AAACR Talk 2009
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AAACR Talk 2009

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Talk form AACR conference in Denver, April 2009

Talk form AACR conference in Denver, April 2009

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AAACR Talk 2009 AAACR Talk 2009 Presentation Transcript

  • Dual Targeting of the Tyrosine Kinase Receptors EGFRvIII and c-Met in Glioblastoma Multiforme
  • Disclosures
    • My laboratory currently receives research funding from Amgen
  • Structure of EGFRvIII * Tyrosine residues mutated in DY5 construct EGFR Homodimer L1 CR1 L2 CR2 992 1068 1086 1148 COOH NH 2 1101 TM L1 CR1 L2 CR2 992 1068 1086 1148 COOH NH 2 1101 TM 974 974 1045 1045 1173 1173 845 845 TK TK P P P P P P P P P Src Shc SHP1 PLC  MAPK/ERK Cascade PKC MAPK/ERK Cascade Gab1 MAPK/ERK Cascade p85 AKT/PKB cascade Grb2 Cbl ubiquitination degradation Eps15 Receptor internalization PKC MAPK/ERK Cascade AP-2 PLC  Src * * * * * Membrane EGFRvIII is formed following deletion of amino acids 6-273 and the concurrent insertion of a glycine residue at the fusion junction K721M mutation in DK construct Δ
  • EGFRvIII Activates Multiple Receptor Tyrosine Kinases (RTKs) Phospho-specific antibody arrays U87MG.  2-7 U87MG.DK 1 2 3 4 5 7 6 1. P-EGFR 2. P-FGFR3 3. P-Axl 4. P-MET 5. P-PDGFR  6.  P-EphA7 7. P-VEGFR3
  • Confirmation that EGFRvIII Phosphorylates c-Met and PDGFR β in U87MG. Δ 2-7 Cells U87MG U87MG + HGF U87MG. Δ 2-7 U87MG.DK Phospho c-Met Total c-Met Phosphorylated PDGFR β U87MG.  2-7 U87MG.DK 0 20 40 60 80 100 Fluorescence intensity (relative units)
  • Panitumumab Inhibits EGFRvIII Activation in U87MG. Δ 2-7 Cells Whole cell lysates probe for total and phosphorylated EGFRvIII p-EGFR (Y1068) p-EGFR (Y1173) Total EGFR Panitumumab - + - +
  • c-Met Phosphorylation is Ligand Independent and Inhibited by Panitumumab but not AMG 102 U87MG Δ 2-7 cells were treated with different antibodies and c-met immunoprecipitated to determine levels of total and phosphorylated c-Met A549 + HGF (400 ng/ml) AMG102 (10 μ g/ml) Panitumumab (20 μ g/ml) Irrelevant Ab (30 μ g/ml) Combination (30 μ g/ml) Phospho c-Met Total c-Met
  • Phosphorylation of PDGFR β is Inhibited by Panitumumab U87MG.DK U87MG.  2-7 AMG102 Panitumumab Combination 0 10 20 30 40 50 60 70 80 90 100 Untreated Treatment Fluorescence Intensity (Relative Units)
  • Levels of pAkt in U87MG Cell Lines Treated with Panitumumab and AMG 102 U87MG.  2-7, pAkt U87MG.  2-7, total Akt U87MG.DK, pAKT U87MG.DK, total Akt Untreated AMG 102 Panitumumab Combination 0 20 40 60 80 100 Treatment Fluorescence intensity (relative units) Untreated AMG 102 Panitumumab Combination 0 50 100 150 200 250 Treatment Fluorescence intensity (relative units) Untreated AMG 102 Panitumumab Combination 0 250 500 750 1000 1250 1500 Treatment Fluorescence intensity (relative units) Untreated AMG 102 Panitumumab Combination 0 20 40 60 80 100 Treatment Fluorescence intensity (relative units)
  • U87MG Xenografts Treated with AMG 102 U87MG U87MG. Δ 2-7 U87MG. wtEGFR
  • U87MG. Δ 2-7 Xenografts Treated with Panitumumab and AMG 102
  • Immunohistochemistry analysis of U87MG. Δ 2-7 xenografts treated with Panitumumab, AMG 102 or combination of both Xenografts were collected one day after second injection (mid-point of therapy) and analyzed for proliferation, blood vessels and apoptosis
  • U87MG.DY5 Xenografts Treated with AMG102 U87MG.DY5 p-EGFR (Y1173)  2-7 DY5 p-EGFR (Y1068)  -tubulin Total EGFR  2-7 DY5
  • Conclusions
    • EGFRvIII activates multiple RTK’s
    • c-Met is activated by EGFRvIII in a ligand independent manner leading to resistance to AMG 102
    • Panitumumab inhibits the EGFRvIII phosphorylation of c-Met and restores response to AMG 102
    • EGFRvIII probably phosphorylates c-Met by direct interaction
    • Combination of Panitumumab and AMG 102 may be an effective therapy in GBM
  • Survival and Proliferation MAP-kinase Akt/PKB PI 3-kinase PIP 3 PDK1 MAP-kinase- kinase-kinase Ras Ras-GEF Grb2 PDGFR β EGFRvIII C-Met U87MG. Δ 2-7 Cells EGFRvIII co-activates other RTKs including c-Met
  • Survival and Proliferation MAP-kinase Akt/PKB PI 3-kinase PIP 3 PDK1 MAP-kinase- kinase-kinase Ras Ras-GEF Grb2 x x Panitumumab Xenografts partially inhibited by Panitumumab but can revert to the HGF/c-Met pathway
  • Survival and Proliferation MAP-kinase Akt/PKB PI 3-kinase PIP 3 PDK1 MAP-kinase- kinase-kinase Ras Ras-GEF Grb2 x x Xenografts inhibited by the combination of Panitumumab and AMG 102 x x
  • Acknowledgements Oncogenic Signalling Laboratory Vino Pillay Terri Burgess, Angela Coxen and Kelly Oliner Amgen Inc.