Dissecting and Modeling Oncogene Dependent Molecular Mechanisms in Lymphoma Genesis and Progression Doctoral Thesis In partial fulfillment of the requirements for the degree “Doctor rerum naturalium (Dr. rer. nat.)” in the Molecular Medicine Study Program at the Georg-August University Göttingen submitted by Elisabeth Hand born in Berlin Göttingen, 2013 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. Mikael Simons E-Mail [email protected] Phone 0049-551-3899533 Postal Address Max-Planck-Institut für experimentelle Medizin Hermann-Rein-Str. 3 37075 Göttingen Prof Dr. Peter Burfeind E-Mail [email protected] Phone 0049-551-397595 Postal Address Universitätsmedizin Göttingen Zentrum Hygiene und Humangenetik Institut für Humangenetik Heinrich-Düker-Weg 12 37073 Göttingen Date of Disputation: Affidavit By this I declare that I independently authored the presented thesis: “Dissecting and modeling oncogene dependent molecular mechanisms in lymphoma genesis and progression” 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”. _______________________________ Elisabeth Hand Göttingen, August 2013 Table of Contents Abstract .................................................................................................................................................... I List of Figures ........................................................................................................................................... II List of Tables ........................................................................................................................................... III 1 Introduction ..................................................................................................................................... 1 1.1 Development and transformation of germinal center B cells in lymph nodes ....................... 1 1.2 Paracrine and autocrine signaling influence B cell development and transformation ........... 4 1.2.1 B cell receptor activated signaling and its implications in lymphomagenesis ................ 4 1.2.2 CD40 mediated signaling ................................................................................................. 6 1.2.3 Hedgehog signaling pathway........................................................................................... 7 1.2.4 NF-kB signaling pathway ................................................................................................. 8 1.2.5 Mitogen activated protein kinase (MAPK) signaling pathways ....................................... 9 1.3 Strategies to describe and stratify lymphoma cases based on gene expression profiling .... 11 1.3.1 Diffuse large B cell lymphoma (DLBCL) ......................................................................... 11 1.3.2 Burkitt’s lymphoma (BL) ................................................................................................ 12 1.3.3 Surrogate markers for oncogenic pathway activation .................................................. 14 Aim of the Study .................................................................................................................................... 15 2 Material and Methods ................................................................................................................... 17 2.1 Biological Material ................................................................................................................. 17 2.1.1 Primary Material ............................................................................................................ 17 2.1.2 Cell Lines ........................................................................................................................ 17 2.2 Chemicals and Consumable Supplies .................................................................................... 18 2.3 Buffers, Solutions and Media ................................................................................................ 21 2.4 Equipment ............................................................................................................................. 23 2.5 Recombinant Proteins ........................................................................................................... 24 2.6 Inhibitors ............................................................................................................................... 25 2.7 Antibodies.............................................................................................................................. 26 2.8 Oligonucleotides .................................................................................................................... 28 2.9 Ready to Use Reaction Systems ............................................................................................ 29 2.10 Software used ........................................................................................................................ 30 2.11 Cell Biology ............................................................................................................................ 30 2.11.1 Cell Culture Techniques ................................................................................................. 30 2.11.2 Activation of B cells with Soluble Stimulating Factors .................................................. 30 2.11.3 Inhibitor Treatment ....................................................................................................... 31 2.11.4 Transfection of siRNA .................................................................................................... 31 2.12 Protein Biochemistry ............................................................................................................. 32 2.12.1 Preparation of Cell Lysates ............................................................................................ 32 2.12.2 SDS-PAGE and Western Blot .......................................................................................... 32 2.13 Molecular Biology .................................................................................................................. 33 2.13.1 mRNA Isolation .............................................................................................................. 33 2.13.2 Reverse Transcription .................................................................................................... 33 2.13.3 qRT-PCR (quantitative Real Time – Polymerase Chain Reaction) .................................. 33 2.14 Microarray Analyses .............................................................................................................. 34 3 Results ........................................................................................................................................... 36 3.1 Temporal pattern of whole genome gene expression changes upon signaling pathway activation ........................................................................................................................................... 36 3.1.1 α-IgM stimulation of BL cells changes the mBL index towards non-mBLs .................... 38 3.1.2 Causality Network of BCR induced gene expression correlations ................................. 39 3.1.3 A mathematically new method to analyze time courses identifies differentially expressed clusters of genes........................................................................................................... 42 3.2 Ptch1 and Hedgehog signaling in lymphoma ........................................................................ 49 3.2.1 Expression profile of Hedgehog signaling components ................................................ 50 3.2.2 Lymphoma cell lines neither secrete nor respond to Sonic hedgehog ......................... 53 3.2.3 PI3K-mediated regulation of PTCH1, c-MYC and LEF1 mRNA expression ..................... 55 3.3 Analyzing intersections in signaling pathways via use of specific chemical inhibitors ......... 58 3.3.1 Inhibitors in use ............................................................................................................. 59 3.3.2 Differential gene expression on the whole genome level upon inhibitor use .............. 63 3.3.3 Nested Effects Models propose a controversial role of Tak1 in CD40 and BCR mediated signaling. 66 3.3.4 NF-κB signaling upon BCR crosslink in Burkitt’s lymphoma .......................................... 73 4 Discussion ...................................................................................................................................... 76 4.1 Analyzing the temporal pattern of gene expression changes ............................................... 76 4.2 A non-canonical role of Hedgehog signaling in lymphoma ................................................... 80 4.3 Analyzing pathway intersections ........................................................................................... 83 5 Conclusion ....................................................................................................................................