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in vivo animal model studies in biological science. Cancer 2. Neuroscience. Cancer research. 2. Neuroscience. Lung Cancer. Cure rate for all patients: 15%. Male. Female. EGFR Expression in NSCLC. Tumours showing high EGFR expression.

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in vivo animal model studies in biological science

  • Cancer

  • 2. Neuroscience

  • Cancer research

2. Neuroscience


Lung Cancer

  • Cure rate for all patients: 15%

Male

Female


EGFR Expression in NSCLC

Tumours showinghigh EGFR expression

High expression associated with

Poor outcome

NSCLC 60-80%


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


Molecular Targeting of EGF Receptor

Cetuximab

Gefitinib (IRESSA)

Erlotinib (Tarceva)


Response to EGFR-TKI in NSCLC patients

Initial diagnosis

2 months after gefitinib

9 months later

Yu CJ. (2005) PloS Med


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%)


Modeling EGFR-TKI responses in pre-clinical model system tyrosine kinase domain exons 18-21

PC9, HCC827

TKI

Response(+)

Response(-)

( Acquired resistance )

EGFR

Mutation(+)

TKI

Response(-)

Primary mutation(s)

NSCLC

TKI

Response(+)

EGFR

Mutation(-)

TKI

Response(-)


Clinical response to EGFR-TKI in NSCLC patients tyrosine kinase domain exons 18-21

Initial diagnosis

2 months after gefitinib

9 months later

Yu CJ. (2005) PloS Med


Mechanisms of the Acquired Resistance tyrosine kinase domain exons 18-21

to EGFR tyrosine kinase inhibitors in NSCLC (2008)

Unknown Mechanisms

T790M secondary mutation

MET amplification


Modeling EGFR-TKI responses in pre-clinical model system tyrosine kinase domain exons 18-21

PC9, HCC827

TKI

Response(+)

Response(-)

( Acquired resistance )

EGFR

Mutation(+)

TKI

Response(-)

Primary mutation(s)

NSCLC

TKI

Response(+)

EGFR

Mutation(-)

TKI

Response(-)


Human cancer cell immunocompromised tyrosine kinase domain exons 18-21

mouse xenograft model


Erlotinib treatment in pc9 orthotopic lung cancer model
Erlotinib treatment in PC9 orthotopic lung cancer model tyrosine kinase domain exons 18-21

vehicle

treated

erlotinib (50mg/kg/day)

treated

Before administration

1 week

2 weeks

3 weeks



Multi-cycle resistance test tumor xenograft model system

Inoculate previously obtained in vivo drug resistant cells to

second animals and subject the animals to repeated drug cycle

PC9TR

PC9


1 tumor xenograft model systemst 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)


The Effect on Cell Viability of si-EGFR in PC-BR clones tumor xenograft model system

scram

si-EGFR

si-EGFR

si-EGFR

si-EGFR

scram

scram

scram

EGFR

Actinin

PC9

#1

#6

#10

EGFR dependent

EGFR independent


Sequencing in PC9 & BR1,6,10 tumor xenograft model system

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


Protein expression and phosphorylation profile tumor xenograft model system

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


#1 tumor xenograft model system

#6

#10

PC

BIBW2992 treatment in inoculated mouse in vivo


in tumor xenograft model systemvivo response to BIBW2992

(25mg/kg)


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%)


EGFR Mutations tyrosine kinase domain exons 18-21

Gefitinib Responders 8/9

Non-responders 0/7 p= 0.00075

Lynch et al, NEJM 2004


Construction of transgenic mouse model tyrosine kinase domain exons 18-21


Mouse EGFR non-small cell lung cancer transgenic mouse model tyrosine kinase domain exons 18-21

CCSP

rtTA

Wong et al. (2006) Cancer Cell


Tet-inducible mutant EGFR expression in mouse lung tyrosine kinase domain exons 18-21

Wong et al. (2006) Cancer Cell


EGFR mutation is oncogenic tyrosine kinase domain exons 18-21

Wong et al. (2006) Cancer Cell


EGFR expression is required tyrosine kinase domain exons 18-21

for the maintenance of tumor

Wong et al. (2006) Cancer Cell


Wong et al. (2006) Cancer Cell tyrosine kinase domain exons 18-21


Lung cancer originated from mutant EGFR tyrosine kinase domain exons 18-21

respond to various EGFR inhibitors

Wong et al. (2006) Cancer Cell


Transgenic mutant EGFR animal model study tyrosine kinase domain exons 18-21

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 tyrosine kinase domain exons 18-21tumorigenesis

Tumor suppressor genes

Oncogenes

metastasis

Immune

Stroma

Angiogenesis

WT cells


Utility of genetically-engineered tyrosine kinase domain exons 18-21

mouse models of cancer

early detection

Genetically

engineered

mouse

prevention

Progression

analysis

Tumor development

chemotherapy


Genetic engineering of mouse genome tyrosine kinase domain exons 18-21

: knock-out and knock-in via homologous recombination


Embryonic stem cell culture tyrosine kinase domain exons 18-21


Homologous recombinant ES cell selection tyrosine kinase domain exons 18-21

and blastocyst injection


Generation of chimera mouse tyrosine kinase domain exons 18-21


Confirmation of germ line transmission tyrosine kinase domain exons 18-21

and generation of knock-out(in) mouse


Conditional activation of p53 tyrosine kinase domain exons 18-21

Advantages:

p53

Native promoters

Temporal, spatial

Regulation of gene expression

unbiased


Conditional knock-out system tyrosine kinase domain exons 18-21


P53 LSL/LSL is a phenocopy of p53-/- tyrosine kinase domain exons 18-21

Cre-recombinase-Oestrogen-Receptor-T2


p53 reactivation mouse model study with tyrosine kinase domain exons 18-21

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


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