TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Genetik Characterization of the Small Molecule Kinase Inhibitor SU11248 (Sunitinib/ SUTENT in vitro and in vivo - Towards Response Prediction in Cancer Therapy with Kinase Inhibitors Michaela Bairlein Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ. -Prof. Dr. K. Schneitz Prüfer der Dissertation: 1. Univ.-Prof. Dr. A. Gierl 2. Hon.-Prof. Dr. h.c. A. Ullrich (Eberhard-Karls-Universität Tübingen) 3. Univ.-Prof. A. Schnieke, Ph.D. Die Dissertation wurde am 07.01.2010 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 19.04.2010 angenommen. FOR MY PARENTS 1 Contents 2 Summary ................................................................................................................................................................... 5 3 Zusammenfassung .................................................................................................................................................... 6 4 Introduction .............................................................................................................................................................. 8 4.1 Cancer ............................................................................................................................................................... 8 4.1.1 The hallmarks of cancer ........................................................................................................................... 8 4.2 Protein kinases and cancer .............................................................................................................................. 11 4.3 Protein kinase inhibitors in targeted cancer therapy ........................................................................................ 18 4.3.1 Classes of small-molecule protein tyrosine kinase inhibitors ................................................................. 22 4.3.2 Multi-targeted small-molecule protein kinase inhibitors ........................................................................ 24 4.4 Sunitinib malate .............................................................................................................................................. 24 4.5 A closer look at targeted cancer therapy and small-molecule kinase inhibitors .............................................. 27 4.5.1 The advantages and drawbacks of targeted cancer therapy with kinase inhibitors ................................. 27 4.5.2 Challenges for multi-targeted kinase inhibitors ...................................................................................... 28 4.5.3 Drug resistance –single versus multi-targeted small-molecule protein kinase inhibitors ....................... 28 4.6 Drug discovery of selective kinase inhibitors.................................................................................................. 30 5 Aims of this PhD thesis .......................................................................................................................................... 32 6 Materials and Methods .......................................................................................................................................... 34 6.1 Materials .......................................................................................................................................................... 34 6.1.1 Laboratory chemicals, biochemicals and inhibitors ............................................................................... 34 6.1.2 Chemicals for SILAC and MS-analysis .................................................................................................. 34 6.1.3 Enzymes .................................................................................................................................................. 35 6.1.4 “Kits“ and other materials ..................................................................................................................... 35 6.1.5 Growth factors and ligands .................................................................................................................... 35 6.2 Media .............................................................................................................................................................. 35 6.2.1 Bacterial media ...................................................................................................................................... 35 6.2.2 Cell culture media .................................................................................................................................. 35 6.3 Stock solutions and commonly used buffers ................................................................................................... 36 6.4 Cells ................................................................................................................................................................ 36 6.4.1 Eukaryotic cell lines ............................................................................................................................... 36 6.5 Antibodies and recombinant proteins .............................................................................................................. 38 6.5.1 Primary antibodies ................................................................................................................................. 38 6.5.2 Secondary antibodies .............................................................................................................................. 38 6.6 Oligonucleotides ............................................................................................................................................. 38 6.6.1 siRNA oligonucleotides .......................................................................................................................... 38 6.7 Methods ........................................................................................................................................................... 39 6.7.1 Cellular Assays ....................................................................................................................................... 39 6.7.2 Affinity chromatography and mass spectrometry ................................................................................... 40 6.7.3 Molecular methods ................................................................................................................................. 42 7 Results ..................................................................................................................................................................... 44 7.1 Profiling of SU11248 activity in cancer cells .................................................................................................. 44 7.1.1 Effect of SU11248 on cancer cell proliferation ...................................................................................... 44 7.1.2 Effect of SU11248 on cancer cell apoptosis and cell cycle distribution ................................................. 52 7.1.3 Effect of SU11248 on cancer cell migration and invasion ..................................................................... 56 7.1.4 Morphological changes of cancer cells after SU11248 treatment .......................................................... 60 7.2 Target-selectivity profiling of SU11248 in cancer cell lines and metastatic renal cell carcinoma (mRCC) tumors ................................................................................................................................................................. 62 7.2.1 Workflow of drug target profiling by affinity chromatography and mass spectrometry ........................ 63 7.2.2 Target identification and functional classification of SU11248 protein interaction partners ................. 65 7.3 Binding-affinity-analysis of SU11248 towards qualitatively identified cellular targets ................................. 72 7.4 Quantification of target-dissociation constants directly from cells ................................................................. 82 7.4.1 Workflow of target affinity measurement based on quantitative mass spectrometry combined with affinity purification experiments ............................................................................................................ 82 7.5 In vitro binding studies and cellular kinase assays .......................................................................................... 87 7.6 Quantification of relative target amount binding to SU11248 in sensitive and insensitive cell lines ................. ......................................................................................................................................................................... 89 7.7 Gene expression analysis of SU11248 sensitive and less responsive cancer cell lines of different cancer types .. ......................................................................................................................................................................... 93 7.8 Phosphoproteomic analysis of cancer cell lines treated with SU11248 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