Identification and functional characterisation of oncogenic pathway signatures in malignant Lymphoma Doctoral Thesis In partial fulfilment of the requirements for the degree “Doctor rerum naturalium (Dr. rer. nat.)” in the Molecular Medicine Study Program at the Georg-August University Göttingen Graduate School 1034 submitted by Alexandra Schrader born in Soest Göttingen, 2011 Thesis Committee Prof Dr. Dieter Kube (Supervisor) E-Mail [email protected] Phone 0049-551-391537 Postal Address Universitätsmedizin Göttingen Zentrum Innere Medizin Abteilung Hämatologie und Onkologie Robert-Koch-Straße 40 37075 Göttingen Prof Dr. Heidi Hahn E-Mail [email protected] Phone 0049-551-39-14010 Postal Address Universitätsmedizin Göttingen Zentrum Hygiene und Humangenetik Institut für Humangenetik Heinrich-Düker-Weg 12 37073 Göttingen Prof Dr. Martin Oppermann E-Mail [email protected] Phone 0049-551-395822 Postal Address Universitätsmedizin Göttingen Zentrum Hygiene und Humangenetik Abteilung Zelluläre und Molekulare Immunologie Humboldtallee 34 37073 Göttingen Date of Disputation: 22.09.2011 Affidavit By this I declare that I independently authored the presented thesis: “Identification and functional characterisation of oncogenic pathway signatures in malignant Lymphoma” and that I did not use other auxiliary means than indicated. Paragraphs that are taken from other publications, by wording or by sense, are marked in every case with a specification of the literary source. Furthermore I declare that I carried out the scientific experiments following the principles of Good Scientific Practice according to the valid “Richtlinien der Georg-August-Universität Göttingen zur Sicherung guter wissenschaftlicher Praxis”. _______________________________ Alexandra Schrader Göttingen, July 2011 Table of Contents Abstract ................................................................................................................ I List of figures ...................................................................................................... II List of tables ...................................................................................................... XII Abbreviations ................................................................................................... XIII 1 Introduction .......................................................................................... 1 1.1 Burkitt Lymphoma (BL) ....................................................................... 2 1.1.1 c-Myc mediates high proliferation of BL cells ......................................... 3 1.1.2 The c-Myc target gene network affects 15% of all genes ....................... 3 1.1.3 Gene expression profiling has enabled the molecular diagnosis of BL .. 4 1.1.4 The microenvironmental factor BAFF might play a role for the survival of BL cells .................................................................................................. 5 1.1.5 Treatment strategy in BL ........................................................................ 5 1.2 Diffuse Large B cell Lymphoma (DLBCL) .......................................... 6 1.2.1 DLBCL are characterised by a complex karyotype involving the translocation of BCL6 ............................................................................. 6 1.2.2 DLBCL is characterised by a high aberrant activity of survival signals including NF-κB ...................................................................................... 7 1.2.3 Global gene expression profiling identified subgroups of DLBCL .......... 7 1.2.4 Treatment strategy in DLBCL ................................................................. 8 1.3 B cell Development and the Germinal Centre Reaction ................... 9 1.4 B cells are dependent on microenvironmental survival signals .... 11 1.4.1 Nuclear factor of kappa B light polypeptide gene enhancer in B-cells signalling .............................................................................................. 12 1.4.2 Mitogen Activated Protein Kinases (MAPKs) ....................................... 12 1.4.3 BCR activation triggers a variety of signalling pathways including Calcium signalling, NF-κB, PI3K/AKT and MAPK activation ................ 13 1.4.4 Toll like receptor mediated activation of NF-κB and MAPK signals ...... 13 1.4.5 CD40 mediates NF-κB and MAPK signals ........................................... 14 1.4.6 B cell activating factor of the TNF superfamily (BAFF/ TNFSF13B) can activate distinct TNF receptors that trigger noncanonical NF-κB and MAPK signalling ................................................................................... 15 1.4.7 IL21 signals via IL21R mediated STAT1 and STAT3 activity ............... 16 Aim of the Study ............................................................................................... 18 2 Materials and Methods ...................................................................... 20 2.1 Biological Material ................................................................................ 20 2.2 Consumable supplies ........................................................................... 21 2.3 Equipment ............................................................................................ 22 2.4 Chemicals ............................................................................................ 24 2.5 Chemical Inhibitors ............................................................................... 26 2.6 Buffers, Solutions and Media ............................................................... 26 2.7 Plasmids .............................................................................................. 30 2.8 Recombinant Proteins and Biological Material ..................................... 30 2.9 Antibodies ............................................................................................. 31 2.10 Oligonucleotides ................................................................................... 33 2.11 Ready to use Reaction Systems .......................................................... 35 2.12 Cell Biology .......................................................................................... 36 2.12.1 Cell culture techniques ......................................................................... 36 2.12.2 Isolation of Tonsillar Mononuclear Cells from human primary tissue .... 36 2.12.3 Enrichment of CD10+ GC B cells ......................................................... 37 2.12.4 Transfection of CD10+ B cells via non-viral DNA transfer ..................... 37 2.12.5 Activation of B cells with soluble stimulating factors ............................. 38 2.12.6 Inhibitor Treatment ............................................................................... 39 2.12.7 Flow cytometry ..................................................................................... 39 2.12.8 Characterisation of cell populations via flow cytometry ........................ 40 2.12.9 Preparative FACS of transfected CD10+ cells ...................................... 40 2.12.10 Cell cycle analysis ................................................................................ 41 2.12.11 Synchronisation of BL cells using Thymidine treatment ....................... 42 2.12.12 Ca2+ Measurement ............................................................................... 42 2.13 Protein Biochemistry ............................................................................. 43 2.13.1 Preparation of cell lysates .................................................................... 43 2.13.2 SDS-PAGE ........................................................................................... 43 2.13.3 Immunoblotting Technique ................................................................... 44 2.14 Molecular Biology ................................................................................. 45 2.14.1 Transformation of E.coli ........................................................................ 45 2.14.2 Isolation of Plasmid DNA ...................................................................... 45 2.14.3 mRNA Isolation .................................................................................... 45 2.14.4 mRNA Amplification .............................................................................. 46 2.14.5 Reverse Transcription .......................................................................... 46 2.14.6 Transcript quantification via qRT-PCR (quantitative Real Time – Polymerase Chain Reaction) ................................................................ 47 2.14.7 Chromatin Immunoprecipitation ............................................................ 48 2.15 Microarray analyses ............................................................................. 50 3 Results ................................................................................................ 54 3.1 High c-Myc activity is an independent negative prognostic factor for diffuse large B cell lymphomas ................................................... 54 3.1.1 Overexpression of c-Myc in primary human GC B cells ....................... 54 3.1.2 Ectopic c-Myc expression triggers a tumour like expression profile in primary GC B cells. ............................................................................... 56 3.1.3 Very high expression of c-Myc target genes is a hallmark of molecular Burkitt lymphomas ................................................................................ 59 3.1.4