Drosophila hematopoietic cells as a model to study in vivo the activity of the human oncogene AML1-ETO

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(1)TH ÈS E En vue de l'obtention du. DOCTORA T DE L’UN I VE RSI TÉ DE TOULOUSE Délivré par l’université Toulouse III – Paul Sabatier Discipline ou spécialité : Gènes, Cellules et Développement Présentée et soutenue par. Dani Osman Le 24 septembre 2009. Titre : "Drosophila hematopoietic cells as a model to study in vivo the activity of the human oncogene AML1-ETO". JURY Pr. David Cribbs, président Dr. Dominique Ferrandon, rapporteur Dr. François Morle, rapporteur Pr. Olaf Heidenreich, examinateur Dr. Lucas Waltzer, directeur de thèse Dr. Marc Haenlin, directeur de thèse Ecole doctorale : Biologie, Santé, Biotechnologies Unité de recherche : Centre de Biologie de développement, CNRS UMR 5547 Directeur(s) de Thèse : Dr. Lucas Waltzer Dr. Marc Haenlin.

(2) “Je. dédie. ce. travail. à. Magali,. mon. adorable épouse, à ma famille, et à ma belle famille, pour tout leur soutien et leur amour”..

(3) « Gratitude is the memory of the heart » (Hans Christian Andersen). « La reconnaissance est la mémoire du cœur » (Hans Christian Andersen).

(4) Acknowledgements First,. I. Dominique. would. like. to. Ferrandon,. thank. François. the. jury. Morle. members:. and. Olaf. David. Cribbs,. Heidenreich. for. accepting to judge and to criticize my PhD work. I am deeply indebted to my supervisors Lucas and Marc as well.. « EEh Lucas, qu’est ce que tu penses de ce résultat, viens voir, c’est encourageant non ? » « fhdhgbnjkdsughzbgmb ». « Pardon Lucas, tu m’as dit quelque chose ? » « ouaiis, ouaais, ça pourrait être intéressant !! Pourquoi pas, mais j’ai rien compris encore, il faut continuer à creuser , …».. Je t’avoue Lucas, ce type de discussion va me manquer énormément ! Je te remercie d’avoir dirigé mes travaux de thèse et pour toute la confiance que tu m’as accordée au cours de ces quatre années. Nombreux sont ceux qui ne croyaient pas au projet, mais grâce à ton soutien, à ta présence au quotidien, à tes précieux conseils, j’ai réussi à mener le projet jusqu’au bout ! Tu m’as appris comment être pragmatique, comment définir les priorités, et comment se lancer quand il le fallait. Merci encore…. Bien évidemment, je remercie aussi notre cher directeur, Marc. Je peux dire que c’est une chance d’avoir un directeur de thèse qui a une vision philosophique ! On ne s’ennuie jamais avec toi Marc,. il. y. a. toujours. de. quoi. discuter. pour. nourrir. l’esprit.. Certaines discussions m’ont permis de voir la science en 3D, ça ne veut. rien. dire. ce. que. je. suis. entrain. de. dire,. mais. je. me. comprends ;). C’était juste très agréable les discussions-café qui m’ont aidé à mûrir scientifiquement et à bien apprécier la recherche fondamentale. Merci aussi de m’avoir convertit en un MAC lover !!. Je. tiens. à. remercier. également. tous. les. membres. de. l’équipe. Waltzer/Haenlin. Je commence par les plus vieux (d’un point de vue chronologique bien sûr).. 4.

(5) « La blouse !! La blouuuuuuse !! Faut la mettre ! Même en se lavant les mains à l’eau !!! Imaginez que quelqu’un se balade avec une bactérie dangereuse (e. coli par exemple) et la renverse sur vous, vous ferez quoi ? ». Oui Benoît, ouiii, t’as raison, faut la mettre.. Merci. à. toi,. pour. ton. soutien. technique,. pour. tes. compétences variées, et ton aide. Je te considère tout simplement comme la mémoire de l’équipe…. « Franchement Dani, t’as de la chance, tu travailles avec une belle fille». C’est Une phrase que j’ai entendue mille et une fois durant ma thèse. Forcément, ils parlent de Karène. De la chance de la. chance…. Ça. fait. aussi. foirer. les. manips. les. gars,. faute. de. concentration ;) . Je te remercie Karène pour la belle ambiance et pour la joie que ta présence impose. Je peux te dire que tu fais partie de la catégorie « special » des gens que je connais…. « Tu es frustré et pas excité », «. on ne peut pas utiliser ce. mot pour dire ceci … ». La liste est trop longue pour tout citer. Je parle bien sûr de ma correctrice personnelle (la classe, c’est pas donné à tout le monde d’en avoir une) : Vanessa. Tu as sans relâche veillé à améliorer mon Français, un grand Merci. Je te remercie aussi pour ta pédagogie remarquable, qui m’a été fort utile tout au long de ma thèse. Je n’oublierai pas le soutien moral que tu m’as accordé lors des moments difficiles. Plein de courage pour d’autres aventures avec AML1-ETO ;).. « Arrête de faire travailler la petite, le labo n’est pas un terrain. de. rugby ».. Si. seulement. ils. savaient. qui. exploitait. l’autre ! C’est vrai que tu m’as supporté comme encadrant, mais j’étais sympa avec toi quand même !!. Même quand tu me faisais des. blagues de genre « Quick » ou « Macdo » ;). Je te remercie Stéphanie pour tous les beaux moments partagés ensemble, pour ta sympathie, ton. amabilité.. Surtout. continue. à. être. championne. de. France. de. Rugby !. 5.

(6) « Tu. veux. un. sandwich ?. Un. gâteau ?. de. la. mousse. au. chocolat ? », Voilà quelqu’un qui a rapidement compris comment il faut me parler…Et oui Amélie, tu l’as rapidement remarqué... Merci pour. ta. disponibilité,. ton. attention,. et. ta. gentillesse. assez. remarquable. Profites bien de ta future vie londonienne, et surtout penses à rentrer chez toi pour dormir.. « Tu. as. confirmé. le. phénotype. avec. un. mutant ?. Et. combien. d’allèle tu as testé ?.». Les questions habituelles de Fernando Roch Mendel.. Je. t’avoue. que. Ca. va. me. manquer. ce. rôle. de. conseiller. génétique que tu as assuré avec grande générosité (je crois que c’est génétique☺). Merci aussi pour les discussions pertinentes que nous avons eu sur l’origine des espèces et l’évolution, ce qui m’as poussé. à. approfondir. mes. connaissances. sur. le. sujet.. Bonne. continuité avec ta famille boudin.. « M2ané2, Évidemment. de. sojo2, Assia. ma3. Boudin. chwayyét Hijazi.. hommos », Allah. de. qui. ykhallilék. je. parle ?. èle. boudin,. yérbou bi 3ézzék w dalélék ». Je sais que j’étais pas à la hauteur de la mission que ton père m’a attribuée, et pourtant j’ai fais de mon mieux. Je te remercie Assia pour l’humour dont tu as fait preuve et aussi un grand merci pour avoir garder mes chats « cat sitter » quand je partais en vacances. Si tu décides de faire un post-doc, surtout ne fait pas tes valises deux jours après ton arrivée ;) .. « Tu vas l’avoir ton papier, tu vas l’avoir ta thèse, tu vas l’avoir. ton. post-doc,. encouragements. etc. « presque ». etc. …).. Ce. n’est. au. quotidien. que. qu’un m’a. exemple apporté. des Mr.. Poleselo. Franchement Cédric c’est très dur de trouver des mots pour te remercier, mais tout ce que je peux dire que tu es quelqu’un très aimable, gentil, intelligent et doué. Les chefs peuvent être fiers (et je suis sûr qu’ils le sont) que tu sois dans l’équipe, pour toutes tes qualités scientifiques et humaines. Un grand merci.. J’ai également Une pensée émue pour l’ensemble des membres du CBD, pour leur amabilité, leurs aides permanentes, et surtout pour les moments de joie formidable et de convivialités passés ensemble. 6.

(7) tout le long de ces quatre années. Je remercie très particulièrement Gwenaëlle (ma grande amie), Aurélie (la talentueuse, félicitations), Nathalie (ma chère tutrice), Dominique (ma pédagogue), Mohammad (mon ami star), François (le grand papa), Phiphi (le petit papa), Yacine (le dragueur), Julie (le grand cœur), Carine (l’adorable), Hélène (la. sympathique),. le. trio. féminin. infernal :. Maëva,. Marine. et. Delphine, le trio masculin mortel : Christophe, Florent et Romain. J’ai une pensée très particulière à Thomas, qui a réussi à être mon collègue, mon ami et mon voisin, tout ça en même temps. Bien sûr Je n’oublie pas les anciens du CBD, Rami, Joanna et Laetitia, pour toute leur amitié et leur sympathie. Je tiens à remercier aussi tous mes amis, et particulièrement Alia, pour toutes les aventures qu’on a vécues ensemble au fil des années.. À vous tous qui avez égayé ces 4 dernières années, je vous souhaite le meilleur pour la suite de vos aventures, où qu’elles vous emmènent.. 7.

(8) Content. ABSTRACT ............................................................................................................................. 10 RÉSUMÉ ................................................................................................................................. 12 FOREWORD ........................................................................................................................... 14 LIST OF ABBREVIATIONS ................................................................................................. 16 LIST OF FIGURES ................................................................................................................ 19 LIST OF TABLES................................................................................................................... 21. CHAPTER I. INTRODUCTION ................................................................................ 22 A. GENERAL DESCRIPTION OF HEMATOPOIESIS AND LEUKEMIA................ 23 1. HEMATOPOIESIS.............................................................................................................. 23 (i) Ontogeny of the mammalian hematopoietic system .................................................. 23 (ii) Blood cell lineages ................................................................................................... 23 (iii) Blood cell functions................................................................................................. 24 2. LEUKEMIA ...................................................................................................................... 25 Leukemia classification ............................................................................................. 26 3. ACUTE MYELOID LEUKEMIA ........................................................................................... 27 (i) Classification............................................................................................................. 27 (ii) Genetic alterations in AML ...................................................................................... 28 B. AML1 IN HEMATOPOIESIS AND LEUKEMIA ...................................................... 29 1. 2. 3. 4. 5.. STRUCTURE AND ISOFORMS OF AML1 PROTEINS ............................................................ 29 ROLE OF AML1 IN HEMATOPOIESIS IN THE EMBRYO....................................................... 31 ROLE OF AML1 IN ADULT HEMATOPOIESIS .................................................................... 31 AML1: A MASTER TRANSCRIPTIONAL REGULATOR OF HEMATOPOIESIS .......................... 33 AML1 IN LEUKEMIA ....................................................................................................... 33. C. THE 8;21 CHROMOSOMAL TRANSLOCATION AND AML DEVELOPMENT34 1. INCIDENCE AND CELL CHARACTERISTICS OF T(8;21) AML ............................................. 34 2. MOLECULAR CHARACTERIZATION OF T(8;21)................................................................. 35 3. STRUCTURE AND TRANSCRIPTIONAL FUNCTION OF ETO AND AML1-ETO PROTEINS .... 36 (i) ETO: a transcriptional co-repressor ........................................................................ 36 (ii) The molecular mechanism of action of AML1-ETO ................................................ 38 4. AML1-ETO IN CELL DIFFERENTIATION, GROWTH AND SURVIVAL.................................. 42 AML1-ETO-related leukemic stem cells ................................................................... 44 D. MURINE MODELS TO INVESTIGATE T(8;21) LEUKEMIA ............................... 46 1. 2. 3. 4.. KNOCK-IN MODELS ......................................................................................................... 46 CHIMERIC MODELS.......................................................................................................... 47 TRANSGENIC MODELS ..................................................................................................... 48 THE TWO HITS MODEL FOR AML1-ETO-INDUCED LEUKEMOGENESIS ............................ 49. 8.

(9) E. ZEBRAFISH AS A MODEL SYSTEM FOR STUDIES OF AML1-ETO-INDUCED LEUKEMOGENESIS............................................................................................................ 51 1. HEMATOPOIESIS IN ZEBRAFISH ....................................................................................... 51 2. EFFECTS OF AML1-ETO IN ZEBRAFISH HEMATOPOIESIS ............................................... 52 3. SCREENING FOR INHIBITORS OF AML1-ETO IN ZEBRAFISH ........................................... 54 F. DROSOPHILA HEMATOPOIETIC SYSTEM: A POTENTIAL MODEL TO STUDY THE ACTIVITY OF AML1-ETO ......................................................................... 55 1. DROSOPHILA BLOOD CELLS: DESCRIPTION AND FUNCTIONS ............................................ 55 Plasmatocytes............................................................................................................ 56 Crystal cells............................................................................................................... 57 Lamellocytes.............................................................................................................. 58 2. HEMATOPOIESIS IN DROSOPHILA .................................................................................... 58 3. MOLECULAR CONTROL OF DROSOPHILA HEMATOPOIESIS ............................................... 60 The GATA transcription factor Serpent .................................................................... 61 The RUNX transcription factor Lozenge................................................................... 62 G. RESEARCH AIMS......................................................................................................... 64. CHAPTER II. RESULTS ............................................................................................... 66 CHAPTER III. DISCUSSION AND SUPLEMENTARY RESULTS............ 67 A. EXPRESSING AML1-ETO IN CIRCULATING LZ+ CELLS INDUCES A PRELEUKEMIC STATE...................................................................................................... 68 B. A GENETIC SCREEN TO IDENTIFY SUPPRESSORS OF AML1-ETO .............. 70 C. PRELIMINARY CHARACTERIZATION OF 8 SUPPRESSORS OF AML1-ETO IN LZ-EXPRESSING CELLS .............................................................................................. 71 D. CALPAINS FAMILY AS MODULATORS OF AML1-ETO ACTIVITY ............... 74 1. GENERALITIES ................................................................................................................ 74 2. DROSOPHILA CALPAINS .................................................................................................. 76 3. POST-TRANSLATIONAL REGULATION OF AML-ETO BY CALPAINS ................................. 77 (i) AML1-ETO: a potential calpains substrate? ............................................................ 77 (ii) Other possible mechanisms of action of calpains .................................................... 78 4. AML1-ETO REGULATES CALPAINB ............................................................................... 80 5. CHARACTERIZATION OF CALPAINS FUNCTION IN AML................................................... 80 E. CONCLUDING REMARKS.......................................................................................... 82. CHAPTER IV. REFERENCES ................................................................................... 84. 9.

(10) Abstract. Hematopoiesis is a complex and dynamic process that leads to the formation and continuous replenishment of blood cells. During this process, a series of transcription factors controls the appearance, commitment and differentiation of stem cells into specific lineages. In particular, in vertebrates, the factor RUNX1/AML1 (for Acute Myeloid Leukemia 1) is required for the emergence of hematopoietic stem cells and for the differentiation of both myeloid and lymphoid lineages. In humans, several genetic alterations affecting AML1 are linked to the development of different hemopathies. Notably, the chromosomal translocation t(8; 21), encoding the fusion protein AML1-ETO, is associated with 10-15% of cases of acute myeloid leukemia. It has been described that AML1-ETO acts essentially by interfering with AML1 function during hematopoietic differentiation. However, the mode of action of this oncogene remains poorly understood and few factors modulating its activity are known.. Recently, numerous studies have shown that several aspects of hematopoietic development are conserved from Drosophila to vertebrates. Notably, transcription factors of the RUNX and GATA families, which are key players in vertebrate’s hematopoiesis, also control the development of blood cells in Drosophila. Taking advantage of the phylogenetic conservation of the genetic circuitry regulating hematopoiesis between humans and Drosophila and of the powerful genetic tools available in Drosophila, we assessed whether Drosophila can provide a suitable model system to study the mechanism of action of the human oncogene AML1-ETO and to identify modulators of its activity.. Our results show that AML1-ETO exerts in Drosophila blood cells expressing a RUNX factor (Lozenge, LZ) similar effects to those observed in human leukemic cells. 10.

(11) carrying the t(8;21), namely a differentiation blockage and an increased proliferation. In addition, by performing a large scale in vivo screen based on RNA interference, we identified calpainB as required for AML1-ETO-induced blood cell disorders in Drosophila. We further showed that calpains inhibition resulted in AML1-ETO degradation and reduced the clonogenic potential of human leukemic cells intrinsically expressing this chimera. Our results establish that Drosophila can be used as an alternative model to better understand AML1-ETO function and to identify new factors regulating its activity.. 11.

(12) Titre: Les cellules hématopoïétiques de la Drosophile comme modèle d’étude in vivo de l’activité de l’oncogène humain AML1-ETO.. Résumé. L’hématopoïèse est un processus complexe et dynamique qui conduit à la formation ainsi qu’au remplacement continu et régulé des cellules sanguines. Au cours de ce processus, une série de facteurs de transcription contrôle l’apparition, l’engagement et la différentiation des cellules souches dans un lignage déterminé. En particulier, chez les vertébrés, le facteur RUNX1/AML1 (pour Acute Myeloid Leukemia 1) est requis pour l’émergence des cellules souches hématopoïétiques ainsi que pour la différentiation des lignées myéloïdes et lymphoïdes. Chez l’homme, différentes altérations génétiques affectant AML1 sont liées au développement de pathologies du système hématopoïétique. Notamment, la translocation chromosomique t(8;21), codant la protéine de fusion AML1-ETO, est associée à 10-15 % des cas de leucémies myéloïde aiguës. Il a été décrit que AML1-ETO agit essentiellement en interférant avec la fonction de AML1 au cours de la différentiation hématopoïétique. Cependant, le mode d’action de cet oncogène reste mal compris et peu de facteurs modulant son activité sont connus.. Récemment, de nombreux travaux ont mis en évidence que divers aspects du développement des cellules hématopoïétiques sont conservés de la Drosophile aux vertébrés. Notamment, les facteurs de transcription des familles RUNX et GATA, acteurs clefs de l’hématopoïèse chez les vertébrés, contrôlent aussi le développement des cellules sanguines chez la Drosophile. Tirant profit d’une part de la conservation phylogénétique des circuits géniques régulant l’hématopoïèse chez l’homme et la Drosophile et d’autre part des puissants. 12.

(13) outils d’analyses génétiques disponibles chez la Drosophile, nous avons cherché à utiliser la Drosophile comme système modèle pour étudier le mécanisme d’action de l’oncogène humain AML1-ETO et identifier des modulateurs de son activité.. Nos résultats montrent que AML1-ETO exerce dans des cellules sanguines de la Drosophile dont la différentiation dépend de l’expression d’un facteur RUNX (Lozenge, LZ), des effets similaires à ceux observés dans des cellules leucémiques humaines portant la translocation t(8;21), à savoir : un blocage de la différenciation des cellules hématopoïétiques exprimant LZ et une augmentation de leur nombre. Grâce à la réalisation d’un crible génétique in vivo par interférence à l’ARN, nous avons identifié calpainB comme requis pour que AML1-ETO induise ces effets. De plus, nous avons pu montrer que l’inhibition des calpaines provoque la dégradation de AML1-ETO et diminue le potentiel clonogénique de cellules leucémiques humaines exprimant cette chimère. Ces travaux montrent que la Drosophile peut être utilisée comme modèle alternatif pour mieux comprendre la fonction de AML1-ETO et identifier de nouveaux facteurs régulant son activité.. 13.

(14) Foreword. Human, during his life, is confronted to many environmental and/or genetic factors that can disrupt the normal functioning of his organism, leading to the development of numerous diseases. Cancer represents more than 100 malignant diseases, which should be treated promptly after diagnosis to avoid fatal consequences. Cancer is characterized by an abnormal growth of cells that invade and destroy nearby normal tissues and thereafter spread throughout the body. Leukemia is a group of cancers that involves essentially the main bloodforming tissue, the bone marrow, which starts producing large numbers of abnormal white blood cells. In the past few years, remarkable efforts have been made to improve the understanding of the molecular pathogenesis of several malignant blood diseases. Acute myeloid leukemia (AML) was particularly the subject of a broad series of studies, which by using cell culture system and vertebrate animal models enabled a better understanding of this deadly blood disorder. However, the cure rate remains low and it is expected that the identification of new key regulatory factors implicated in the development of AML may allow the elaboration of new efficient and less toxic therapeutic strategies. The establishment of tractable genetic model like Drosophila can be useful to further dissect genetic pathways that are potentially relevant to the pathogenesis and treatment of AML. Accordingly, during my PhD, I developed a system to explore the mode of action of the human leukemogenic fusion protein AML1-ETO in Drosophila.. First, I will overview human blood cell development and functions and introduce the AML malignant disease, one of the most common hematological malignancies in the world. I will focus subsequently on one particular genetic abnormality found frequently in AML: the. 14.

(15) 8;21 translocation. Then I will introduce Drosophila hematopoietic system and finally I will present and discuss the main results of my PhD work.. 15.

(16) List of abbreviations AGM : Aorta-Gonade-Mesonephros ALL : Acute Lymphoblastic Leukemia AML : Acute Myeloid Leukemia AML1-ETOtr : AML1-ETO truncated Bc : Black cell Bgb : Big brother bHLH : basic Helix-Loop-Helix BrdU : Bromodeoxyuridine Bro : Brother Calp : Calpain CBF : Core Binding Factor CBFß : Core Binding Factor ß subunit CFU : Colony Forming Unit CLL: Chronic Lymphoblastic Leukemia CML: Chronic Myeloid Leukemia Col : Collier COX : Cyclooxygenase inhibitors CRQ : Croquemort Dpn : Deadpan dsRNA : double strand RNA EBF : Early B-cell Factor ECM : Extra-Cellular Matrix ENU : N-ethyl-N-nitrosourea ETO : Eight Twenty One FAB : French-American-British FAK : Focal Adhesion Kinase FLT3 : Fms-Like Tyrosine kinase 3 FPD : Familial Platelet Disorder Gcm : Glial cell missing GFP : Green Fluorescent Protein GM-CSF-R: Granulocyte/Macrophage Colony Stimulation Factor Receptor. 16.

(17) HDAC : Histone Deacytelase HEB : Hela-E-box-Binding protein Hs : Heat shock HSCs : Hematopoietic Stem Cells ICM: Intermediate Cell Mass ICSBP: Interferon Consensus Binding Protein IL-3: Interleukin 3 JAK/STAT : Janus Kinase/Signal Transducer and Activators of Transcription LSCs : Leukemic Stem Cells LZ : Lozenge MDR1 : Multidrug Resistance 1 MDS : Myelodysplastic Syndrome MPD : Myeloproliferative Disorder MTG8 : Myeloid Tumor Gene 8 NCoR : Nuclear receptor co-Repressor NHR : Nervy Homology Region NMTS : Nuclear-Matrix-attachment Signal NOD/SCID : Nonobese Diabetic-Severe Combined Immunodeficient PDGF : Platelet-Derived Growth Factor PEST : proline (P), glutamic acid (E), serine (S) and threonine (T) PO : Phenoloxidase proPOs : prophenoloxidase PROS : Prospero PSC : Posterior Signaling Center Pvr : Drosophila homolog of receptor for cytokines of Platelet-Derived Growth Factor (PDGF) family and Vascular Endothelial Growth Factor (VEGF) receptor RBI : Rostral Blood Island RHD : Runt Homology Domain RNAi : RNA interference RUNX : Runt related protein SCL : Stem Cell Leukemia SMRT : Silencing Mediator for Retinoid and Thyroid hormone receptor SRP : Serpent SVP : Sevenup 17.

(18) TCRß : T-cell Receptor ß chain enhancer TEL-PDGFßR : receptor tyrosine kinase, Platelet-Derived Growth Factor Receptor ß TLE : Transducin-Like Enhancer of split TSA : Trichostanin A UAS : Upstream Activating Sequence Upd : Unpaired USH : U-shaped VEGF : Vascular Endothelial Growth Factor WHO : World Health Organization WT1 : Wilms tumor. 18.

(19) List of figures Figure 1. A summary of the process of blood development. Figure 2. A model that depicts hematopoiesis as a continuum of lineage relationships between hematopoietic stem cells (HSCs) and their oligopotent progeny. Figure 3. Estimated proportion of new cases (%) in 2009 for types of leukemia. Figure 4. Multistep model for AML pathogenesis. Figure 5. Structure of AML1 protein. Figure 6. Schematic representation of alternative splicing of the AMLI transcripts. Figure 7. AML1 expression in haematopoietic sites in the E10.5 embryo. Figure 8. AML1 transcriptional mechanisms. Figure 9. Structure of fusion proteins generated in AML1-related leukemia. Figure 10. Leukemic blast with an Auer body. Figure 11. Genomic structure of t(8;21). Figure 12. Stucture of AML1-ETO fusion protein. Figure 13. Schematic representation of ETO/MTG8 illustrating the position of the four nervy homology regions (NHR1–4). Figure 14. A model for AML1-ETO repression mechanism. Figure 15. A model of a high DNA-binding activity of tetrameric AML1-ETO to duplicated AML1-binding sites. Figure 16. Model illustrating how different cells can be at the origin of the leukemic transformation events. Figure 17. Primitive hematopoiesis in the zebrafish embryo. Figure 18. Drosophila blood cells. Figure 19. Two waves of Drosophila hematopoiesis. Figure 20. Lymph gland structure.. 19.

(20) Figure 21. AML1-ETO inhibits PO45-lacZ reporter transgene expression. Figure 22. LZ+ circulating blood cells persist massively to pupal stage in presence of AML1ETO. Figure 23. CalpainB protein is expressed specifically in crystal cells in the embryo. Figure 24. Absolute number of circulating LZ-GFP+ cells in third instar larvae in knockdown conditions of AML1-ETO suppressors. Figure 25. Ratio of circulating progenitors (GFP+, PO45-) to differentiated (GFP+, PO45+) crystal cells in third instar larvae in knock-down conditions of AML1-ETO suppressors. Figure 26. CG3759 is required for AML1-ETO function in the embryo. Figure 27. Schematic representation of the different Drosophila calpains domains. Figure 28. PESTfind analysis shows that AML1-ETO contains two predicted strong PEST regions. Figure 29. Calpain1 cleaves AML1-ETO in vitro. Figure 30. CalpainB is not required for AML1-ETOtr function in LZ-GFP+ cells. Figure 31. CalpainB expression and cellular localization is regulated by AML1-ETO.. 20.

(21) List of tables. Table 1. The FAB classification of AML. Table 2. Suppressors of AML1-ETO. Table 3. Expression patterns of calpains. Table 4. Functional diversity of calpains. 21.

(22) Chapter I. INTRODUCTION. 22.

(23) Figure 1. A summary of the process of blood development. (adapted from Smith, 2003)..

(24) A. General description of hematopoiesis and leukemia. 1. Hematopoiesis. (i) Ontogeny of the mammalian hematopoietic system. Mammalian hematopoietic development consists of two distinct waves (Dzierzak and Medvinsky, 1995; Medvinsky and Dzierzak, 1996). The first wave is called primitive hematopoiesis and takes place in the yolk sac at embryonic day 7.5 in the mouse. Primitive cells are mainly erythroid cells that express embryonic hemoglobin. The second wave is known as definitive hematopoiesis and occurs at embryonic day 9,5 with multipotent hematopoietic stem cells (HSCs) arising in the aortic-gonadal-mesonephros (AGM) region and in the placenta. Definitive HSCs seeds different hematopoietic sites of the embryo (fetal liver and thymus) and give rise to all blood cell types. At the end of fetal development, HSCs migrate to the bone marrow, which becomes the major site of postnatal hematopoiesis through the life span.. (ii) Blood cell lineages. The marrow, a spongy tissue located in the medullar cavity of bone, is the major site for blood cell production during the adult life. Blood cell formation and maturation is a complex and dynamic hierarchical process. It relies on a few HSCs or “mother” cells, which are capable throughout the life to self-renew or expand into proliferative committed progenitors that give rise to more committed progeny and ultimately differentiate to mature cells. The classical scheme of hematopoiesis can be viewed as a tree, in which the HSCs represent the trunk and the two main branches represent the derived myeloid and lymphoid 23.

(25) Figure 2. A model that depicts hematopoiesis as a continuum of lineage relationships between hematopoietic stem cells (HSCs) and their oligopotent progeny. This model proposes a series of invariant pairwise developmental relationships between the various hematopoietic lineages. Arcs indicate the known oligopotent progenitor cells. Some of the arcs overlap: in particular, the arrowheads on two of the arcs indicate that dendritic cells (DCs) can be derived from both megakaryocyte–monocyte progenitors and B-cell–T-cell progenitors. EPLM, early progenitor with lymphoid and myeloid potential; FLT3, FMSrelated tyrosine kinase 3; LMPP, lymphoid-primed multipotent progenitor. NK cell, natural killer cell (adapted from Ceredig et al., 2009)..

(26) lineages (Figure 1) (Smith, 2003). Myeloid cells include lineages that give rise to erythrocytes, megakaryocytes, monocytes/macrophages and granulocytes (neutrophils, eosinophils, basophils). Lymphoid cells regroup B, T and natural killer cells. Yet, this binary cell fate tree is still debated and recent studies have proposed new hierarchical trees or circle/arc models of hematopoiesis (Figure 2). The new models suggest that intermediate progenitors have both lymphoid and myeloid potential (Ceredig et al., 2009). For instance, in contrast to the classical view, it was shown that pro-B cells are multipotent and can give rise to myeloid cells, natural killer and T cells. Thus, HSCs can reach a specific cell fate through more than one type of intermediate progenitors; this reflects the high complexity of the hematopoietic system.. (iii). Blood cell functions. When the fully developed and functional cells are formed, they leave the marrow and enter the blood circulating system. The mature blood cells circulating in the body perform several specialized functions (Hartenstein, 2006):. - The erythrocytes make up about 45% of the blood. They transport oxygen to tissues and partly recover carbon dioxide produced as waste.. - Platelets, which are small cell fragments produced from megakaryocytes, are mainly implicated in blood clotting. After wound and hemorrhage, they aggregate and release factors that promote blood coagulation.. - Granulocytes and Macrophages are the principal actors of the innate immune system, which provide immediate protection against infection. They contain a wide variety of 24.

(27) enzymes involved in the attack and digestion of bacteria and other pathogens invading the body. Besides their phagocytic activity of infected cells, macrophages process proteins of the microbes and expose them on the membrane. They serve an essential antigen-presenting role, which allows the more specialized adaptative immune response to occur during the days that follow.. - The lymphocytes are the major players of the adaptative immunity. In infection conditions, they expand and yield highly specific and clonal responses to microbial antigens presented by the macrophages: whereas B lymphocytes mass produce antibodies against a specific antigen, T lymphocytes present direct cytotoxic effects on recognized infected cells. Like all blood cells, T cells originate in the bone marrow but mature in the thymus, a small organ in the upper chest. Lymphocytes are considered as the blood cell subtypes responsible of the immunological memory of the organism.. 2. Leukemia. Over the last 60 years, many genetic disorders affecting blood cell development and functions have been identified. In some cases, they lead to a wide array of clonal (neoplastic) malignant diseases called leukemia. The term leukemia is derived from the Greek words leukos, meaning “white”, and aima, meaning “blood”. Hence, leukemia is the cancer of white blood cells. The causes of leukemia are not well known. Chemical (e.g. benzene) exposure, smoking, ionizing radiation, heredity and certain cytotoxic anti-cancer drugs are suspected to cause some forms of leukemia (Estey and Dohner, 2006; Segel and Lichtman, 2004).. 25.

(28) Figure 3. Estimated proportion of new cases (%) in 2009 for types of leukemia, (adults and children). CML: Chronic Myeloid Leukemia, AML: Acute Myeloid Leukemia, CLL: Chronic Lymphoblastic Leukemia, ALL: Acute Lymphoblastic Leukemia. Source: Cancer Facts & Figures 2009, American Cancer Society; 2009 (total is more than 100% due to rounding)..

(29) Leukemia classification. Leukemia are classified by two factors; how quickly the illness develops and what cell subtypes are affected.. - Acute leukemia are rapidly progressing diseases that can overrun the body within a few days or weeks resulting in the accumulation of immature, non-functional cells in the marrow and blood. These cells eventually crowd out normal blood cells and spread to other organs where they cause damages. Chronic leukemia progress more slowly over time allowing greater numbers of functional healthy cells to be made. It is a less dramatic illness than acute leukemia. In the absence of cure after chemical treatment, chronic leukemia may progress to acute leukemia (Kelly and Gilliland, 2002).. - According to the type of blood cells that are affected, leukemia is divided into myeloid or lymphoid subtypes. When the cancerous cells arise primarily from the bone marrow lymphocytes, the disease is categorized as lymphoblastic leukemia. Whereas the cancer is called myeloid (or myelogenous) leukemia if the abnormal cells derived from the myeloid cell lines of the bone marrow.. Next, I will focus on AML, which is the predominant fatal form of acute leukemia. In 2009, the estimated proportion of new AML cases in the United States is around 29% of all types of leukemia (Figure 3). Older people are more likely to develop AML (80 % of acute leukemia cases) than younger adults or children (15-20 %). This disease disturbs essentially the homeostasis of the innate immune system. In healthy situation, the components of the innate immune system in the peripheral blood are described as “end. 26.

(30) FAB subtype. Common name. Frequency (%). M0. Acute myeloblastic leukemia with minimal differentiation. M1. Acute myeloblastic leukemia without maturation. 15-20. M2. Acute myeloblastic leukemia with granulocytic maturation. 25-30. M3. Acute promyelocytic leukemia (APL). 5-10. M4. Acute myelomonocytic leukemia, Acute myelomonocytic with bone marrow eosinophilia (M4eo ). 20. M5. Acute monoblastic leukemia (M5a) Acute monocytic leukemia (M5b). 2-9. M6. Acute erythroid leukemias - erythroleukemia (M6a) - very rare pure erythroid leukemia (M6b). 3-5. M7. Acute megakaryoblastic leukemia. 3-12. Table 1. The FAB classification of AML (Lowenberg et al., 1999).. 3.

(31) cells”, which are highly differentiated and incapable of proliferation (Nemeth and Bodine, 2007). In AML, these two essential features of myeloid cells are seriously disturbed.. 3. Acute myeloid leukemia AML is a heterogeneous group of clonal disorders that share the characteristic of arising as the consequences of acquired somatic mutations in multipotential progenitors or in more differentiated precursor cells (Frohling et al., 2005). These mutations result in a rapid uncontrolled accumulation in both marrow and bloodstream of myeloid progenitors, called AML blasts, which lose the ability to differentiate to functional cells. Moreover, AML blasts release cytokines that interfere with the production, from normal blasts, of healthy differentiated myeloid cells (Youn et al., 2000). AML patients thus suffer from severe anemia, bleeding and infections as consequences of decreased number of functional erythrocytes, platelets and granulocytes.. (i) Classification. There are two commonly used classification schemata for AML. The first is the older French-American-British (FAB) classification described in the 1970s (Lowenberg et al., 1999), the second is the newer World Health Organization (WHO) classification system (Gilliland and Tallman, 2002). FAB classification remains the foundation on which the morphological diagnosis of AML is based. It divides AML into subtypes, M0 through M7, based on the type of cells from which leukemia developed and how mature the cells are (Table 1). WHO classification takes into account other factors such as cytogenetics, molecular genetics, the presence of antecedent hematological disorder and immunophenotypic features. The subclassification of AML provides important information about the expected 27.

(32) Figure 4. Multistep model for AML pathogenesis. Class I mutations confer a proliferative and/or survival advantage to hematopoietic cells. Class II mutations result in loss of function of transcription factors that are important for normal hematopoietic differentiation. When expressed alone, these mutations may confer phenotypes most like MDS (Myelodysplastic Syndrome) or MPD (Myeloproliferative Disorder) (Gilliland and Tallman, 2002; Kelly and Gilliland, 2002)..

(33) course of the disease. It helps in assessing the patients treatment options and allows the separation of patients into favorable, intermediate and adverse prognostic categories (Frankfurt et al., 2007).. (ii) Genetic alterations in AML. Recently, significant progress in the understanding of the genetic basis of AML allowed to set the paradigm that a crucial cooperation between two classes of molecular events is required to promote the development of AML (Downing, 2003; Estey and Dohner, 2006; Frohling et al., 2005; Kelly and Gilliland, 2002; Steffen et al., 2005). The first type of genetic events comprises mutations affecting genes encoding for growth/survival related factors, whereas the second type of genetic aberrations appears to affect genes that code for particular transcriptional regulatory factors involved in myeloid differentiation (Figure 4). Since the type I mutations are not specific to an AML subtypes, they are largely considered as secondary events required for progression to overt AML (Moore, 2005). Class I mutations are exemplified by constitutively activated tyrosine kinases and their downstream effectors, such as BCR/ABL, RAS, C-KIT and FLT3. When the class I mutations are expressed alone, they result in a myeloproliferative disorder (MPD) with leukocyctosis and normal differentiation (Kelly and Gilliland, 2002). Conversely, when class II mutations are expressed alone, they may induce phenotypes most like myelodysplastic syndrome (MDS), i.e. ineffective differentiation (Kelly and Gilliland, 2002). Only when these two classes mutations are coexpressed, they result in AML phenotypes.. Some of the most frequent class II genetic damages considered as initiating the pathogenesis of AML are non-random chromosomal aberrations (e.g. reciprocal translocations, deletions, inversions) (Frohling et al., 2005; Look, 1997). In particular, 28.

(34) numerous cytogenetic abnormalities that involve gene encoding AML1 (Acute Myeloid Leukemia 1) have been found in many subtypes of AML (Sawyers, 1997). AML1 (also referred as RUNX1, CBPα2, CBFA2 and PEBP2αB) is a member of the Runt-like conserved family of transcription factors (RUNX) that also include RUNX2 and RUNX3 in human. RUNX genes are involved in a wide variety of developmental processes, ranging from segmentation and sex determination in insects to skeletal and blood formation in mammals. AML1 gene on chromosome 21 was cloned and identified basically through its involvement in one particular AML associated-translocation (see below). Subsequently, several studies have demonstrated that AML1 is crucial for the regulation of normal hematopoiesis.. Historically, cytogenetic aberrations have been extensively used to classify AML subtypes and to identify genes involved in this pathogenesis. Yet most genetic events that induce this disease remain unknown. More recently, whole genome sequencing was used to identify genes mutated in cases of cytogenetically normal AML (Ley et al., 2008). This technique opens the way to the unbiased identification of genetic alterations in AML.. B. AML1 in hematopoiesis and leukemia. 1.. Structure and isoforms of AML1 proteins. AML1/RUNX1 belongs to the RUNX family of genes, and encodes the DNA-binding-α subunit of the heterodimeric Core Binding Factor (CBF) complex. All RUNX proteins bind DNA via a Runt homology domain (RHD), which is highly homologous to the DNA binding domain of the Drosophila nuclear protein RUNT (Canon and Banerjee, 2000; Duffy and Gergen, 1994; Duffy et al., 1991; Kania et al., 1990). Furthermore, the Runt domain mediates the heterodimerization of RUNX proteins with the single common Core Binding Factor β. 29.

(35) Figure 5. Structure of AML1 protein. In its N-terminal part, AML1 contains a DNA binding domain called Runt domain. Other important sequences are located at the carboxy-terminal part defining a transactivation domain, a nuclear-matrix-attachment signal (NMTS) and a VWRPY conserved motif that is required for the interaction with transcriptional corepressors of the Groucho family..

(36) subunit (CBFβ), which facilitates efficient DNA binding to the consensus DNA sequence TGYGGTY in RUNX target gene promoters (de Bruijn and Speck, 2004; Ferjoux et al., 2007; Ogawa et al., 1993a; Ogawa et al., 1993b; Wang et al., 1993). RUNX factors contains also other important sequences at the carboxy-terminal part defining a transactivation domain, a nuclear-matrix-attachment signal (NMTS) and a VWRPY conserved motif that is required for the interaction with transcriptional corepressors of the Groucho family (Figure 5) (Aronson et al., 1997; Durst and Hiebert, 2004; Levanon et al., 1994; Zeng et al., 1997). Despite their structural similarity, RUNX genes have divergent functions in mammalian development. While AML1 is essential for hematopoiesis, RUNX2/AML3/CBFA1 is required in osteogenesis and RUNX3/AML2/CBFA3 in neurogenesis (Blyth et al., 2005).. AML1 gene is known to be regulated by two alternative promoters conserved in vertebrates, the P1 or distal, and the P2 or proximal, which are juxtaposed by their corresponding first coding exons (Ghozi et al., 1996; Miyoshi et al., 1995). This transcription generates at least three alternatively spliced proteins isoforms, called AML1a, AML1b and AML1c (Figure 6). AML1a (from P2) lacks a C-terminal transcriptional activation domain and acts as inhibitor of AML1b (from P2) and AML1c (from P1) functions. AML1a expression level is found abnormally high in ALL and AML-M2 patients, indicating a potential role in leukemogenesis (Liu et al., 2009). AML1b and AML1c contain the same Cterminal domain but have distinct amino-terminal sequences, due to the activity of alternative promoters. It has been shown that AML1b mouse homologue is expressed in the early developmental stage (day 7), in contrast to the gradual upregulation and maintenance of AML1c expression during embryogenesis. Thus far, except a diverged effect on the growth of neutrophil cells (Telfer and Rothenberg, 2001), no data has been reported regarding any difference in biological properties of AML1b and AML1c isoforms.. 30.

(37) Figure 6. Schematic representation of alternative splicing of the AMLI transcripts. A schematic representation of the exons is shown at the top. Solid boxes represent the coding regions, and open boxes represent the 5'- and 3' untranslated regions. Positions of polyadenylation signals are shown by A (adapted from Miyoshi et al., 1995)..

(38) 2.. Role of AML1 in hematopoiesis in the embryo. The biological function of AML1 during normal hematopoietic development was characterized essentially by using mouse models. AML1-deficient mice exhibited normal embryo morphogenesis and had normal active yolk sac-derived primitive erythropoiesis. However, they failed to develop fetal liver-derived definitive hematopoiesis (Okuda et al., 1996). As secondary effects, hemorrhages were revealed in the central nervous system and these mice died during midembryonic development. Similar phenotypes were obtained with another independent AML1-knock-out mice engineered with different strategy (Wang et al., 1996). These first studies suggested that AML1 is required for the generation or further differentiation of HSCs, allowing the establishment of definitive hematopoiesis of all lineages. Many subsequent studies have confirmed that the defects resided in the emergence of definitive hematopoietic stem and progenitors cells, which arise from several distinct sites where AML1 is normally expressed during the embryo development (Mukouyama et al., 2000; North et al., 1999; Yokomizo et al., 2001). These sites include yolk sac, the umbilical and vitelline arteries, and AGM region (Figure 7).. 3.. Role of AML1 in adult hematopoiesis. AML1 is also expressed in the bone marrow where it plays important role in adult hematopoiesis. To establish its expression pattern in the bone marrow, two mouse strains have been developed in which GFP or LACZ markers were knocked into the AML1 locus (Lorsbach et al., 2004; North et al., 2004). These mice as well as other expression studies showed that AML1 is expressed at high levels in postnatal hematopoietic stem and progenitor cells. Moreover, and except the erythrocyte lineage where it is undetectable, AML1 is dynamically expressed in the other bone marrow cell lineages. Its expression generally. 31.

(39) Figure 7. AML1 expression in haematopoietic sites in the E10.5 embryo. (a) AML1 is expressed (blue) in a small population of endothelial cells and haematopoietic cells that are scattered throughout the yolk sac (ys), in endothelial cells lining the vitelline (v) and umbilical (u) arteries, and in endothelial cells, mesenchymal cells and intra-aortic haematopoietic clusters in the ventral portion of the dorsal aorta within the aorta/gonad/mesonephros (AGM) region. The fetal liver (fl) contains AML1 + haematopoietic cells. AML1 is also expressed in several sites that are unrelated to haematopoiesis. (b) Detailed view of AML1 expression in endothelial cells (e), mesenchymal cells (m) and a haematopoietic cluster (hc) in the ventral AGM region (adapted from Speck and Gilliland, 2002)..

(40) decreases when the cells are in terminal differentiation stage. The function of AML1 in the bone marrow was well characterized using a conditional knock-out approach in adult mice (Ichikawa et al., 2004). Based on the Cre/loxP-mediated recombination system, Ichikawa et al. engineered mice lacking AML1 in adult HSCs and their progenitors. In contrast to the embryo, deletion of AML1 in adult did not abolish HSCs emergence and function as hematopoiesis still established in the bone marrow. This indicates that the molecular mechanisms implicated in the generation of the hematopoietic system in the embryo differ from those required for its maintenance. In addition, and despite its strong expression, AML1 did not seem to be crucial for granulocytes and monocytes development as their morphology and maturation appears normal in AML1-deficient mice. However, these mice exhibited markedly reduced numbers of mature B and T cells as well as a deficiency of platelets and megakaryocyte differentiation. Interestingly, AML1-haploinsufficiency in human was shown to cause a familial platelet disorder (FPD), characterized by thrombocytopenia (a decrease in the number of platelets) and impaired platelets function, which prevents efficient and rapid cicatrisation. Moreover, some patients with FPD syndrome showed a markedly increased risk of developing AML during their lifetime (Ho et al., 1996). Taken together, these results demonstrated that AML1 is necessary for the maturation of lymphoid lineages and megakaryocytes, and mutations affecting AML1 contribute to abnormalities in hematopietic development and malignancy.. 32.

(41) Figure 8. AML1 transcriptional mechanisms. (A) AML1 binds to a consensus DNA sequence present in the cis-elements of target genes. The AML1 binding site is often adjacent to binding sites for other DNA-binding proteins, including Ets, Myb, and C/EBPα. AML1 regulates gene expression in cooperation with these lineage-specific transcription factors. AML1 mediates transcriptional activation (A) or repression (B) upon recruitment of non-DNA-binding coactivators (p300/CBP) or corepressors (mSin3A, HDAC, and TLE/Groucho) (adapted from Kurokawa and Hirai, 2003)..

(42) 4.. AML1: a master transcriptional regulator of hematopoiesis. AML1 has been shown to act as a master nuclear transcriptional orchestrator of a large number of genes involved in hematopoietic differentiation. These genes include those encoding. T-cell. receptor. β. chain. enhancer. (TCRβ),. cytokines. such. as. granulocyte/macrophage colony stimulation factor receptor (GM-CSF-R) and interleukin 3 (IL-3), and other granulocyte factors such as neutrophil elastase, myeloperoxidase, TNFα and granzyme B (Lutterbach and Hiebert, 2000; Peterson and Zhang, 2004). AML1 binding sites in the promoter of these targets is often adjacent to the binding sites for other DNA-binding proteins (Figure 8). In fact, various cooperating factors are required for AML1 to regulate efficiently the transcription of its targets (Blyth et al., 2005; Peterson and Zhang, 2004). In addition to generic transcription-co-activators such as histone acetyltransferases (p300/CREBbinding protein), other AML1 partners are specific-lineage DNA binding transcription factors including C/EBPα, PU.1, PAX5, GATA1, and ETS family members (Ets-1, MEF) (Elagib et al., 2003; Goetz et al., 2000; Gu et al., 2000; Hanai et al., 1999; Kim et al., 1999; Kitabayashi et al., 1998b; Libermann et al., 1999; Mao et al., 1999; Pardali et al., 2000; Petrovick et al., 1998; Westendorf et al., 1998; Zhang et al., 1996). For instance, C/EBPα and PU.1 are twotranscription factors required for granulocyte/monocyte lineages development. AML1 interacts with C/EBPα and PU.1 that cooperatively bind to DNA and regulate the promoter activity of human M-CSF receptor (Zhang et al., 2001a). Hence, AML1 appears to function as a transcriptional organizer that recruits other factors and together they form complexes which stimulate lineage-restricted transcription.. 5.. AML1 in leukemia. Mutations affecting AML1 have been identified in several blood diseases beside FPD. 33.

(43) Figure 9. Structure of fusion proteins generated in AML1-related leukemia. t(8;21) results in fusion of the AML1 and ETO genes, and is predominantly associated with the M2 subtype of AML (for details, see in the text and figure 12). The TEL–AML1 fusion gene is the most common gene rearrangement associated with childhood cancer. The TEL gene product is a transcriptional repressor that contains a pointed (PNT) oligomerization motif, which might serve, in part, to recruit the nuclear corepressor complex to AML1 promoters. The AML1–EVI1 fusion gene is associated with a small percentage of cases of CML in blast crisis and in MDS. The structure is similar to that of AML1–ETO, and the fusion protein might serve properties of transcriptional repression. TA, transactivation domain; ZF, zinc finger (adapted from Speck and Gilliland, 2002)..

(44) While point mutations detected throughout the full length of AML1 are mainly associated with MDS (that can progress in rare cases to AML) (Harada and Harada, 2009), chromosomal translocations affecting this gene are found in human leukemia (Lo Coco et al., 1997). Thus far, more than ten different chromosomal translocations in acute leukemia cases that involved AML1 are known. Relevant examples of these molecular aberrations include t(8;21) (AML1ETO), t(12;21) (TEL-AML1) and t(3;21) (AML1-EVI1) (Figure 9). They are associated to the development of either myeloid or lymphoid leukemia. For instance, the t(8;21) and t(12;21) are among the most frequent chromosomal translocations in patients with acute myeloid or acute lymphoid leukemia, respectively. In addition, the inv(16), which affects CBFβ, the partner of AML1, is also found in 15% cases of AML. It is largely believed that the resulting fusion proteins dominantly inhibit AML1 function. The t(8;21), which was the first identified in AML and most studied translocation, will be described below in details.. C. The 8;21 chromosomal translocation and AML development. 1.. Incidence and cell characteristics of t(8;21) AML The 8;21 chromosomal translocation [t(8;21)(q22;22)] is associated with 10 to 15% of. all cases of AML and 40% of cases of the M2 AML subtype (Peterson and Zhang, 2004). It has also been detected, at lower frequencies, in AML-M1 and M4, and in rare cases of MDS and MPD (Downing, 1999). As stated above, and according to the WHO classification, cytogenetic abnormalities are closely associated with morphologically distinct subtypes of AML and serve as an important prognostic factors in predicting response to treatment (Bain, 2001). Leukemic blasts from patients with t(8;21) AML are morphologically distinguishable by the typical presence of prominent aurer rods (Figure 10) and abnormal cytoplasmic granules (Downing, 1999). They are characterized by myelomonocytic differentiation and. 34.

(45) Figure 10. Leukemic blast with an Auer body. (adapted from Lowenberg et al., 1999)..

(46) increased eosinophils with a favorable prognostic in adults after therapeutic treatment (relatively low risk of relapse (30-40%) and overall 5-year survival of 70% in adults) (Mrozek et al., 2001).. 2.. Molecular characterization of t(8;21). The molecular dissection of the 8;21 translocation by several groups in the early 1990s led to the cloning of two genes located at the breakpoints, AML1 on chromosome 21q22 and ETO (Eight Twenty One) on chromosome 8q22 (Erickson et al., 1992; Kozu et al., 1993; Maseki et al., 1993; Miyoshi et al., 1993; Miyoshi et al., 1991; Nisson et al., 1992; Nucifora et al., 1993; Shimizu et al., 1992). Most studies described that AML1 and ETO are rearranged in the leukemic cell DNAs from t(8;21) AML patients resulting in the juxtaposition in frame of their coding sequences. In t(8;21) leukemic patient cells, one allele of AML1 and one allele of ETO are affected, the others are still intact. While the t(8;21) generates two fusion genes called AML1-ETO and ETO-AML1, only AML1-ETO mRNAs and proteins have been detected; no ETO-AML1 transcripts have been identified in AML patients (Peterson et al., 2007a).. As shown in Figure 11, the breakpoints within the AML1 locus are clustered in the intron 5, downstream of AML1 promoters. Hence, the expression of AML1-ETO fusion gene is under the regulation of AML1 promoters. The commonly known AML1-ETO transcripts are found to originate from the proximal promoter regulating AML1 in patient cells (Miyoshi et al., 1993) and surprisingly, no mRNAs generated from the distal one have been identified. It is believed that the t(8;21) occurs at the level of HSCs and thus AML1-ETO is expressed and functions very early during hematopoieisis (discussed below). 35.

(47) P1. P2. Figure 11. Genomic structure of t(8;21). On chromosome 8, the ETO gene is made up of 13 exons spanning approximately 87 kb, which can give four alternative splice forms and is regulated by two promoters. Chromosome 21 contains the AML1 gene with nine exons that give various alternative splice forms and is regulated by two promoters (P1 and P2) and spans 260 kb. The crossing lines between ETO and AML1 denote the breakpoint cluster areas. Owing to the absence of a splice acceptor in exon 1b of ETO, the mRNA of the fusion transcripts does not include this exon. White boxes and black boxes indicate translated and untranslated exon sequences, respectively. Underlined numbers in the AML1-ETO mRNA denote exons contributed by the ETO gene. (adapted from Peterson and Zhang, 2004)..

(48) Figure 12. Stucture of AML1-ETO fusion protein. AML1-ETO contains the N-terminal 177 amino acids of AML1 including the Runt domain fused in frame to the almost entire ETO protein. AML1-ETO lacks the C-terminal transactivation domain of AML1..

(49) 3.. Structure and transcriptional function of ETO and AML1-ETO proteins. AML1 binds DNA and several of its partners via the Runt homology domain, which still present in AML1-ETO. However, AML1 C-terminal transactivation domain is lost in the chimera and the majority of the coding region of ETO replaces it. AML1-ETO contains the N-terminal 177 amino acids of AML1 fused in frame to the almost entire ETO protein (575 amino acids) (Figure 12) This structure led to believe that AML1-ETO has different properties from wild type AML1, a hypothesis demonstrated through several studies. These studies have led to better understanding of AML1-ETO-induced leukemogenic process and have given insights into the normal role of the ETO family of proteins.. ETO: a transcriptional co-repressor. ETO (also known as MTG8 [myeloid tumor gene 8],or RUNX1T1) belongs to the ETO evolutionarily conserved family of nuclear factor formed by three members in human. They share four homologous domains (NHR1-4) with the Drosophila Nervy protein (NHR, Nervy Homology Region) (Feinstein et al., 1995; Hug and Lazar, 2004) (Figure 13). The first NHR region (NHR1) shares similarity with Drosophila TAF110 and related TAF transcriptional proteins and may have a role in sub-nuclear localization (Erickson et al., 1994; Hug and Lazar, 2004). The NHR2 has a predicted coiled structure with heptad repeat of hydrophobic amino acids and is required for homo- and heterodimerization between the ETO family members. NHR3 has notable homology with Nervy but little is known about its function. NHR4, termed also the MYND domain, has two non-DNA binding zinc fingers (Hildebrand et al., 2001). These two zinc-finger motifs are involved in protein-protein interaction and appear to be essential for ETO function. It has been shown that NHR2 works. 36.

(50) NHR 1. NHR2. NHR3. NHR4. 98%. 88%. 84% 90%. MTG16. 95%. 79%. 66% 87%. 50%. 51%. 46% 70%. MTGR 1 Nervy. ETO. Figure 13. Schematic representation of ETO/MTG8 illustrating the position of the four nervy homology regions (NHR1–4). Numbers indicate the percent of identity between ETO and MTG16, MTGR1 and Drosophila nervy for each NHR (Davis et al., 2003)..

(51) synergistically with NHR4 to ensure multiple interactions that are crucial for some of the best-studied characteristics of ETO (Gelmetti et al., 1998; Kitabayashi et al., 1998a; Zhang et al., 2001b). The identification of proteins interacting with ETO domains, notably the NHR4, led to the conclusion that ETO acts as a transcriptional co-repressor (Hug and Lazar, 2004; Lausen et al., 2004; Lutterbach et al., 1998b). Indeed, it was clearly shown by several groups that ETO interact with nuclear receptor co-repressor (NCoR), silencing mediator for retinoid and thyroid hormone receptor (SMRT), histone deacytelases (HDAC 1, 2 and 3) and Sin3A corepressor (Amann et al., 2001; Gelmetti et al., 1998; Lutterbach et al., 1998b; Wang et al., 1998). Thus, ETO is assimilated to a scaffold protein for different co-repressor complexes that ensure the repressive activity of transcription factors. ETO contains also two N- and C- terminal NMTS that mediate targeting of this repressor to intra-nuclear compartments that are different than those occupied by AML1. Importantly, it has been demonstrate that AML1-ETO, which lacks the NMTS of AML1, shares the same nuclear foci of ETO that are transcriptionally inactive (Barseguian et al., 2002; McNeil et al., 1999; Odaka et al., 2000). Such aberrant sub-nuclear targeting of AML1 DNA binding activity by AML1-ETO may be critical for the accurate regulation of AML1 target genes. Finally, the ETO protein has a high content of proline, serine and threonine rich sequence (PST) in the N and C terminus of the protein that could affect its stability (Hildebrand et al., 2001).. ETO expression was detected in a variety of normal human tissues with the highest mRNA levels occurring in brain and heart (Wolford and Prochazka, 1998). It is also expressed in almost all of CD34+ compartment (hematopoietic progenitors) of human bone marrow, in megakaryocytes (Erickson et al., 1996) and in peripheral blood leukocytes (Erickson et al., 1994). ETO has been predominantly detected in the nucleus of leukemic cells. 37.

(52) Figure 14. A model for AML1-ETO repression mechanism. While native AML1 activates target gene expression (A), AML1-ETO interferes with AML1 binding to DNA and recruits transcriptional complexes that contain N-CoR, mSin3A, and HDAC (B). These complexes in turn repress the expression of genes normally regulated by the native AML1 complex (adapted from Kurokawa and Hirai, 2003)..

(53) and in the nucleus and cytoplasm of neurons. ETO mutant mice exhibit complete regression of the midgut suggesting an important role of ETO in the gastrointestinal system (Calabi et al., 2001). Yet, its normal function in hematopoiesis is unknown.. The molecular mechanism of action of AML1-ETO. a) Direct transcriptional repression. As a first general model concerning the leukemogenic mechanism of AML1-ETO, it was proposed that AML1-ETO antagonizes AML1 function by binding via the Runt domain to AML1-responsive promoters, but instead of supporting transcription, AML1-ETO recruits co-repressor and HDACs through its ETO moiety. Consequently, it represses target genes expression and block myeloid differentiation (Figure 14).. Indeed, many studies using transient transfection of reporter genes revealed that AML1-ETO is not simply a competitor for AML1, but rather a dominant repressive form that actively blocks AML1-dependant transactivation of target genes such as GM-CSF, IL-3, multidrug resistance 1 (MDR1) and the tumor suppressor p14ARF (Frank et al., 1995; Hwang et al., 1999; Linggi et al., 2002; Lutterbach et al., 1998a; Meyers et al., 1995; Uchida et al., 1997). The effects of these transcriptional block on cellular behaviors are described below. These works on AML1 target genes analyzed also the involvement of each AML1-ETO domain in transcriptional repression. Notably, they described the role of NHR4 in the recruitment of N-CoR complex and the requirement of the NHR2 for the oligomerization of AML1-ETO, which is crucial to its transcriptional function (Liu et al., 2006).. 38.

(54) Figure 15. A model of a high DNA-binding activity of tetrameric AML1-ETO to duplicated AML1-binding sites. Runt homology DNA-binding domain (Runt), 4 Nervy homology regions (NHR1-4), and Nterminus (N) of AML1-ETO are marked. The oligomerization provides an advantage to AML1-ETO to duplicated binding sites (TGT/CGGTspaceTGT/CGGT) compared with AML1 (adapted from Okumura et al., 2008)..

(55) More recently, genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1-ETO dependent transcriptional regulation. This study showed that AML1 and AML1-ETO often bind to the same DNA binding regions, without necessarily displacing the wild type AML1 (Gardini et al., 2008). Furthermore, it has been shown that 70% of genes regulated by AML1-ETO in U937 human myeloid cells are repressed by AML1-ETO and contain AML1 consensus binding sites in their promoters (Gardini et al., 2008). However, the 30% of remaining genes are induced by AML1-ETO and their promoters regions show enrichment in binding sites for other transcription factors (see below). Moreover, another study revealed that AML1-ETO preferentially binds to DNA sequences with duplicated AML1-binding consensus sites, indicating the chimera may selectively regulate a subset of AML1 target genes (Okumura et al., 2008). In addition, the preference for duplicated AML1 DNA binding sites by AML1-ETO depended on the NHR2 oligomerization domain (Okumura et al., 2008). Thus oligomerized AML1-ETO may have a stronger binding affinity for target genes with several AML1 binding sites in their promoter regions (Figure 15). This might lead to higher competitive binding for certain AML1 target genes.. b) Transcriptional activation. Although characterized as a repressor of transcription, AML1-ETO exhibits also the ability to induce gene expression. It was shown that AML1-ETO synergizes with AML1 to up-regulate the transcription of the M-CSF receptor gene, an AML1 target gene (Rhoades et al., 1996). Given that the interactions of AML1-ETO with the co-repressor complexes described above appeared very stable, the recruitment of co-activators by AML1-ETO seems. 39.

(56) unlikely. This suggests that AML1-ETO may act indirectly on M-CSF receptor promoter by titrating a co-repressor like Sin3A, which can bind AML1 as well as AML1-ETO.. The chimera regulates also positively the transcription of genes even when they are not AML1 targets. BCL-2, a major known anti-apoptotic gene, contains an AML1-binding motif in its own promoter that is not regulated by wild type AML1. In transient transfection assays, AML1-ETO was able to activate the transcription of a BCL-2 promoter construct; an activity not shared with the wild type AML1 or ETO proteins (Klampfer et al., 1996). Otherwise, two established cell lines carrying the 8;21 translocation showed elevated levels of endogenous BCL-2 protein compared to myeloid leukemia cell lines that did not harbor the t(8 ;21). The levels of BCL-2 were correlated to the levels of AML1-ETO protein present in these two cell lines (Klampfer et al., 1996). This suggested that high level of BCL-2 promote the survival of t(8;21)-bearing AML cells. However, the phenotypes of murine progenitor cells overexpressing BCL-2 or AML1-ETO are very different suggesting that BCL-2 is not the primary target of AML1-ETO (Kohzaki et al., 1999). The molecular basis and the biological function beside AML1-ETO-induced BCL-2 transcriptional transactivation remain to be elucidated. Survivin, another anti-apoptotic gene that is not regulate by AML1, is induced by AML1-ETO in both cell line model and in primary human hematopoietic CD34+ cells (Balkhi et al., 2008). Survivin expression is undetectable in normal adult tissues but upregulated in certain tumors as well as in AML (Wagner et al., 2006). AML1-ETO acts as an activator of survivin transcription and binds physically to the only AML1 binding site in the survivin promoter. Importantly, it has been shown that depletion of survivin expression abolished AML1-ETO-mediated inhibition of C/EBPα, MPO and G-CSFR in myeloid cell lines, which leads to their growth arrest and granulocytic differentiation (Balkhi et al., 2008).. 40.

(57) Together, these data as well other studies on p21 (see murine models) highlight the role of AML1-ETO in the transcriptional regulation of genes required for cell survival.. c) Indirect transcriptional functions through protein-protein interactions In addition to directly regulating gene transcription, AML1-ETO might contribute to leukemogenesis by targeting several hematopoietic transcription regulators. Notably, by interacting with C/EBPα, E protein, PU.1, MEF, SMAD, and PLZF, AML1-ETO impairs their transcriptional activity and thereby their function (Jakubowiak et al., 2000; Mao et al., 1999; Melnick et al., 2000; Pabst et al., 2001a; Vangala et al., 2003; Zhang et al., 2004). For example, C/EBPα is a crucial transcription factor for the differentiation of granulocytes. It has been shown that C/EBPα expression is undetectable in AML patients carrying the (8;21) translocation or it is downregulated upon ectopic expression of AML1-ETO in myeloid cell lines (Pabst et al., 2001a; Pabst et al., 2001b). It was proposed that AML1-ETO, which can physically bind to C/EBPα (Westendorf et al., 1998), represses C/EBPα expression through the inhibition of C/EBPα positive auto-regulation. Moreover, it was demonstrated that AML1-ETO inhibits C/EBPα-dependent activation of the myeloid cell-specific, rat defensin (antimicrobial peptide) NP-3 promoter (Westendorf et al., 1998), suggesting that AML1-ETO acts as inhibitor of C/EBPα-mediated activation of target genes required for differentiation of myeloid blasts. Physical interactions of AML1-ETO with other transcription factors can influence the DNA-binding profile of both AML1-ETO and these factors. It was shown that through the conserved TAF4 homology domain, AML1-ETO interacts with transcription factors of the E protein family, particularly the basic helix-loop-helix (bHLH) HEB factor (Hela-E-boxbinding protein) (Zhang et al., 2004) which has roles in lymphocyte differentiation (Quong et. 41.

(58) al., 2002) and is associated to leukemogenesis and tumorigenesis (Sjogren et al., 2000). Such interactions have two consequences (Gardini et al., 2008; Zhang et al., 2004). First, it was proposed that AML1-ETO could be redirect through this interaction to E-box motifs resulting in silencing of E protein function through an aberrant co-factor (p300) exchange mechanism. Second, it was showed that HEB DNA binding profile is grossly rearranged upon AML1ETO expression. Thus, through protein-protein interactions, AML1-ETO and/or its partners can reach specific repositioning in the nucleus, resulting in aberrant gene expression profile and cellular behavior.. All together, these data highlight an important role of AML1-ETO in the induction of an aberrant transcriptional regulation of genes required for hematopoietic cell differentiation, proliferation and survival. This is likely a crucial step for the development of t(8;21)-induced leukemia.. 4.. AML1-ETO in cell differentiation, growth and survival. As stated above, AML is a disease characterized by an important accumulation in the body of myeloid cells that are arrested in their maturation. To better understand the mode of action of t(8;21) in AML pathogenesis, many groups have analyzed in vitro the effects of AML1-ETO on cellular behavior/properties using primary cell culture or hematopoietic cell lines that retain some capacity to (terminally) differentiate. In 1994, Sakakura et al. were the first to examine the function of AML1-ETO in the proliferation and differentiation of Kasumi-1 and SKNO-1, two leukemic cell lines derived from patients carrying the (8;21) chromosome translocation (Asou et al., 1991; Sakakura et al., 1994). By over-expressing an AML1 truncated form containing the Runt domain or using antisense oligonucleotides complementary to the fusion transcript, they observed a clear growth inhibition and induction 42.