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Phosphoproteomics and Cancer

Phosphoproteomics and Cancer. Scott A. Gerber, PhD Departments of Genetics and Biochemistry Norris Cotton Cancer Center Geisel School of Medicine at Dartmouth. Kinases, cell division and cancer. CDK1 Plk1 Aurora A. CDK1 Plk1 Aurora A. CDK1 Plk1 Aurora A/B. PP1. Prophase.

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Phosphoproteomics and Cancer

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  1. Phosphoproteomics and Cancer • Scott A. Gerber, PhD • Departments of Genetics and Biochemistry • Norris Cotton Cancer Center • Geisel School of Medicine at Dartmouth

  2. Kinases, cell division and cancer CDK1 Plk1 Aurora A CDK1 Plk1 Aurora A CDK1 Plk1 Aurora A/B PP1 Prophase Prometaphase PP2A/PP1 Interphase Metaphase PP2A Cytokinesis PP1 CDK1 Plk1 Plk1 Aurora B Anaphase Telophase Aurora B

  3. Kinases, cell division and cancer spindle defects multipolar spindle aberrant cytokinesis Prophase Prometaphase Interphase Metaphase Cytokinesis chromosome missegregation Anaphase Telophase

  4. Kinases, cell division and cancer spindle defects chromosome instability & aneuploidy multipolar spindle aberrant cytokinesis Prophase Prometaphase Interphase Metaphase Cytokinesis chromosome missegregation Anaphase Telophase

  5. Phosphoproteomics and cancer: opportunities, challenges & progress • Genomics-based targeted treatments: • EGFR (~10%) – gefitinib/erlotinib • ALK (~4%)- crizotinib • RAS/RAF (~28%) – MEK inhibitors adapted from Pao and Girard, Lancet 2011

  6. Phosphoproteomics and cancer: opportunities, challenges & progress Plk1 AurkA Plk1 AurkA Plk1 PLK1 mRNA expression T N T N N T COSMIC* Drug(s) N – normal tissue T – tumor tissue 8,300 tumors 61 (0.7 %) mutations BI6727 (volasertib) Phase III 9,200 tumors 35 (0.4 %) mutations MLN8237 (alisertib) Phase III *http://cancer.sanger.ac.uk/cancergenome/projects/cosmic,La Locano 2011, J Trans Med; Wolf 1997, Oncogene

  7. Phosphoproteomics and cancer: opportunities, challenges & progress NIH3T3 U-2OS U-251MG A-432 Schwanhäusser et al. 2011, Nature; Lundberg et al. 2010, Mol. Sys. Biol

  8. Phosphoproteomics and cancer: opportunities, challenges & progress • Polo-like kinase 1 (Plk1): protein vs mRNA vschemosensitivity α-Plk1 H23 H522 H1650 H1838 H2170 H1975 H1395 r2 = 0.04 r2 = 0.17 relative Plk1 protein abundance relative LD50 Plk1 inhibitor relative Plk1 mRNA abundance relative Plk1 protein abundance Kettenbach & Gerber, Nature Protocols (2011)

  9. Phosphoproteomics and cancer: opportunities, challenges & progress Cdk1 activity cdk1 P P ATP ADP P PP1 activity kinase PP1 PP1 “active” S G2 M G1 G1 Cell cycle progression phosphatase Tumor phosphoproteomes represent the balance of opposing activities

  10. Quantitative Proteomics: SILAC eachtryptic peptide ends in Lys or Arg & can be quantified 13C & 15N Lys & Arg (heavy) 12C & 14N Lys & Arg (light) • - mix conditions • - lyse • IP A/A’ • SDS-PAGE & digest Experiment mixing cells allows multi-step, subcellular fractionation & accurate quantitation 100% condition (1): light condition (2): heavy Quantities are ratios: 50% Information e.g. heavy / light m/z Condition 1 Condition 2 P P P P P anti-A antibody, etc. A P OH A’ P Biology C’ P C B B Stable Isotope Labeling by Amino acids in Cell culture Ong et al., Molecular & Cellular Proteomics 2002

  11. Spike-in SILAC analysis of human lung cancer tumors Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

  12. Spike-in SILAC analysis of human lung cancer tumors Two non-small cell lung cancer (NSCLC) tumors Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

  13. Spike-in SILAC analysis of human lung cancer tumors Target discovery Dimensionality reduction Motif-based biomarkers Motif-X – http://motif-x.med.harvard.edu/

  14. Spike-in SILAC analysis of human lung cancer tumors

  15. Spike-in SILAC analysis of human lung cancer tumors previously: control 12C14N combine lyse trypsin digestion LC-MS/MS + inhibited 13C15N phosphopeptide enrichment SCX chromatography TiO2 Plk1 inhibitor > 700 Plk1 candidate substrate phosphorylation sites in these lung tumors: Kinase-specific “substrate-omes” may be reflective of in vivo activity Kettenbach et al., Science Signaling (2011); Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

  16. Spike-in SILAC analysis of human lung cancer tumors What about protein abundance differences? Tumor phosphoproteomes contain greater dynamic information than proteomes Schweppe, Rigas & Gerber, Journal of Proteomics (2013)

  17. Translational phosphoproteomics: Status • Currently moving forward with the analysis of 40 NSCLC lung tumors for phosphoproteomic analysis • Study population • Subjects undergoing thoracic surgery for presumed lung cancer • Determine if significant correlation exists between Aurora A and/or Plk1 substrates and progression-free survival • Correlate global phosphoproteomics patterns with disease-free survival, time to disease recurrence, lung cancer-specific survival and overall survival • New instrumentation affords deeper coverage • New instrumentation enables multiplexed analyses

  18. Acknowledgements The Gerber Lab proteomics.dartmouth.edu Dr. Lilian Kabeche Dr. Devin Schweppe (past) Jason Gilmore Jeffrey Milloy Sierra Cullati Katelyn Cassidy Andrew Grassetti Mark Adamo The Kettenbach Lab www.kettenbachlab.org Dr. ArminjaKettenbach Adam Petrone Scott Rusin Kate Schlosser NCCC Thoracic Oncology Dr. James Rigas Dr. Konstantin Dragnev Funding American Cancer Society NIH P20-GM103413 R01-CA155260 S10-OD016212 Cambridge Isotopes ThermoFisher Scientific GL Sciences

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