1) The document discusses targeting the EGFRvIII and c-Met tyrosine kinase receptors in glioblastoma multiforme (brain cancer). 2) It finds that the truncated EGFRvIII receptor activates other receptor tyrosine kinases like c-Met in a ligand-independent manner, leading to resistance to drugs targeting c-Met alone. 3) Treatment with panitumumab, which inhibits EGFRvIII, blocks EGFRvIII from phosphorylating c-Met and restores the effectiveness of AMG102, an anti-c-Met drug. The combination treatment is a potentially effective therapy for glioblastoma.

1. Dual Targeting of the Tyrosine Kinase Receptors EGFRvIII and c-Met in Glioblastoma Multiforme

3. 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

4. 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

5. 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)

6. 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 - + - +

7. 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

8. 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)

9. 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)

10. U87MG Xenografts Treated with AMG 102 U87MG U87MG. Δ 2-7 U87MG. wtEGFR

11. U87MG. Δ 2-7 Xenografts Treated with Panitumumab and AMG 102

12. 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

13. U87MG.DY5 Xenografts Treated with AMG102 U87MG.DY5 p-EGFR (Y1173)  2-7 DY5 p-EGFR (Y1068)  -tubulin Total EGFR  2-7 DY5

15. 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

16. 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

17. 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

18. Acknowledgements Oncogenic Signalling Laboratory Vino Pillay Terri Burgess, Angela Coxen and Kelly Oliner Amgen Inc.