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Talk form AACR conference in Denver, April 2009
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Dual Targeting of the Tyrosine Kinase Receptors EGFRvIII and c-Met in Glioblastoma
Multiforme
Disclosures
My laboratory currently receives research funding from Amgen
Structure ofEGFRvIII
* Tyrosine residues mutated in DY5 construct
EGFR Homodimer
L1
CR1
L2
CR2
992
10681086
1148
COOH
NH2
1101
TM
L1
CR1
L2
CR2
992
10681086
1148
COOH
NH2
1101
TM
974 974
1045 1045
1173 1173
845845
TK TK
P
P
P
P
P
P
P
P
P
SrcShc
SHP1
PLC
MAPK/ERK Cascade
PKC MAPK/ERK Cascade
Gab1
MAPK/ERK Cascade
p85 AKT/PKB cascade
Grb2Cbl 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 constructΔ
U87MG.2-7
U87MG.DK
1 2 34 5
76
1. P-EGFR 2. P-FGFR3 3. P-Axl 4. P-MET 5. P-PDGFR6.P-EphA7 7. P-VEGFR3
EGFRvIII Activates Multiple Receptor Tyrosine Kinases (RTKs)
Phospho-specific antibody arrays
Confirmation that EGFRvIII Phosphorylates c-Met and PDGFRβ in U87MG.Δ2-7 Cells
U87M
G
U87M
G +
HG
FU87
MG
.Δ2-
7U87
MG
.DK
Phospho c-Met
Total c-Met
Phosphorylated PDGFRβ
U87MG. 2-7 U87MG.DK0
20
40
60
80
100
Flu
ore
scen
ce i
nte
nsi
ty (
rela
tive
un
its)
Panitumumab Inhibits EGFRvIII Activation in U87MG.Δ2-7 Cells
p-EGFR(Y1068)
p-EGFR(Y1173)
Total EGFR
Panitumumab - + - +
Whole cell lysates probe for total and
phosphorylated EGFRvIII
c-Met Phosphorylation is Ligand Independent and Inhibited by Panitumumab but not AMG 102
A549 + HGF(400 ng/ml)
AMG102 (10 μg/ml)
Panitumumab(20 μg/ml)
Irrelevant Ab(30 μg/ml)
Combination(30 μg/ml)
Phospho c-Met
Total c-Met
U87MGΔ2-7 cells were treated with different antibodies and c-met immunoprecipitated to determine levels of total and
phosphorylated c-Met
Phosphorylation of PDGFRβ is Inhibited by Panitumumab
U87MG.DK U87MG.2-7 AMG102 Panitumumab Combination0
10
20
30
40
50
60
70
80
90
100
UntreatedTreatment
Flu
ore
sc
en
ce
In
ten
sit
y (
Re
lati
ve
Un
its
)
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
Flu
ore
scen
ce i
nte
nsi
ty (
rela
tive
un
its)
Untreated AMG 102 Panitumumab Combination0
50
100
150
200
250
Treatment
Flu
ore
scen
ce i
nte
nsi
ty (
rela
tive
un
its)
Untreated AMG 102 Panitumumab Combination 0
250
500
750
1000
1250
1500
Treatment
Flu
ore
scen
ce i
nte
nsi
ty (
rela
tive
un
its)
Untreated AMG 102 Panitumumab Combination0
20
40
60
80
100
Treatment
Flu
ore
scen
ce i
nte
nsi
ty (
rela
tive
un
its)
28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 600
250
500
750
1000
1250
1500
1750ControlAMG102
U87MG
Number of days post inoculation
Tu
mo
ur
volu
me
(mm
3)
6 7 8 9 10 11 12 13 14 15 16 17 18 190
250
500
750
1000
1250
1500
1750
2000
ControlAMG102
U87MG.2-7
Number of days post inoculation
Tu
mo
ur
volu
me
(mm
3)
U87MG Xenografts Treated with AMG 102U87MG
U87MG.Δ2-7
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 480
250
500
750
1000
1250
1500ControlAMG 102
U87MG.WT
Number of days post inoculation
Tu
mo
ur
volu
me
(mm
3 )
U87MG.wtEGFR
U87MG.Δ2-7 Xenografts Treated with Panitumumab and AMG 102
7 9 11 13 15 17 19 21 23 25 27 29 31 33 350
250
500
750
1000
1250
1500
1750
2000
2250
Control
PanitumumabCombination
AMG 102 (100 g)
U87MG.2-7
Number of days post inoculation
Tu
mo
ur
volu
me
(mm
3)
7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 370
250
500
750
1000
1250
1500
1750
2000
Control
PanitumumabCombination
AMG 102 (30 g)
U87MG.2-7
Number of days post inoculation
Tu
mo
ur
volu
me
(mm
3)
Survival of Data U87MG.2-7
0 4 8 12 16 20 24 28 32 360
25
50
75
100Control
AMG102 (30 g)PanitumumabCombination
Days
Per
cen
t su
rviv
al
Immunohistochemistry analysis of U87MG.Δ2-7 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
U87MG.DY5 Xenografts Treated with AMG102
p-EGFR(Y1173)
2-7 DY5
p-EGFR(Y1068)
-tubulin
Total EGFR
2-7 DY5
U87MG.DY5
Conclusions
EGFRvIII activates multiple RTK’s c-Met is activated by EGFRvIII in a ligand independent
manner leading to resistance to AMG 102 Panitumumab inhibits the EGFRvIII phosphorylation of c-
Met and restores response to AMG 102 EGFRvIII probably phosphorylates c-Met by direct
interaction Combination of Panitumumab and AMG 102 may be an
effective therapy in GBM
Survival and Proliferation
MAP-kinase Akt/PKB
PI 3-kinase
PIP3
PDK1
MAP-kinase-kinase-kinase
Ras
Ras-GEF
Grb2
PDGFRβ
EGFRvIII C-MetU87MG.Δ2-7 Cells
EGFRvIII co-activates other RTKs including c-Met
Survival and Proliferation
MAP-kinase Akt/PKB
PI 3-kinase
PIP3
PDK1
MAP-kinase-kinase-kinase
Ras
Ras-GEF
Grb2
x xPanitumumab
Xenografts partially inhibited by Panitumumab but can revert to the HGF/c-Met pathway
Survival and Proliferation
MAP-kinase Akt/PKB
PI 3-kinase
PIP3
PDK1
MAP-kinase-kinase-kinase
Ras
Ras-GEF
Grb2
x x
Xenografts inhibited by the combination of Panitumumab and AMG 102
x
x
Acknowledgements
Oncogenic Signalling Laboratory
Vino Pillay
Terri Burgess, Angela Coxen and Kelly Oliner
Amgen Inc.