Receptor protein-tyrosine phosphatases controlling activity of the oncoprotein FLT3 ITD Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Medizinischen Fakultät der Friedrich-Schiller-Universität Jena vorgelegt von M. Sc. Anne Kresinsky geboren am 13.05.1990 in Elsterwerda Gutachter: 1. PD Dr. rer. nat. habil. Jörg Paul Müller, Universitätsklinikum Jena 2. Apl. Prof. Dr. rer. nat. habil. Frank-Dietmar Böhmer, Universitätsklinikum Jena 3. Prof. Dr. med. habil. Carsten Müller-Tidow, Universitätsklinikum Heidelberg Tag der öffentlichen Verteidigung: 15.01.2019 Table of Contents Table of Contents TABLE OF CONTENTS .................................................................................................. I ABBREVIATIONS .......................................................................................................... V ZUSAMMENFASSUNG ............................................................................................... VII SUMMARY .................................................................................................................... IX 1. INTRODUCTION ........................................................................................................ 1 1.1 Hematopoietic system ............................................................................................ 1 1.2 Acute myeloid leukemia (AML) .............................................................................. 2 1.2.1 General aspects of AML ..................................................................................... 2 1.2.2 Therapeutic strategies for AML .......................................................................... 4 1.3 Receptor-type tyrosine kinases ............................................................................. 4 1.3.1 General aspects on receptor-type tyrosine kinases ............................................ 4 1.3.2 FMS-like tyrosine kinase 3 ................................................................................. 5 1.3.2.1 Structure and expression profile of FLT3 ..................................................... 5 1.3.2.2 FLT3 activation, signaling and function in hematopoiesis ............................. 6 1.3.3 FLT3 ITD ............................................................................................................ 7 1.3.3.1 Altered signaling quality and transforming capacity of oncogenic FLT3 ITD . 7 1.3.3.2 Clinical impact of FLT3 ITD .......................................................................... 8 1.4 Protein-tyrosine phosphatases ............................................................................. 9 1.4.1 Receptor protein-tyrosine phosphatase J (Ptprj) ................................................10 1.4.1.1 Structure and expression profile of Ptprj .....................................................10 1.4.1.2 Ptprj mode of action ....................................................................................11 1.4.1.3 Substrates of Ptprj ......................................................................................12 1.4.1.4 Phenotype of Ptprj-/- mice ............................................................................13 1.4.1.5 Role of Ptprj on FLT3 and FLT3 ITD ...........................................................13 1.4.1.6 Ptprj as clinical target ..................................................................................14 1.4.2 Receptor protein-tyrosine phosphatase C (Ptprc) ..............................................15 1.4.2.1 Structure and expression profile of Ptprc ....................................................15 1.4.2.2 Ptprc mode of action ...................................................................................16 1.4.2.3 Ptprc substrates and signaling ....................................................................17 1.4.2.4 Consequences of Ptprc inactivation ............................................................18 1.4.2.5 Clinical impact of Ptprc ...............................................................................18 2. AIMS OF THE STUDY ..............................................................................................21 3. MATERIAL ................................................................................................................23 3.1 Cell work ................................................................................................................23 I Table of Contents 3.1.1 Cell lines ...........................................................................................................23 3.1.2 Cell culture ........................................................................................................23 3.1.3 Plasmids ...........................................................................................................24 3.1.4 Enzymes ...........................................................................................................24 3.1.5 gRNA ................................................................................................................25 3.2 Antibodies ..............................................................................................................25 3.2.1 Flow cytometric Antibodies ................................................................................25 3.2.2 Western Blot Antibodies ....................................................................................25 3.2.3 Immunohistochemistry Antibodies .....................................................................26 3.3. Buffers and Solutions ..........................................................................................27 3.4 Staining Solutions .................................................................................................28 3.5 Mouse models ........................................................................................................29 3.6 Reaction Kits, Chemicals and Consumables .......................................................29 3.6.1 Reaction Kits .....................................................................................................29 3.6.2 Chemicals .........................................................................................................30 3.6.3 Consumables ....................................................................................................30 3.7 Software .................................................................................................................31 4. METHODS ................................................................................................................32 4.1 DNA work/cloning ..................................................................................................32 4.1.1 Restriction .........................................................................................................32 4.1.2 CRISPR/Cas9 oligonucleotide annealing ..........................................................32 4.1.3 Ligation .............................................................................................................33 4.1.4 Transformation ..................................................................................................33 4.1.5 Plasmid preparation ..........................................................................................33 4.2 Cell culture .............................................................................................................34 4.3 Transfection of HEK293T cells .............................................................................34 4.4 Transduction of 32D muFLT3 ITD .........................................................................35 4.5 Polymerase chain reaction (PCR) .........................................................................35 4.5.1 Amplification for genotyping ..............................................................................35 4.5.2 Amplification for sequencing..............................................................................36 4.6 Flow cytometric analysis ......................................................................................36 4.6.1 Verification of CRISPR/Cas9 ko clones .............................................................36 4.6.2 B and T cell analysis .........................................................................................37 4.6.3 Analysis of committed myeloid progenitors and LSK cells .................................37 4.7 Cell lysis .................................................................................................................38 4.8 SDS-PAGE and Western blotting ..........................................................................39 II Table of Contents 4.9 Viability and Proliferation Assays ........................................................................40 4.9.1 Colony forming unit (CFU) assay ......................................................................40 ® 4.9.2 The Cell Titer-Blue Assay (IC50) .......................................................................40 4.10 Mouse work ..........................................................................................................41 4.10.1 Genotyping ......................................................................................................41 4.10.2 Isolation and characterization of mouse organs ...............................................41 4.10.3 Blood analysis .................................................................................................42