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.