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

Lung Cancer Cure rate for all patients: 15% MaleFemale

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

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

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

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

GxGxxGK E746- A750 R 768 LR G719A/C (5%) 861 L858R (~40%) L861Q (4%) Deletions (~45%) 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

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

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

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

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

Human cancer cell immunocompromised mouse xenograft model

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

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

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

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)

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

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

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 BIBW2992 for24H PC9 #10# #1 20

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

in vivo response to BIBW2992 (25mg/kg)

GxGxxGK E746- A750 R 768 LR G719A/C (5%) 861 L858R (~40%) L861Q (4%) Deletions (~45%) 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

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

Construction of transgenic mouse model

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

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

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

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

Wong et al. (2006) Cancer Cell

Lung cancer originated from mutant EGFR respond to various EGFR inhibitors

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

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

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

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

Embryonic stem cell culture

Homologous recombinant ES cell selection and blastocyst injection

Generation of chimera mouse

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

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

Conditional knock-out system

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

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