AML-associated mutations of the transcription factor C/EBPα: studies in a neutrophil differentiation model Inaugural-Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) an der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Isabel Mölter geboren in Pforzheim Freiburg im Breisgau Juli 2017 Die Untersuchungen zur vorliegenden Arbeit wurden von August 2012 bis Juli 2017 am Institut für Medizinische Mikrobiologie und Hygiene des Universitätsklinikums der Albert-Ludwigs-Universität Freiburg unter der Leitung von Prof. Dr. Georg Häcker durchgeführt. Dekan der Fakultät für Biologie: Prof. Dr. Bettina Warscheid Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner Betreuer der Arbeit: Prof. Dr. Georg Häcker Referent: Prof. Dr. Georg Häcker Koreferentin: Prof. Dr. Heike Pahl Drittprüfer: Prof. Dr. Tilman Brummer Datum der mündlichen Prüfung: 27.09.2017 I. Table of contents I. Summary .............................................................................................................. 8 II. Zusammenfassung............................................................................................... 9 III. Introduction ..................................................................................................... 11 Neutrophil granulocytes ......................................................................................... 11 C/EBPα, the master transcription factor in granulopoiesis .................................... 14 Lymphoid-enhancer binding factor 1 (Lef-1) and severe congenital neutropenia .. 18 Neutrophil granulocytes in inflammation ................................................................ 18 Granules ............................................................................................................ 20 NETs .................................................................................................................. 22 Neutrophil cell death .............................................................................................. 23 Acute myeloid leukaemia (AML) ............................................................................ 24 The Hoxb8 system ................................................................................................ 27 IV. Objectives ....................................................................................................... 29 V. Results ............................................................................................................ 30 Generation of Hoxb8 neutrophils ........................................................................... 30 Conditional C/EBPα-/- ER-Hoxb8 neutrophils do not differentiate but are lost over time ....................................................................................................................... 31 Lef-1-/- Hoxb8 neutrophils have heterogeneous phenotypes ................................. 32 Retroviral overexpression of C/EBPα mutants in ER-Hoxb8 neutrophils as a model to study their implication in AML ............................................................................ 35 Proliferation of Hoxb8 neutrophils expressing AML-associated C/EBPα mutations is not significantly altered .......................................................................................... 38 Proliferation in suspension ................................................................................. 38 Proliferation in semi-solid media: methylcellulose colony assay ........................ 40 ER-Hoxb8 neutrophils expressing N- and C-terminal mutations of C/EBPα display differentiation defects ............................................................................................ 41 Effector functions of Hoxb8 neutrophils expressing C-terminal mutations of C/EBPα are reduced ........................................................................................................... 48 Expression of the granule protein neutrophil elastase is reduced in C/EBPα K313 and C/EBPα BRM2 expressing ER-Hoxb8 neutrophils ...................................... 48 Secretion of the pro-inflammatory cytokines TNF and IL-6 by C/EBPα K313 expressing Hoxb8 neutrophils ............................................................................ 49 C/EBPα K313 retains transactivation capacity: luciferase reporter assay .......... 50 Cell death of Hoxb8 neutrophils ............................................................................ 53 C/EBPα expression on mRNA and protein levels .................................................. 55 C/EBPα mRNA-levels are slightly higher in wt C/EBPα expressing Hoxb8 neutrophils than in C/EBPα K313 expressing cells ............................................ 56 C/EBPα protein levels of C/EBPα K313/BRM2 expressing ER-Hoxb8 cells are strongly increased .............................................................................................. 56 Protein stability of C/EBPα K313 is not significantly increased compared to wt C/EBPα .............................................................................................................. 58 C/EBPα K313 protein levels are decreased in HEK293FT cells upon transient transfection ........................................................................................................ 61 In vivo differentiation of ER-Hoxb8 neutrophils expressing C/EBPα wt/K313 ........ 61 Upon adoptive transfer, Hoxb8 neutrophils expressing C/EBPα K313 are detectable for longer times in the bone marrow ................................................. 62 Hoxb8 neutrophils expressing C/EBPα K313 showing delayed maturation are able to differentiate in vivo ................................................................................. 65 C/EBPα protein levels are increased in AML patient samples ............................... 67 VI. Discussion ...................................................................................................... 68 Regulation of C/EBPα expression ......................................................................... 68 C/EBPα and its influence on proliferation of Hoxb8 neutrophil progenitor cells ..... 69 Differentiation of ER-Hoxb8 cells is impaired upon expression of N- or C-terminally mutated C/EBPα.................................................................................................... 71 Hoxb8 neutrophil effector functions are altered dependent on different C/EBPα mutations ............................................................................................................... 75 Cell death of Hoxb8 neutrophils ............................................................................ 78 Conclusion............................................................................................................. 78 VII. Material and Methods ..................................................................................... 80 1. Material ........................................................................................................... 80 1.1. Mice.......................................................................................................... 80 1.2. Cell lines ................................................................................................... 80 1.3. Culture media and amendments .............................................................. 81 1.4. Viral Constructs ........................................................................................ 82 1.5. Inhibitors, cytokines and other reagents ................................................... 84 1.6. Blocking solutions and antibodies ............................................................ 85 1.7. Buffers and solutions ................................................................................ 87 1.8. Electronic Devices .................................................................................... 91 2. Methods .......................................................................................................... 92 2.1. Cell Culture .............................................................................................. 92 2.2. Generation of Hoxb8 neutrophil progenitor cells ...................................... 92 2.3. Retrovirus production and transduction of Hoxb8 neutrophil progenitor cell lines ....................................................................................................... 93 2.4. Transfection of HEK293FT cells ............................................................... 94 2.5. Proliferation of Hoxb8 neutrophil progenitors in liquid culture .................. 94 2.6. Proliferation of Hoxb8 neutrophil progenitors in semi-solid medium ......... 94 2.7. In vitro differentiation of Hoxb8 neutrophil progenitor cells ....................... 94 2.8. FACS staining of cell surface markers ..................................................... 95 2.9. Cell death staining .................................................................................... 95 2.10. Giemsa staining .................................................................................... 95 2.11. Lysate preparation, SDS-Page and Western Blotting ........................... 95 2.12. Inhibition of translation using cycloheximide ......................................... 96 2.13. RNA extraction and cDNA synthesis ..................................................... 96 2.14. Quantitative real-time PCR (qRT-PCR)................................................. 97 2.15.