1. in vivo animal model studies in biological science 1.Cancer 2. Neuroscience 1.Cancer research 2. Neuroscience

2. Lung Cancer Cure rate for all patients: 15% MaleFemale

3. EGFR Expression in NSCLC NSCLC 60-80% Tumours showing high EGFR expression Poor outcome High expression associated with

4. EGFR signaling pathways HER PLC g PI3K Shc Sos Ras Raf Mek PKC AKT CyclinD1 Elk1 Myc Sp1 PDK-1 p70 S6K PTEN FKHR-L1 JAK STAT Grb2 p27 KIP1 Jun/Fos E2F PEA3 Bad/Bcl2 GSK3 Erk Citri A, et al. Exp Cell Res 2003

5. Molecular Targeting of EGF Receptor Gefitinib (IRESSA) Erlotinib (Tarceva) Cetuximab

6. Response to EGFR-TKI in NSCLC patients Yu CJ. (2005) PloS Med Initial diagnosis 2 months after gefitinib 9 months later

7. GxGxxGK E746- A750 R 768 LR 858 719 G719A/C (5%) 861 L858R (~40%) L861Q (4%) 18192021222324 Deletions (~45%) 766- 768 Insertions (3%) 776 S768I (2%)R776C (2%) EGF Ligand Binding Tyrosine Kinase TM Primary activating mutations are mainly found in EGFR tyrosine kinase domain exons 18-21

8. NSCLC EGFR Mutation(+) EGFR Mutation(-) TKI Response(+) TKI Response(-) TKI Response(+) TKI Response(-) Primary mutation(s) ( Acquired resistance ) Modeling EGFR-TKI responses in pre-clinical model system PC9, HCC827

9. Clinical response to EGFR-TKI in NSCLC patients Yu CJ. (2005) PloS Med Initial diagnosis 2 months after gefitinib 9 months later

10. MET amplification T790M secondary mutationUnknown Mechanisms Mechanisms of the Acquired Resistance to EGFR tyrosine kinase inhibitors in NSCLC (2008)

11. NSCLC EGFR Mutation(+) EGFR Mutation(-) TKI Response(+) TKI Response(-) TKI Response(+) TKI Response(-) Primary mutation(s) ( Acquired resistance ) Modeling EGFR-TKI responses in pre-clinical model system PC9, HCC827

12. Human cancer cell immunocompromised mouse xenograft model

13. Erlotinib treatment in PC9 orthotopic lung cancer model Before administration1 week 2 weeks3 weeks vehicle erlotinib (50mg/kg/day) treated

14. Tumorigenic animals Treatment duration Culture succeeded Tumor regression Culture failed Continue treatment PC9 lung86 months3140 PC9 S. C.135 months1039 HCC827 lung43 months0400 HCC827 S. C.13 months0001 Data summary of erlotinib treatment in pre-clinical animal tumor xenograft model system

15. PC9TR PC9 Multi-cycle resistance test Inoculate previously obtained in vivo drug resistant cells to second animals and subject the animals to repeated drug cycle

16. 1 st generation EGFR-TKI gefitinib, erlotinb : reversible EGFR blocker 2 nd generation EGFR-TKI e.g. pan-erbB blocker, multi-target EGFR blocker, irreversible EGFR blocker (BIBW2992)

18. The Effect on Cell Viability of si-EGFR in PC-BR clones EGFR Actinin scram si-EGFR PC9#10#6#1 EGFR dependentEGFR independent

19. Q L I T(790) PC9 Sequencing in PC9 & BR1,6,10 Q L I T(790) BR#1 Q L I T(790) BR#6 Q L I T(790) BR#10

20. Protein expression and phosphorylation profile in PC9-BR clones EGFR Actinin AKT ERK p-EGFR p-AKT p-Her3 p-ERK STAT3 MET p-MET p-STAT3 p70 S6K BIM 0 0.2 2 20 200 0 0.2 2 20 200 BIBW2992 for24H 0 0.2 2 20 200 PC9 #10#6 0 0.2 2 20 200 #1 20

21. BIBW2992 treatment in inoculated mouse in vivo #1 #6 PC #10

22. in vivo response to BIBW2992 (25mg/kg)

23. GxGxxGK E746- A750 R 768 LR 858 719 G719A/C (5%) 861 L858R (~40%) L861Q (4%) 18192021222324 Deletions (~45%) 766- 768 Insertions (3%) 776 S768I (2%)R776C (2%) EGF Ligand Binding Tyrosine Kinase TM Primary activating mutations are mainly found in EGFR tyrosine kinase domain exons 18-21

24. EGFR Mutations Gefitinib Responders 8/9 Non-responders0/7 p= 0.00075 Lynch et al, NEJM 2004

25. Construction of transgenic mouse model

26. CCSPrtTA Wong et al. (2006) Cancer Cell Mouse EGFR non-small cell lung cancer transgenic mouse model

27. Wong et al. (2006) Cancer Cell Tet-inducible mutant EGFR expression in mouse lung

28. Wong et al. (2006) Cancer Cell EGFR mutation is oncogenic

29. Wong et al. (2006) Cancer Cell EGFR expression is required for the maintenance of tumor

30. Wong et al. (2006) Cancer Cell

31. Lung cancer originated from mutant EGFR respond to various EGFR inhibitors

32. Transgenic mutant EGFR animal model study 1. mutant EGFR is oncogenic 2. continued expression of EGFR is required for the maintenance of tumor 3. mutant EGFR is a therapeutic target

33. Factors controlling tumorigenesis Immune Oncogenes Tumor suppressor genes Stroma Angiogenesis WT cells metastasis

35. Utility of genetically-engineered mouse models of cancer Genetically engineered mouse Tumor development Progression analysis early detection prevention chemotherapy

36. Genetic engineering of mouse genome : knock-out and knock-in via homologous recombination

38. Embryonic stem cell culture

39. Homologous recombinant ES cell selection and blastocyst injection

40. Generation of chimera mouse

41. Confirmation of germ line transmission and generation of knock-out(in) mouse

42. Conditional activation of p53 Advantages: p53 Native promoters Temporal, spatial Regulation of gene expression unbiased

43. Conditional knock-out system

44. P53 LSL/LSL is a phenocopy of p53-/- Cre-recombinase-Oestrogen-Receptor-T2

50. p53 reactivation mouse model study with conditional gene expression mouse 1.p53 inactivation is required for the maintenance of p53 mutant tumors 2.p53 gene delivery or other ways to reactivate p53 in p53 mutant tumor could be a therapeutic option