Inhibitor-sensitive fibroblast growth factor receptor ... · Inhibitor-sensitive fibroblast growth...

Inhibitor-sensitive fibroblast growth factor receptor mutations in lung squamous cell carcinoma

Rachel G. Liao Laboratory of Matthew Meyerson MD, PhD

TCGA Symposium November 28, 2012

S

 

 

Squamous cell carcinoma of the lung: a disease without treatment options

S Adenocarcinoma of the lung has seen many targeted therapy advances in the past decade (EGFR, EML4-ALK, ERBB2), while

S Squamous cell carcinoma had few targets and no targeted therapies—and the clinical burden is great

 

 

 

 

FGFR events in the TCGA Lung Squamous Cell Carcinoma sequencing project

S ~10% focal amplification of FGFR1

S ~8% mutation across the four receptors

S 3% FGFR2, 3% FGFR3

S Not significantly mutated across the dataset

A

Liao.et.al..Figure.1

FGFR2 and FGFR3 mutations are observed in lung SqCC

W290C

S320C

E471Q

K660E

K660N

T787K

FGFR2 1 200 400 600 800

FGFR3

R248C S249C

K717M

S435C

Ig)like.domain Transmembrane.domain Kinase.domain Alternate.exon

1 200 400 600 800

FGFR2 and FGFR3 mutations do not repeatedly co-occur with other events except

TP53 mutation

Trevor Pugh

R248C

S249C

S435C

K717M

WT03

EGFR0insNPG

pBp0GW

FGFR30monomer

FGFR30monomer

FGFR30dimer

actin

Unreduced

Reduced

FGFR2%monomer

FGFR2%monomer

Unreduced

Reduced

actin

W290Cb

W290Cc

S320C

K660Eb

K660Ec

WT2b

WT2c

FGFR2%dimer

A

B

Liao et al. Figure 2.

FGFR2/3 mutations are transforming in an anchorage-independent growth assay

W29

0Cb

S320C

b

E471Q

b

K660E

b

K660N

b

T787K

b

WT 2b

EGFR insN

PG

pBpG

W

0

5

10

15

20

F G F R 2 b m u t a t i o n

F o l d

o v e

r W T 2

b

W29

0Cc

E471Q

c

K660E

c

K660N

c

T787K

c

WT 2c

EGFR insN

PG

pBpG

W

0

2

4

6

8

F G F R 2 c m u t a t i o n

F o l d

o v e

r W T 2

c

R248C

S249C

S435C

K717M

3c W

T

pBpG

W

0

5

10

F G F R 3 m u t a t i o n

F o l d

o v e

r W T

FGFR3

R248C S249C WT-3cAP24534-(uM):------0-------.01------.1 -------0------.01------.1 ------0-------.01------.1

pFGFR

FRS2

pFRS2

Erk-1/2

pErk-1/2

actin

FGFR2

K660Nb K660Nc WT-2b WT-2c

FRS2

actin

AP24534-(uM):------0-----.01----.1 ------0-----.01----.1 ------0----.03----.1 ------0----.03----.1

pFGFR

pFRS2

Erk-1/2

pErk-1/2

FGFR2

W290Cb W290Cc S320C K660Eb K660EcAP245344(uM):444444044444.0144444.1 444444044444.0144444.1 444444044444.014444.1 444444044444.014444.1 444444044444.014444.1

pFGFR

FRS2

pFRS2

Erk41/2

pErk41/2

actin

A

B


010100100010000

W290C

b

W290C

c

S320C

b

K660E

b

K660E

c

K660N

b

K660N

c

R248C

S249C

EGFR inSNPG

0.5

W29

0Cb

W29

0Cc

S320C

K660E

b

K660E

c

K660N

c

R248C

S249C

EGFR insN

PG

Rel

ativ

e co

lony

form

atio

n

FGFR2/3 transformation can be blocked by FGFR inhibitors

Relative colony formation in response to Ponatinib BGJ398AP24534

0 nM+1.0 +1.0

Rel

ativ

e co

lony

form

atio

n

10 nM 100 nM 1000 nM 10000 nM

0.5

0.0 0.0

cDNA construct cDNA construct

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Loss of transformation correlates with loss of phosphorylation

FGFR2

W290Cb

W290Cc

S320C

K660Eb

K660Ec

K660Nb

K660Nc

WT2b

WT2c

R248C

S249C

WT3c

EGFR4mut

Ba/F3

FGFR3

pFGFR

FRS2

actin

pFRS2

A

B

C


-3 -2 -1 0 10

50

100W290CbW290CcS320CbK660EbK660EcK660NbK660NcWT2bEGFR insNPGBa/F3 +IL3

Log uM AP24534

Gro

wth

inhi

bitio

n

-3 -2 -1 0 10

50

100

W290CbW290CcS320CbK660EbK660EcK660NbK660NcWT2bEGFR insNPGBa/F3 +IL3

Log uM BGJ398

Gro

wth

inhi

bitio

n

FGFR2

W290Cb

W290Cc

S320C

K660Eb

K660Ec

K660Nb

K660Nc

WT2b

WT2c

R248C

S249C

WT3c

EGFR4mut

Ba/F3

FGFR3

pFGFR

FRS2

actin

pFRS2

A

B

C


BGJ398

W29

0Cb

W29

0Cc

S320C

b

K660E

b

K660E

c

K660N

b

K660N

cW

T2b

EGFR insN

PG

Ba/F3 +

IL31

10

100

1000

10000

mutation

IC50

(nM

)

AP24534

W29

0Cb

W29

0Cc

S320C

b

K660E

b

K660E

c

K660N

b

K660N

cW

T2b

EGFR insN

PG

Ba/F3 +

IL31

10

100

1000

10000

mutation

IC50

(nM

)

IC50

(nM

)

W290CbW290CcS320CbK660EbK660EcK660NbK660NcWT2bEGFR insNPGBa/F3 +IL3

W29

0Cb

W29

0Cc

S320C

b

K660E

b

K660E

c

K660N

b

K660N

cW

T2b

EGFR insN

PG

Ba/F3 +

IL3

W29

0Cb

W29

0Cc

S320C

b

K660E

b

K660E

c

K660N

b

K660N

cW

T2b

EGFR insN

PG

Ba/F3 +

IL3

0

50

100

Cells exhibiting dependency on the FGFR pathway are sensitive to FGFR inhibitors

Gro

wth

inhi

bitio

n W290Cc 100 S320Cb K660Eb K660Ec

Gro

wth

inhi

bitio

n

W290Cb

50 K660Nb K660Nc WT2b

0 EGFR insNPG -3 -2 -1 0 1 -3 -2 -1 0 1 Ba/F3 +IL3

Log uM AP24534 Log uM BGJ398

AP24534 BGJ398

1000010000

10001000

IC50

(nM

)

100100

1010

11

mutation mutation

A clinical case

FGFR2 mutation in the coding sequence at p.P253R

An FGFR2-positive tumor regresses upon pazopanib treatment

 

 

 

 

Conclusions

S FGFR2/3 mutations observed in lung SqCC are sufficient to drive transformation in the NIH-3T3 cell line model, and the transformation phenotype can be reversed by FGFR small molecule inhibition

S Ba/F3 cells dependent on FGFR2/3 signaling for proliferation can be growth inhibited by FGFR small molecule inhibition

S A clinical success confirms that these findings provide a rationale for further study of patients with FGFR events in their tumors

S TCGA data have been used effectively to find new driving, targetable events in tumors (though these events do not always meet the threshold of statistical significance)

 

 

     

Acknowledgements

S

S

Matthew Meyerson

Peter Hammerman

Josh Francis

Heidi Greulich

Ami Bhatt

Tzu-Hsiu Chen

Bethany Kaplan

Tanaz Sharifnia

Luc de Waal

Alice Berger

Trevor Pugh

Joonil Jung

All lab members

S Novartis S Diana Grauss-Porta S Ralph Tiedt

Cory Johannesson

Jesse Boehm

Ben Munoz

Robert Haddad

Matt Wilkerson (UNC)

David Ornitz (WashU)

Pamela Pollock (QIT)

FGFR biology

Ras/ MAPK

PI3K/ AKT/ mTOR

Stat3

FRS2 GRB2

GAB1

FGFR

FGF

nucleus

HSPG

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Disulfide bonding observed in ECD mutations to Cys

Inhibitor-sensitive fibroblast growth factor receptor ... · Inhibitor-sensitive fibroblast growth...

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