in vivo animal model studies in biological science

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

2. Lung Cancer • Cure rate for all patients: 15% Male Female

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

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

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

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

7. Primary activating mutations are mainly found in EGFR tyrosine kinase domain exons 18-21 EGF Ligand Binding TM Tyrosine Kinase L858R (~40%) S768I (2%) R776C (2%) G719A/C (5%) L861Q (4%) 719 768 776 858 861 GxGxxG 18 K 19 20 R L 21 R 22 23 24 E746-A750 766-768 Deletions (~45%) Insertions (3%)

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

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

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

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

12. Human cancer cell immunocompromised mouse xenograft model

13. Erlotinib treatment in PC9 orthotopic lung cancer model vehicle treated erlotinib (50mg/kg/day) treated Before administration 1 week 2 weeks 3 weeks

14. Data summary of erlotinib treatment in pre-clinical animal tumor xenograft model system

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

16. 1st generation EGFR-TKI gefitinib, erlotinb : reversible EGFR blocker 2nd 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 scram si-EGFR si-EGFR si-EGFR si-EGFR scram scram scram EGFR Actinin PC9 #1 #6 #10 EGFR dependent EGFR independent

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

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

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

22. invivo response to BIBW2992 (25mg/kg)

23. Primary activating mutations are mainly found in EGFR tyrosine kinase domain exons 18-21 EGF Ligand Binding TM Tyrosine Kinase L858R (~40%) S768I (2%) R776C (2%) G719A/C (5%) L861Q (4%) 719 768 776 858 861 GxGxxG 18 K 19 20 R L 21 R 22 23 24 E746-A750 766-768 Deletions (~45%) Insertions (3%)

24. EGFR Mutations Gefitinib Responders 8/9 Non-responders 0/7 p= 0.00075 Lynch et al, NEJM 2004

25. Construction of transgenic mouse model

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

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

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

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

30. Wong et al. (2006) Cancer Cell

31. Lung cancer originated from mutant EGFR respond to various EGFR inhibitors Wong et al. (2006) Cancer Cell

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 Tumor suppressor genes Oncogenes metastasis Immune Stroma Angiogenesis WT cells

35. Utility of genetically-engineered mouse models of cancer early detection Genetically engineered mouse prevention Progression analysis Tumor development 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 • p53 inactivation is required for the maintenance of p53 mutant tumors • p53 gene delivery or other ways to reactivate p53 in p53 mutant tumor • could be a therapeutic option