, M. Riera Piqué Borràs, Paresh Vyas, Eric Soler, Alexis Caulier, Thomas Mercher, and J.-B. Micoll

Subjects
Cancer research, Acute erythroid leukemia, medicine, Hematology, Biology, medicine.disease
Published
2019
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32. ETO2-GLIS2 Controls Differentiation Arrest and Self-Renewal through Aberrant Enhancers Regulation in Pediatric Leukemia

Author

Benjamin T. Kile, Lou Le Mouel, Aurelie Siret, William Vainchenker, Philippe Rameau, Catherine Carmichael, Clarisse Thiollier, Sébastien Malinge, Hana Raslova, Mehdi Khaled, M'Boyba Diop, Olivier A. Bernard, John D. Crispino, Eric Soler, Cécile K. Lopez, Thomas Mercher, Philippe Dessen, Cathy Ignacimouttou, Camille Lobry, Zakia Aid, Francoise Pfumio, Cécile Thirant, Juerg Schwaller, Paola Ballerini, Arnaud Petit, Guy Leverger, Celine Lefebvre, Nathalie Droin, Julie Riviere, and Christian Wichmann

Subjects
Genetics, Gene knockdown, Immunology, Myeloid leukemia, Cell Biology, Hematology, Biology, medicine.disease, Biochemistry, Cell biology, Gene expression profiling, Fusion gene, Acute megakaryoblastic leukemia, medicine, Transcriptional regulation, Enhancer, Transcription factor
Abstract

Deregulated gene expression due to genetic alterations, such as gene fusions affecting transcription and/or epigenetic factors is the hallmark of acute myeloid leukemia and the basis for the differentiation block of hematopoietic progenitors. Acute megakaryoblastic leukemia (AMKL) is a subtype of poor prognosis acute myeloid leukemia (AML) affecting primarily young children. Recently, the ETO2-GLIS2 fusion has been identified in 20-30% of de novo AMKL and associated with the worst prognosis in this subtype of AML. To characterize the transformation induced by ETO2-GLIS2, we first defined the consequences of ETO2-GLIS2 expression on hematopoietic progenitors and the contribution of ETO2 and GLIS2 on differentiation and self-renewal. Using methylcellulose replating assays and phenotype characterization, we show that the GLIS2 moiety drives the megakaryocytic phenotype whereas both the ETO2 and GLIS2 moieties are required for maintaining self-renewal. Global expression profiling and comparison to patients' signature consistently identify ETO2-GLIS2-mediated deregulation of major transcriptional regulators of hematopoiesis and leukemogenesis, including overexpression of the ERG oncogene. ChIP-seq analysis reveals that ETO2-GLIS2 is recruited at normal ETO2 complexes sites and also at GLIS2-specific targets through binding via GLIS2 DNA-binding domain. We demonstrate that ETO2-GLIS2 fusion localize at half of H3K27Ac-dense enhancers, so called super-enhancers, to control transcription of associated genes. We show that interaction of ETO2-GLIS2 with ETO2 complexes is an essential node for the transcriptional control by the fusion at enhancer elements. Indeed, ETO2-GLIS2 dimerizes and interacts with endogenous ETO2 via its NHR2 domains. An NHR2 peptide-interference strategy inhibits oligomerization, reverses the transcriptional activation at enhancers, promotes megakaryocytic differentiation and abrogates human AMKL cells maintenance in vivo. Finally, upregulation of ERG by ETO2-GLIS2 further strengthen enhancers formation as ERG is co-recruited generating a feed forward loop at these elements and its knockdown or genetic inactivation downregulates expression of ETO2-GLIS2 targets required for leukemic cells survival. We propose that the megakaryocytic differentiation arrest and self-renewal controlled by ETO2-GLIS2 results from an imbalance in the expression of master transcription factors imposed by aberrant chromatin structures at enhancers that may be disrupted by targeting the NHR2 interface. Disclosures No relevant conflicts of interest to declare.

Published
2016
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