pFRS2 Y196

Debio1347TAS-120BGJ398ICC13-7

FRS2pSHP2 Y542

SHP2pMEK1/2 S217/221

MEK1/2pERK1/2 T202/Y204

ERK1/2

Supplemental Figure 1

OPTNExon 9Exon 17

FGFR2

Chr10q26.13 p13

5’ 3’

C T C ACA A C CA A T G A G G A AG G A G A A G C AG A AGluAsnThrLeu

764 765 766 767 768 185 186 187 188 189Thr GluAlaGlyGlu Glu

OPTNFGFR2

KIAA1217

Exon 7Exon 17

FGFR2

Chr10q26.13 p12.2

5’1 768 283 1943 amino acid

3’

A ICC13-7

F G

1.5 CCLP-1

Rel

ativ

e Ex

pres

sion

1.0

0.5

0

FGFR

1

FGFR

1b

FGFR

1c

FGFR

2

FGFR

3

Rel

ativ

e Ex

pres

sion 1.5 FGF20 expression

1.0

0.5

0

CC

LP-1

HuC

CT1

SNU

1079

SSP2

5

RBE

ICC

13-7

FGFR1

SG23

1R

BESN

U10

79SS

P25

MM

NK1

CC

LP-1

CC

SW1

Huh

-28

ICC

2IC

C4

β-actin

B

EDC

pFRS2 Y196

FRS2

β-actin

CC

LP-1

ICC

13-7

ICC

15IC

C16

ICC

17IC

C2

ICC

4H

uCC

T1SS

P25

SG23

1M

MN

K1R

BESN

U10

79H

uh-2

8

short

long

Supplementary Figure S1. Characterization of FGFR-driven ICC models

A, Immunoblot of lysates from the indicated biliary tract cancer cell lines and immortalized bile

duct cells (MMNK-1). The two FGFR inhibitor sensitive ICC cell lines, CCLP-1 and ICC13-7 (red),

show constitutive FRS2-Y196 phosphorylation.

B, Structure of FGFR2-OPTN chromosomal fusion in ICC13-7 cells.

C-E, Examination of FGF pathway components in CCLP-1 cells. C, Immunoblot for FGFR1 in

lysates from a set of human biliary tract cancer cell lines, including CCLP-1. MMNK-1 cells are

shown as a reference. D, qRT-PCR analysis showing that FGFR1 is expressed as the IIIc isoform

in CCLP-1 cells. E, Results of qRT-PCR analysis showing FGF20 overexpression in CCLP-1

cells.

F, Immunoblot showing dose-response of the indicated FGFR inhibitors on downstream signaling

pathways (dose range: BGJ398: 0.4 nM-250 nM, TAS-120: 0.08-50 nM, Debio1347: 8 nm-5000

nM; for each drug, doses were increased at 5-fold increments).

G, Structure of the FGFR2-KIAA1217 chromosomal fusion in the MG69 PDX model.

pFRS2 Y196

ParentEmpty vector

300 nM Debio1347

75 nM TAS-120

75 nM BGJ398

ICC13-7

FRS2pSHP2 Y542

SHP2pMEK1/2 S217/221

MEK1/2pERK1/2 T202/Y204

ERK1/2β-actin

FGFR2-PHGDH WT

FGFR2-PHGDH V565F

FGFR2-PHGDH L618V

FGFR2-PHGDH K660M

FGFR2-PHGDH K715R

-

-

-

+

-

-

-

-

+

-

+

-

-

-

-

+

-

-

-

-

+

-

+

-

-

-

-

+

-

-

-

-

+

-

+

-

-

-

-

+

-

-

-

-

+

-

+

-

-

-

-

+

-

-

-

-

+

-

+

-

-

-

-

+

-

-

-

-

+

-

+

-

-

-

-

+

-

-

-

-

+

-

+

-

DMSO

Parental

Empty vector

WT

V565F

L618V

K660M

K715R

ICC13-7

FGFR

2-PH

GD

H

75nM

BGJ3

98

75nM

TAS-

120

300n

M De

bio13

47

Emptyvector

DMSOCCLP-1

FGFR

2-PH

GD

H

50 nM

BGJ3

98

50 nM

TAS-

120

200 n

M De

bio13

47

WT

V565F

E566A

N550K

N550H

M538I

L618V

K660M

H683L

K715R

Supplemental Figure 2

A

F

B

E

D

C

FGFR2-PHGDHshort

longNon-specific

FGFR2β-actin

FGFR2β-actin

Vect

orW

TM

538I

N55

0HN

550K

V565

FE5

66A

L618

VK6

60M

H68

3LK7

15R

Vect

orW

T

Vect

orPa

rent

al

WT

V565

FL6

18V

K660

MK7

15R

CCLP-1

CCLP-1

Day 0 Day 14

ICC13-7

FGFR2-PHGDH shortlong

Non-specific

Non-specific

Non-specific

V565F

M538IN550H

N550KE566A L618V K660M

H683L K715R WT

Supplementary Figure S2. Activity of FGFR inhibitors against different FGFR2 kinase

domain mutations in ICC cells

CCLP-1 cells (A-C) and ICC13-7 cells (D-F) were established to express the FGFR2-PHGDH

fusion with a WT kinase domain or with the indicated mutations or expressing empty vector

control. In B-D and F, cells were treated with the indicated FGFR inhibitors or DMSO vehicle. A,

Immunoblot analysis of lysates of CCLP-1 derivative lines grown in the absence of drug treatment.

B, Crystal violet staining of CCLP-1 cells after 5 days treatment. C, Pooled CCLP-1 cell clones of

all FGFR2 fusion variants were grown for 14 days in the absence of drug selection. The individual

clones were monitored using genomic DNA extracted at day 0 and day 14, using a ddPCR assay

specific to each mutation. Data are generated from two independent experiments. D, Crystal violet

staining of ICC13-7 cells after 14 days treatment. E, Immunoblot analysis of lysates from ICC13-

7 derivative lines grown in the absence of drug treatment. CCLP-1 cell lysates are shown as a

reference. F, immunoblot analysis of ICC13-7 cells after 6-8 hour treatment.

All figures for revision v3 6All figures for revision v3 7