Technische Universität München Fakultät für Medizin The molecular function of MLL/AF4 for patient derived acute leukemias growing in mice Birgitta Christine Heckl Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Jürgen Ruland Prüfer der Dissertation: 1. Prof. Dr. Marc Schmidt-Supprian 2. apl. Prof. Dr. Arnd Kieser Die Dissertation wurde am 29.03.2018 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 10.10.2018 angenommen. Für meine Eltern Life is like riding a bicycle. To keep your balance, you must keep moving. Albert Einstein Parts of this work have been published in: Birgitta Christine Heckl*, Michela Carlet*, Binje Vick, Catrin Roolf, Ameera Alsadeq, Michaela Grunert, Wen-Hsin Liu, Andrea Liebl, Wolfgang Hiddemann, Rolf Marschalek, Denis Martin Schewe, Karsten Spiekermann, Christian Junghanss, Irmela Jeremias: “Frequent and reliable engraftment of certain adult primary acute lymphoblastic leukemias in mice” Leuk Lymphoma 2018 Sept 20:1-4. Table of contents Table of contents 1. Summary ........................................................................................................................ 1 Zusammenfassung ......................................................................................................... 2 2. Introduction .................................................................................................................... 4 2.1 Acute leukemia is a cancer of the hematopoietic system ............................................ 4 2.1.1 Acute lymphoblastic leukemia .............................................................................. 5 2.1.2 Acute myeloid leukemia ....................................................................................... 5 2.2 Epigenetic regulators in acute leukemia ..................................................................... 6 2.2.1 Classes of epigenetic regulators .......................................................................... 7 2.2.2 Mutations in epigenetic regulators ....................................................................... 9 2.2.3 Epigenetic regulators as drug targets in acute leukemia .....................................10 2.2.4 Mixed lineage leukemia as a global epigenetic regulator ....................................11 2.3 MLL rearrangements in acute leukemia .....................................................................13 2.4 RNA interference .......................................................................................................18 2.4.1 Inhibition of gene expression by RNA interference ..............................................19 2.4.2 An inducible CreERT2 system ..............................................................................20 2.4.3 Delivery systems for therapeutic RNA interference .............................................23 2.5 Patient derived xenograft mouse models in acute leukemia ......................................24 2.6 Genetically engineered mouse models ......................................................................25 2.7 Objectives..................................................................................................................26 3. Material .........................................................................................................................27 3.1 Patient material .........................................................................................................27 3.2 Mice ..........................................................................................................................27 3.3 Cell lines ...................................................................................................................27 3.4 Bacterial strains ........................................................................................................28 3.5 Plasmids ...................................................................................................................28 3.6 Antibodies .................................................................................................................29 3.7 Enzymes ...................................................................................................................30 3.7.1 Restriction endonucleases ..................................................................................30 3.7.2 Enzymes .............................................................................................................31 3.8 Standard size ladders ................................................................................................31 3.9 Oligonucleotides ........................................................................................................31 3.9.1 Oligonucleotides for short-hairpin RNA ...............................................................31 3.9.2 PCR primer for finger printing of mitochondrial DNA ...........................................32 I Table of contents 3.9.3 qPCR primer .......................................................................................................32 3.10 Buffers .....................................................................................................................32 3.11 Media and solutions ................................................................................................33 3.12 Chemicals and reagents .........................................................................................34 3.13 Kits ..........................................................................................................................37 3.14 Consumables ..........................................................................................................37 3.15 Hardware and Equipment........................................................................................38 3.16 Software ..................................................................................................................40 4. Methods ........................................................................................................................41 4.1 Ethical issues ............................................................................................................41 4.1.1 Working with patient derived material .................................................................41 4.1.2 Working with animals ..........................................................................................41 4.2 The xenograft mouse model of individual acute leukemia .........................................41 4.2.1 Engraftment and amplification of primary patient cells ........................................42 4.2.2 Sacrification of mice ............................................................................................42 4.2.3 PDX cell isolation from bone marrow and spleen ................................................43 4.2.4 Mitochondrial fingerprinting .................................................................................43 4.2.5 Monitoring leukemia growth in vivo by blood measurement ................................45 4.2.6 Bioluminescence in vivo imaging ........................................................................45 4.2.7 Tamoxifen treatment in vivo in PDX cells ............................................................45 4.2.8 Constitutive competitive transplantation assay in vivo .........................................46 4.2.9 Inducible competitive transplantation assay in vivo .............................................46 4.3 In vitro cell culture of PDX cells and cell lines ............................................................47 4.3.1 Freezing of PDX cells and cell lines ....................................................................47 4.3.2 Thawing of PDX cells and cell lines ....................................................................47 4.3.3 Determination of cell numbers and staining of apoptotic cells .............................48 4.3.4 In vitro cultivation of PDX cells and cell lines ......................................................48 4.3.5 Ex vivo cultivation of transduced PDX ALL cells in co-culture .............................49 4.3.6 Tamoxifen treatment in vitro in cell lines .............................................................50 4.3.7 Constitutive competitive assay in vitro in cell lines ..............................................50 4.3.8 Inducible competitive assay in vitro in cell lines...................................................50 4.4 Genetic engineering of PDX cells and cell lines .........................................................51 4.4.1 Enrichment of PDX cells .....................................................................................51 4.4.2 Lentivirus production using HEK-293 packaging cells .........................................51 4.4.3 Lentiviral titer determination ................................................................................53 4.4.4 Lentiviral transduction of PDX cells and cell lines ...............................................53 II Table of contents 4.4.5 Enrichment of GEPDX cells and cell lines by fluorescence-activated cell sorting ...........................................................................................................................54 4.4.6 Analysis of PDX cells and cell