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RESEARCH ARTICLE RESEARCH ARTICLE

Cell-Autonomous and Non–Cell-Autonomous Mechanisms of Transformation by Amplifi ed FGFR1 in Lung Cancer Florian Malchers 1 , Felix Dietlein 1 , Jakob Schöttle 1 , 2 , Xin Lu 1 , Lucia Nogova 5 , Kerstin Albus 3 , Lynnette Fernandez-Cuesta 1 , Johannes M. Heuckmann 6 , Oliver Gautschi 8 , Joachim Diebold 9 , Dennis Plenker 1 , Masyar Gardizi 5 , Matthias Scheffl er 5 , Marc Bos 1 , 5 , Danila Seidel 1 , Frauke Leenders 1 , André Richters 7 , Martin Peifer 1 , Alexandra Florin 3 , Prathama S. Mainkar 11 , Nagaraju Karre 11 , Srivari Chandrasekhar 11 , Julie George 1 , Steffi Silling 4 , Daniel Rauh 7 , Thomas Zander 5 , Roland T. Ullrich 2 , H. Christian Reinhardt 5 , Francois Ringeisen 10 , Reinhard Büttner 3 , Lukas C. Heukamp 3 , Jürgen Wolf 5 , and Roman K. Thomas 1 , 3

Research. on January 19, 2021. © 2014 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst December 3, 2013; DOI: 10.1158/2159-8290.CD-13-0323

http://cancerdiscovery.aacrjournals.org/

FEBRUARY 2014�CANCER DISCOVERY | 247

ABSTRACT The 8p12 locus (containing the FGFR1 tyrosine kinase gene) is frequently amplifi ed

in squamous cell lung cancer. However, it is currently unknown which of the 8p12-

amplifi ed tumors are also sensitive to fi broblast growth factor receptor (FGFR) inhibition. We found

that, in contrast with other recurrent amplifi cations, the 8p12 region included multiple centers of

amplifi cation, suggesting marked genomic heterogeneity. FGFR1 -amplifi ed tumor cells were depend-

ent on FGFR ligands in vitro and in vivo . Furthermore, ectopic expression of FGFR1 was oncogenic,

which was enhanced by expression of MYC. We found that MYC was coexpressed in 40% of FGFR1 -

amplifi ed tumors. Tumor cells coexpressing MYC were more sensitive to FGFR inhibition, suggesting

that patients with FGFR1- amplifi ed and MYC-overexpressing tumors may benefi t from FGFR inhibitor

therapy. Thus, both cell-autonomous and non–cell-autonomous mechanisms of transformation modu-

late FGFR dependency in FGFR1 -amplifi ed lung cancer, which may have implications for patient selec-

tion for treatment with FGFR inhibitors.

SIGNIFICANCE: Amplifi cation of FGFR1 is one of the most frequent candidate targets in lung cancer.

Here, we show that multiple factors affect the tumorigenic potential of FGFR1 , thus providing clinical

hypotheses for refi nement of patient selection. Cancer Discov; 4(2); 246–57. ©2013 AACR.

See related commentary by Lockwood and Politi, p. 152.

Authors’ Affi liations: 1 Department of Translational Genomics, University of Cologne; 2 Max-Planck-Institute for Neurological Research; Institutes of 3 Pathology and 4 Virology, University of Cologne; 5 Department I of Inter-nal Medicine and Center for Integrated Oncology, University Hospital of Cologne; 6 Blackfi eld AG, Cologne; 7 Technical University Dortmund, Dort-mund, Germany; 8 Medical Oncology and 9 Institute of Pathology, Cantonal Hospital, Luzern; 10 Novartis Pharma AG, Basel, Switzerland; and 11 Division of Natural Products Chemistry, CSIR–Indian Institute of Chemical Technol-ogy, Tarnaka, Hyderabad, India

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

F. Malchers and F. Dietlein contributed equally to this work.

Data deposition: Sequencing data are available at http://www.translational-genomics.uni-koeln.de/scientifi c-resources

Corresponding Author: Roman K. Thomas, Department of Translational Genomics, University of Cologne, Weyertal 115b, 50931 Cologne, Germany. Phone: 49-221-478-96861; Fax: 49-221-478-97902; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-13-0323

©2013 American Association for Cancer Research.

INTRODUCTION The discovery of genomic alterations in kinase genes has

changed the clinical care of patients with lung adenocarcino-

mas bearing such alterations ( 1, 2 ). However, the landscape

of genome aberrations differs dramatically between the most

common lung cancer subtypes lung adenocarcinoma, squa-

mous cell lung cancer, and small-cell lung cancer. Only a

few therapeutically tractable genome alterations have so far

been found in squamous cell lung cancer ( 3, 4 ) and small-

cell lung cancer ( 5 ). Of these, amplifi cations of 8p12 ( FGFR1 ,

WHSC1L1 ) and mutations of DDR2 have been associated with

preclinical sensitivity to kinase inhibition ( 6–9 ). Further-

more, early signs of clinical activity of fi broblast growth fac-

tor receptor (FGFR) inhibitors in FGFR1 -amplifi ed small and

squamous cell carcinomas underscore the validity of such

approaches ( 10, 11 ). Unfortunately, though, it is presently

unclear which of the tumors bearing FGFR1 amplifi cations

are also sensitive to FGFR inhibition.

The FGFR family consists of four receptor tyrosine kinases,

which are common targets of deregulation by translocation,

point mutation, and amplifi cation in cancer ( 12–14 ). FGFRs

are activated via 22 different FGF ligands resulting in down-

stream FRS2 and extracellular signal-regulated kinase (ERK)

phosphorylation ( 13 ); their signaling is modulated endog-

enously by multiple negative intrinsic feedback loops ( 15–17 ).

Furthermore, alternative splicing of FGF receptors mediates

different responses to FGF ligands in epithelial (expressing

IIIb splice variants) and mesenchymal tissues (expressing IIIc

splice variants) in development ( 18 ). In particular, alternative

splicing of exon 8 determines the difference between epithe-

lial and mesenchymal variants of FGFR1 ; the mesenchymal

FGFR1-IIIc-β variant differs from full-length FGFR1-IIIc-αby skipping exon 2 (Ig1 loop; ref. 12 , 19 ).

Several recent studies described the 8p12 amplicon ( FGFR1 ,

WHSC1L1 ) as recurrently amplifi ed in lung cancer; the ampli-

con spans approximately 10 Mbps and contains about 50

genes in total ( 5, 6 ). Here, we sought to characterize in detail

the structure of the 8p12 amplicon to identify mechanisms of

oncogenic transformation induced by amplifi ed FGFR1 and

to determine modulators of FGFR dependency in lung cancer.

RESULTS Genomic Heterogeneity of the 8p12 Amplicon in Squamous Cell Lung Cancer

In lung cancer, amplifi cation of 8p12 occurs most fre-

quently in the squamous cell and small-cell subtypes ( 5–7 ).

We analyzed single-nucleotide polymorphism (SNP) array data

( 20–23 ) of 306 surgically removed squamous cell carcinoma

specimens that were generated as part of a large international

lung cancer genomics consortium project (24). Most sam-

ples revealed multiple individual broad alterations (termed




248 | CANCER DISCOVERY�FEBRUARY 2014 www.aacrjournals.org

Malchers et al.RESEARCH ARTICLE

macro lesions , size ≥ 20 Mbp), including gains of entire chromo-

somal arms (36% of copy-number segments), so that focal copy-

number alterations were masked ( Fig. 1A left). We therefore

developed a novel approach, termed Focal Amplifi cation Peak

Purifi cation (FAPP), which smoothens broad alterations and

thus extracts the primary focal amplifi cation peak ( Fig. 1 ). We

subsequently performed a signifi cance analysis (using GISTIC;

ref. 25 ) on this purifi ed dataset, resulting in 16 regions of recur-

rent focal amplifi cations affecting a total of 206 genes ( Fig. 1A ,

right, 1B and C , and Supplementary Fig. S1). We identifi ed

highly focal and signifi cant recurrent amplifi cation peaks for

3p11 (containing EPHA3 ) and 12p15 (containing the FGFR

adaptor gene FRS2 ), not previously described in squamous cell

lung cancer ( Fig. 1D ). The 12p15 locus ( FRS2 ) included 8 genes

and was amplifi ed at high amplitude (copy number ≥4) in 2%

of all cases ( Fig. 1A and D bottom). Because FRS2 encodes

the central adaptor protein downstream of FGFRs ( 15 ), FRS2-

amplifi ed lung tumors may depend on signaling downstream

of FGF receptors. Overall, FAPP dramatically reduced the

number of genes (n Gene ) that occurred in broad amplifi cation

peaks ( Fig. 1B and C ), such that in particular the 3q26 and

8q24 amplicons gained focality ( Fig. 1A ).

As an independent validation of our fi ndings, we applied the

same computational approach to another cohort of squamous

cell lung cancer specimens [ n = 299, The Cancer Genome Atlas

(TCGA); ref. 3 ] and obtained similar results (Supplementary

Fig. S2A). In the TCGA cohort, we also observed a marked

heterogeneity of the 8p12 amplicon (Supplementary Fig. S2B).

Furthermore, we found similar amplifi cation patterns of

EPHA3 and FRS2 ( Fig. 1D , right). Thus, the 8p12 amplicon in

squamous cell lung cancer is profoundly heterogeneous on the

genomic level; FGFR1 was not even included in the amplicon of

several tumor specimens.

We next assessed co-occurrence of amplifi cation events and

performed hierarchical clustering of purifi ed copy-number

data ( 26 ). Using this approach, we were able to sort 210 of

306 squamous cell lung cancer primary tumor samples (69%)

into clusters (Supplementary Fig. S3). We found that 8p12

( FGFR1 ) clustered together with the 11q13 ( CCND1 , FGF4 ,

FGF19 ) amplicon in some cases, a fi nding that has been

recently associated with FGFR inhibitor sensitivity in the

presence of beta-klotho expression ( 27 ). Most of the ampli-

fi ed regions formed single clusters [e.g., 3q26 ( SOX2 ), 4q12

( KIT , PDGFRA ), 8q24 ( MYC )] or were split into minor sub-

clusters [7p11 ( EGFR ), 12q15 ( FRS2 ), 19q12 ( CCNE1 )]. The

8p12 ( FGFR1 , WHSC1L1 ) amplicon was, however, identifi ed as

the most heterogeneous alteration, covering 5 segments and

forming 4 subclusters (Supplementary Fig. S3).

We subsequently plotted the segmented copy-number data

sorted by the genomic coordinate of their amplifi cation

center in a 15-Mbp region. This approach revealed the full

extent of the heterogeneity of the 8p12 amplicon ( Fig. 1C and

Supplementary Figs. S2B, S3, and S4); the 8p12 amplicons of

only 28% of all specimens centered on FGFR1 (Supplementary

Fig. S4). In marked contrast, 90% of the amplifi cations of

7p11 were centered on EGFR and displayed a homogeneous

amplifi cation pattern ( Fig. 1C, bottom , and Supplementary

Figs. S3 and S4). Similarly, CCND1 , SOX2 , and MYC were each

in the center of the respective amplicon in the vast majority

of the cases (Supplementary Fig. S4).

Finally, we analyzed chromosomal regions bearing high

copy-number fl uctuations that indicate the presence of

genomic breakpoints. Accordingly, 8p12-amplifi ed samples

were frequently found to harbor chromosomal breakpoints in

a 10-Mbp region that contained the FGFR1 gene. In addition,

breakpoints of the 8p12 locus occurred in non–8p12-amplifi ed

samples, too. In contrast, the homogeneous amplifi cations of

3q26 ( SOX2 ), 7p11 ( EGFR ), and 11q13 ( CCND1 ) revealed chro-

mosomal breakpoints only within a 5-Mbp range around the

respective oncogene (data not shown). Conclusively, this data

emphasizes that FGFR1 lies in a genomic locus with a highly

heterogeneous pattern of amplifi cation.

In summary, the 8p12 amplifi cation pattern exhibits strik-

ing differences from other recurrent amplicons in squamous

cell lung cancer; only a fraction of the amplifi ed samples har-

bor FGFR1 in the epicenter of the amplicon. Thus, beyond the

absolute copy-number amplitude, the geographic amplicon

extension may be biologically relevant for diagnostic purposes.

Ligand Dependency of FGFR Signaling in Lung Tumor Cells Bearing Focal FGFR1 Amplifi cations

Because many of the FGFR ligands are present in the tumor

microenvironment of malignant tumors ( 28 ), we next sought

to determine whether FGFR1 -amplifi ed lung tumor cell lines

are dependent on ligand binding. To this end, we analyzed

the genomic features of 148 lung cancer lines to identify cell

lines that capture the genomic pattern that we observed in the

primary tumors (Supplementary Fig. S5). We tested in par-

ticular for the presence or absence of genome alterations that

occur specifi cally in different lung cancer subtypes (24). Because

only few FGFR1 -amplifi ed cell lines exist that are of squamous

cell histology (HCC95 and H520 in this study), we addition-

ally included the 8p12-amplifi ed lines H1581 (large-cell) and

DMS114 (small-cell; Supplementary Fig. S5). We note, how-

ever, that large-cell carcinomas can be mostly reclassifi ed to the

other lung tumor subtypes (24). Because FGFR1 amplifi cations

are almost entirely restricted to squamous cell and small-cell

carcinomas ( 3 , 5 , 6 , 29 , 30 ), we postulate that these cell lines are

valid models of FGFR1- amplifi ed lung cancers (Supplemen-

tary Fig. S5). Furthermore, the two FGFR-dependent cell lines,

H1581 and DMS114, both include FGFR1 in the main peak of

the amplicon.

We found that H1581 cells showed high basal activation

of the FGFR1 pathway, which was completely antagonized

by FGFR inhibition ( Fig. 2A ). Basal levels of phospho-FGFR

(p-FGFR) were lower in the other FGFR inhibitor-sensitive

line, DMS114 ( Fig. 2A ), and phospho-ERK (p-ERK) levels

were enhanced by the addition of FGFs 1, 2, and 4. Both cell

lines exhibited high levels of FGF secretion under steady-

state conditions and increased ligand secretion under serum

starvation ( Fig. 2B ). Thus, FGFR-dependent lung tumor cells

may be sustained in their growth through autocrine and/

or paracrine FGFR activation. High baseline levels of p-ERK

were unchanged under FGFR inhibition and starvation in

the NRAS-mutant cell line HCC15; in FGFR1 -amplifi ed but

inhibitor-insensitive H520 cells, p-ERK could be activated by

FGFs 1, 2, and 4, but p-FGFR levels were unaffected by the

addition of inhibitor (Supplementary Fig. S6). Thus, FGFR1 -

amplifi ed lung cancer cells display a variable degree of autoac-

tivation of FGFR signaling, which might be due to autocrine





FGFR1 Signaling in Lung Cancer RESEARCH ARTICLE

Figure 1. Genomic heterogeneity of the 8p12 amplicon in squamous cell lung cancer. A, recurrence of copy-number aberrations [Genomic Identifi cation of Signifi cant Targets in Cancer (GISTIC) algorithm] in raw segmented inferred copy-number (CN) data (Affymetrix SNP 6.0) of 306 squamous cell lung cancer tumor samples (left, red) and in macro lesion purifi ed CN index (right, green). B, signifi cant reduction of n Gene in broad amplicons from raw CN data to CN index. Averages of log-transformed n Gene were tested by the Welch t test. C, representative screenshots of segmented copy-number data before (left) and after (right) processing with FAPP algorithm. Genomic regions (15 Mbps) containing the EGFR (7p11) and FGFR1 (8p12) genes are displayed. Samples were sorted by the genomic coordinate of the highest inferred copy number value. Positions of EGFR and FGFR1 are highlighted in green. D, repre-sentative screenshots of recurrent amplifi cations of EPHA3 (3p11, top) and FRS2 (12p15, bottom) in CLCGP (left, Hg18 annotation) and TCGA (right, Hg19 annotation) squamous cell lung cancer datasets. Genomic positions of FRS2 and EPHA3 are highlighted in green. FAPP, Focal Amplifi cation Peak Purifi cation; CLCGP, Clinical Lung Cancer Genome Project; TCGA, The Cancer Genome Atlas; EGFR , EGF receptor.

A Raw copy-number

before FAPP Purified copy-number index

after FAPP

1p12 (0)

2p16 (13)

2p15 (21)

2q32.3 (0)

3q26.33 (339)

4q12 (11)

5q15.2 (11)

5p12 (6)

7p11.2 (24)

8p12 (45)

8p24.2 (20)

9p13.3 (29)

11p13.3 (27)

12p12.1 (27)

12p15 (19)

13p22.1 (0)

13q34 (21)

14q21.1 (7)

18p11.2 (7)

18p12.1 (0)

19p12 (38)

19p13.2 (147)

22q11.21 (19)

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94

58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

1p12 (11)

3p11.1 (2)

3q26.33 (10)

4q12 (4)

5q11.2 (1)

7p11.2 (18)

8p12 (41)

8p24.21 (5)

9q13.3 (40)

11q13.2 (27)

12p12.1 (3)

12p15 (8)

13p22 (0)

18p11.2 (6)

q value

19p12 (5)

19p13.2 (12)

22p11.21 (16)

FGFR1MYC

EGFR

CCND1

SOX2

FRS2KRAS

KIT, PDGFR

EPHA3

Raw copy-number amplification

Copy-number index amplification

Chromosomal location (nGene)

1

4

16

64

256

1,024P = 0.008 P = 0.679

Broad ampliconin raw data

Raw

CN Data

Raw

CN Data

Purified

CN Index

B

Raw copy-number (before FAPP) Purified copy-number index (after FAPP)

Mbp

Chr. 8

Mbp

Chr. 7

FGFR1 FGFR1

EGFR EGFR

FRS2 FRS2

EPHA3

EPHA3

C

Mbp

Chr. 3

Mbp

Chr. 12

D CLCGP TCGA

1,02410–35 10–3510–12 10–1210–4 10–40.05 0.05

Focal Amplicon in raw dataBroad

256

64

16

4

1

1 4 16

nGene

in raw CN data peaks

nG

en

e in p

uri

fied C

N index p

eaks

MYCKRAS

SOX2

EGFRFRS2

CCND1FGFR1

DefocusedFocus

64 256 1,024

Purified

CN Index

Focal ampliconin raw data

1

2

3

4

5

6

7

8

9

10

11

12

13

1415161718

nG

en

e in C

N p

eaks






Figure 2. Ligand binding is essential for signaling perpetuation of FGFR-dependent cell lines. A, stimulation immunoblots for H1581 and DMS114 (both FGFR1 amp ). Not all bands were detected on the same membrane due to overlapping protein sizes. B, upregulation of normalized FGF-2 concentrations (c Norm ) by 48-hour normal (RMPI + 10% FCS) culture conditions (FCS + ), serum starvation (FCS − ) or with (PD173074, 1 μmol/L) FGFR inhibition (FCS − , PD) for H1581, DMS114, HCC95, H520 (all FGFR1 amp ) and HCC15 (NRAS mut ). C, interaction between the IgG2-IgG3-interloop domain of FGFR1 (green), FGFR1β Arg161 (red) and FGF-2 (yellow). Physiologic intra- and intermolecular interactions of FGFR1β Arg161 with FGF-2 Asn95 and His93 as derived from crystal structures (bottom left). PyMOL software predicts loss of interaction if Arg161 is substituted (site-directed mutagenesis, SDM) by Gln161 (bot-tom right). D, for each FGFR1β mutant, FGFR inhibitor sensitivity (PD173074) was assessed under increasing concentrations of FGF-2 ( x -axis, logarithmic) by measuring cellular ATP content after 9 hours. FGF-2–GI 50 dependencies ( y -axis, logarithmic) were fi tted to logistic functions. E, reconstitution of ERK phosphorylation for the ligand-binding–defi cient L76T mutation under high doses of FGF-2 as assessed by immunoblotting. F, individual tumor volumes of H1581 (top) and A549 (bottom) in waterfall blot representation after 8 weeks. Tumors of mice in the H1581 control virus group, which had to be sacrifi ced before the end of observation, are marked (+). Mice were exposed to either control virus (AdCMV-null, middle), FGF-trapping virus (AdsFGFR, right), or non-infectious supernatants (left). Column widths refl ect group sizes, and signifi cance values were derived from the Student t test. G, Representative images of nude mice 8 weeks after tumor cell injection; subcutaneous tumors are highlighted (arrows). FCS, fetal calf serum; PD, progressive disease.

FGFR1

FGF-2

SDM FGFR1ββR161Q

FGFR1 FGFR1

FGF-2 FGF-2

A B

D

p-ERK

t-ERK

–

–

+

–+

+–

–+

–+

+–

–+

–+

+FGF-2 (25 ng/mL)

PD173074 (1 μmol/L)

wt

L76T,

V472M V472M

5 10 15 20

V472M

K83E + V472M

D157N + V472M

D193N + V472M

L76T + V472M

Parental

A78L + V472M

*: Grubb outlier test to

parental and V472M

10

5

1

0.5

0.1

0.05

1005010510.5

0.10.05

0.010.005

No FG

F-2

C

5,0002,500

500

500

250

250

5025

52.5

5025

52.5

No virusControl virus

AdsFGFR virusH1581 [FGFR1amp]

A549 [KRASmut]

+

+ + + +

+

P = 0.016

P = 0.299 P = 0.008

E

F

DMS114 [FGFR1amp]

– – – – – – – – + +FG

F-1

FGF-2

FGF-4

FGF-8

FGF-1

9

KGF-2

FCS 1

0%

none

FGF-1

FGF-2

– – – – – – – – + +FG

F-2

FGF-4

FGF-8

FGF-1

9

KGF-2

FCS 1

0%

none

FGF-1

FGF-2

t-ERK

p-AKT

p-FGFR

Control virus AdsFGFR virus

H1581

A549

G

H15

81

DM

S114

HCC95

H52

0

HCC15

0

3

6

9

P = 0.007

P = 0.019

FCS+

HCC95

H15

81

DM

S114

p-FRS2α

FGF-1

p-ERK

t-FGFR1

Stimulation

PD173074, 1 µmol/L

Co

nce

ntr

atio

n r

atio

× 1

0–

9

HCC15

FCS–, PDFCS–

R161Q + V472M

GI50

variation factor25

H52

0HCC95

HC

C15H

520

P = 0.008

DM

S114

H15

81

H1581 [FGFR1amp]

GI 5

0 (

PD

17

30

74

) in

µm

ol/

L

Tu

mo

r v

olu

me

lo

g s

ca

le

(% o

f c

on

tro

l)

c (FGF-2) in ng/mL






secretion of FGFs in the most inhibitor-sensitive cell lines,

H1581 and DMS114 ( Fig. 2B ).

We therefore sought to determine formally whether secre-

tion of extracellular ligands was required for the viability of

FGFR-dependent cells (Supplementary Fig. S7). To this end,

we ectopically expressed FGFR1 mutants, which have reduced

affi nity to FGF ligands ( Fig. 2C , extracellular domain; ref. 31 ) in

cis with the V472M “gatekeeper” mutation that induces resist-

ance to FGFR inhibition ( 6 ). As expected, ectopic expression of

the V472M gatekeeper mutation alone resulted in resistance

to FGFR inhibition in H1581 cells ( Fig. 2D ). We next cloned

the gatekeeper mutant V472M together with the extracellular

FGFR1β mutant R161Q ( Fig. 2C and D ), which is analogous to

the R251Q mutation of FGFR2. This mutation strongly inter-

feres with ligand binding to FGFR2 ( 31 ). We further predicted

fi ve other extracellular mutation sites in the Inter–IgG2–IgG3

domain (ref. 32 ; L76T, A78L, K83E, D157N, D193N) to poten-

tially interfere with ligand binding ( Fig. 2D ). The combination

of the gatekeeper mutant V472M with any of the extracellular

mutants K83E, D157N, and D193N did not alter the resist-

ance to inhibitor treatment ( Fig. 2D ). In contrast, expression of

either L76T or R161Q abrogated the resistance induced by the

V472M mutation expressed in cis . However, increasing doses

of recombinant FGF-2 restored inhibitor resistance induced

by V472M in these cells ( Fig. 2D ). Thus, exogenously added

ligands overcome the reduction in ligand affi nity induced by

L76T or R161Q. As a consequence, ERK signaling of the dou-

ble mutant was reduced by FGFR inhibition and restored by

high doses of FGF-2 (25 ng/mL, Fig. 2E ; ref. 31 ). As expected,

under FGFR inhibition by PD173074, FGF-2 was insuffi cient

to reactivate ERK signaling in the case of wild-type FGFR1β

( Fig. 2E ). These experiments support the notion that autocrine

FGFR activation is required for survival of H1581 cells that

exhibit focal amplifi cation of FGFR1 .

Finally, we tested whether ligand dependency of FGFR1 -

amplifi ed cells was also relevant for tumor formation in vivo . To

this end, we applied adenoviruses expressing a soluble version

of the extracellular domain of FGFR fused to the immunoglob-

ulin heavy chain (AdsFGFR; ref. 33 ) that competes with cellular

FGFRs for soluble ligand ( 34 ). Subcutaneous injection of these

viruses inhibited tumor formation in H1581 xenografts ( Fig. 2F

and G ), but not the development of KRAS -mutant A549 tumors

( Fig. 2F and G ). Treatment with AdsFGFR, but not empty-

vector virus, delayed a gain in weight in both the mice bearing

H1581 ( P = 0.04) and A549 ( P = 0.012) tumors, thus suggesting

that FGF signaling is required for body weight maintenance

( 35 ). Effi cacy of viral infection was confi rmed by adenovirus-

specifi c PCR from fi xed liver tissue of all treatment groups.

In summary, we provide evidence that FGFR1 -amplifi ed

and inhibitor-sensitive H1581 cells depend on ligand binding

in vitro and in vivo .

Cell-Autonomous Transformation by FGFR1 and MYC

As a next step, we sought to test whether wild-type FGFR1

was oncogenic when overexpressed and analyzed the onco-

genic phenotype of NIH3T3 cells ectopically expressing

FGFR1 in soft agar assays. Whole-transcriptome sequenc-

ing (RNAseq) of six primary FGFR1- amplifi ed squamous cell

lung cancer tumors as well as four amplifi ed cancer cell lines

(Supplementary Fig. S8A) revealed that mesenchymal splice

variants of FGFR1 were predominantly expressed in the FGFR

inhibitor-sensitive cell lines. We therefore cloned these splice

variants (FGFR1-IIIc-α, FGFR1-IIIc-β) from H1581 cells and

transduced NIH3T3 cells with these variants of FGFR1 either

alone or together with six additional genes ( REL , SOX2 , MYC ,

CCND1 , DYRK1B , AKT2 ) with a possible role in squamous cell

lung cancer biology ( Fig. 3A and Supplementary Fig. S9). The

latter genes are located in or close to recurrent amplicons in

this lung tumor subtype. Both FGFR1 variants reproducibly

induced mild transformation of NIH3T3 cells to anchorage-

independent growth ( q = 8 × 10 −9 ; Fig. 3A and B ). In our

hands NIH3T3 cells did not survive transduction with MYC

alone. However, transduction of NIH3T3 cells with MYC and

FGFR1 ( q = 2 × 10 −5 ) was strongly oncogenic as determined by

the number and size of colonies in soft agar ( Fig. 3B ).

Similar to FGFR-dependent H1581 cells ( 6 ), treatment

with the FGFR inhibitor PD173074 induced apoptosis in

these FGFR1 – MYC cotransduced NIH3T3 cells, but not

in cells expressing FGFR1 alone ( Fig. 3C ). Thus, FGFR1 -

amplifi ed cells coexpressing MYC may be more susceptible

to FGFR inhibition, which has been similarly reported for

FGFR2- mutant breast cancer ( 36 ).

Injection of NIH3T3 FGFR1-IIIc-α and -β cells into nude

mice led to palpable subcutaneous tumors after a median

of 20 days ( Fig. 3D , top, and Supplementary Fig. S10A).

HEK293 cells, transduced with FGFR1 , similarly induced

subcutaneous tumors in vivo (Supplementary Fig. S10B), and

intravenous injection of NIH3T3 FGFR1α cells led to tumor

growth in the lungs (data not shown). Treatment with the

FGFR inhibitor BGJ398 (15 mg/kg, q.d.) repressed tumor

growth of NIH3T3 cells expressing either of the mesenchymal

FGFR1 splice variants ( Fig. 3D and Supplementary Fig. S10A).

Thus, the catalytic activity of FGFR1 was required for tumor

formation in vivo . However, FGFR inhibition by BGJ398 did

not induce tumor shrinkage in tumors expressing FGFR1

alone. In contrast, this treatment led to regressions of tumors

coexpressing FGFR1 and MYC ( Fig. 3D ; P < 0.001).

Of note, the tumors expressing FGFR1 alone also exhib-

ited low nuclear expression levels of MYC ( Fig. 3E ). However,

MYC was expressed at much higher nuclear levels in the

double-transduced cells, which was subject to FGFR-depend-

ent regulation ( Fig. 3E and Supplementary Fig. S11). Thus,

FGFR1-expressing tumors upregulate MYC in vivo , but only

very high levels of MYC expression are likely to govern sus-

ceptibility to FGFR inhibition.

FGFR1 Dependency and MYC Expression Supporting the notion that MYC may interplay with FGFR1

signaling, we found it to be strongly regulated by FGFR1 in

the FGFR-dependent cell lines H1581 and DMS114. Accord-

ingly, levels of MYC and of cyclin D1 decreased upon FGFR

inhibition within 24 hours ( Fig. 4A ). In contrast, expression lev-

els remained relatively stable in FGFR1 -amplifi ed HCC95 and

H520 cells, which are resistant to FGFR inhibition ( 6 ), as well as

in the NRAS-mutant HCC15 cells ( Fig. 4A and Supplementary

Fig. S10C). MYC was also highly regulated on the transcrip-

tional level in H1581, but not in H520 cells (data not shown).

To formally test whether MYC expression levels dictate sen-

sitivity to FGFR inhibition, we stably silenced MYC in H1581






Figure 3. Overexpression of FGFR1 induces oncogenic transformation, which is enhanced by expression of MYC . A, NIH3T3 cells were retrovirally (pBabe) (co)-transduced with FGFR1 and eight further cancer genes. Colony formation in a 21-day soft agar assay was compared with empty vector con-trols by the Benjamini–Hochberg corrected t test and classifi ed into strong (++), mild (+; <10 colonies per well), and no (0) transformation. NIH3T3 cells did not survive transduction with MYC alone (X). *, the Benjamini–Hochberg correction is not signifi cant. B, protein expression and phosphorylation of transduced cells were analyzed by immunoblotting (top). Mesenchymal FGFR1α (full length) could be differentiated from FGFR1β by protein size. Rela-tive colony counts of a 21-day soft agar assay were compared by the Benjamini–Hochberg corrected t test (bottom). Error bars display SD of average counts of three independent experiments. C, Induction of apoptosis (Annexin-V/PI, fl ow cytometry) in NIH3T3 cells, (co-) transduced with FGFR1β ± MYC, by 72-hour FGFR inhibition (PD173074, 1 μmol/L). FGFR-dependent H1581 cells (PD173074, 1 μmol/L) as well as ALK-dependent NIH3T3–EML4–ALK cells (TAE684, 1 μmol/L) were used as positive controls. Resistant HCC15 and NIH3T3-e.V. cells served as negative controls. *, Signifi cant induction of apoptosis. D, nude mice, engrafted with retrovirally transduced NIH3T3 cells, received BGJ398 (15 mg/kg, q.d., lower curve) or 5% glucose (upper curve), respectively, upon formation of palpable tumors. Volumes of tumors formed by NIH3T3–FGFR1β cells (top) and NIH3T3–FGFR1β–MYC cells (bottom) were assessed every second day and compared by the t test. Error bars display SD of three independent experiments. E, representative immunohisto-chemical MYC stains (×40 magnifi cation) of subcutaneous mouse tumors after 14-day FGFR inhibitory therapy (right) and vehicle (left). Highest nuclear expression levels are indicated.

q = 2 × 10–5

0.25

0.5

0.75

1

0N

orm

alized

co

lon

y c

ou

nt

(%)

q = 0.18 × 10–9

q = 8.1 × 10–9

q = 0.02

p-FGFR

EML4–ALK

t-FGFR1

MYC

Actin

1

10

100

1,000

10,000

0 2 4 6 8 10 12 14 16 18 20

Rel. t

um

or

vo

lum

es (

log

scale

%)

Duration of treatment (days)

Xenograft mouse model NIH3T3 FGFR1β

BGJ398 (15 mg/kg)

Vehicle

P = 0.11

1

10

100

1,000

10,000

0 2 4 6 8 10 12 14

Rel. t

um

or

vo

lum

es (

log

scale

%)

Duration of treatment (days)

BGJ398 (15 mg/kg)

Vehicle

P < 0.001

0.25

0.5

0

Ind

ucti

on

of

ap

op

tosis

(%

of

co

ntr

ol)

0.05

0.1

0.15

0.2

0.3

0.35

0.4

0.45

NIH3T3

e.V.

e.V.

EML4

–ALK

FGFR

1β

FGFR

1β + M

yc

HCC15

[NRAS

mut ]

H15

81 [F

GFR1am

p ]

TAE684, 1 µmol/L PD173074, 1 µmol/L

*

*

*

FGFR1

MYC (8p24)

CCND1 (11q13)

SOX2 (3q27)

DYRK1B (19q13)

AKT2 (19q13)

FGFR1 (8p12)

– +

+

0

0

X

0

0

0

KRASG12V

EML4–ALK

++

++

++

REL (2p15) +*

0

0

0

0

A B C

D E

FGFR1β

FGFR1β+ MYC

KRASG12V

EML4

–ALK

Score 2 Score 1

Score 3 Score 1

Score 0 Score 0

Score 0 Score 0

Vehicle Therapy

Xenograft mouse model NIH3T3 FGFR1β + MYC

EML4

–ALK e.

V.

FGFR

1α

FGFR

1β

FGFR

1α + M

yc

FGFR

1β + M

yc






Figure 4. MYC in FGFR signaling and inhibitor response. A, FGFR1 -amplifi ed H1581, DMS114, and HCC95 cells as well as HCC15 (NRAS mut ) controls were treated with PD173074 (1 μmol/L, 24 hours). Expression levels of MYC, cyclin D1, and actin as well as ERK phosphorylation were monitored by immunoblotting. Con.: positive control NIH3T3-FGFR1 β cells. B, protein expression of MYC was silenced by stable lentiviral transduction of FGFR-dependent H1581 cells as well as HCC15, H2882, and HCC95 controls. Knockdown effi ciency was validated by immunoblotting for H1581, H2882, and HCC15 cells (top). FGFR dependency was determined by measuring cellular ATP content after 96 hours (bottom). C, relative RNA expression levels of FGFR1-4 (black, blue, green, gray) and MYC (red) in a cohort of 14 cancer cell lines enriched for FGFR1 amplifi cation. Correlation of FGFR dependency and FGFR1 × MYC expression levels (inset). Signifi cance of correlation was derived from Student t distribution. D, segregation of FGFR1 amplifi ca-tion with RNA expression levels of MYC . Cancer cell lines were divided into an FGFR-dependent (H1581, DMS114, and HCC1599) GI 50 < 500 nmol/L, PD173074) versus resistant group (A427, H520, H1703, HCC15, H358, HCC95, H187, SW1271, H526, and DMS153 cells). Expression levels were compared by the Student t test. wt, wild-type.

0

0.25

0.5

0.75

1

1.25

Via

bilit

y (

% o

f co

ntr

ol)

c (PD173074) in μmol/L

3010510.50.10.050.010.0005

Empty

vector

H1581

shMYC

HCC15

HCC95

H2882

H1581

e.V.

MYC

Actin

H2882 HCC15

e.V.

e.V.

shM

yc

shM

yc

shM

yc

A

0

0.25

0.5

0.75

1

4.75

H1581DMS114

HCC1599A427

H1703H520 HCC15H358

HCC95H187

SW1271H526

DMS153

4.3 5.5 3.8 2 4.13.3 1.4 2.2 3.9 1.4 0.8 1.4 25.7

Cell lines

Exp

ressio

n (

% o

f co

ntr

ol)

FGFR1

Copy

number

GI50

values (PD173074)

FGFR4FGFR3FGFR2FGFR1c-MYC

10510.50.1

FG

FR

1 ×

c-m

yc

ex

pre

ssio

n

H1581

DMS114

r = –0.63

P = 0.02

GI50

values of PD173074 (μμmol/L)

SBC-7

B

C D

p-ERK

MYC

Cyclin D1

0 2 8 16 24

H1581 [FGFR1amp]

Hours:

PD173074, 1 μmol/L

0 2 8 16 24

DMS114 [FGFR1amp]

Con.

0 2 8 16 24

HCC95 [FGFR1amp]

Hours:

PD173074, 1 μmol/L

0 2 8 16 24

HCC15 [NRASmut]

Con.

Con.

Con.

p-ERK

MYC

Cyclin D1

Actin

Actin

P = 0.02

Sensitive Resistant

0.25

0.5

0n = 3 n = 10

No

rmalized

exp

ressio

n

(% o

f co

ntr

ol) FGFR1

c-MYC

3 3

70

amp

wt

sens res

FG

FR

1

PD1730741

0.75

0.5

0.25

0

cells. This manipulation led to FGFR inhibitor resistance

( Fig. 4B ). Unfortunately, we could not test this hypothesis in

DMS114 cells because they did not tolerate MYC knockdown.

We next examined the regulation of downstream effectors in

MYC signaling and found that the mitochondrial apoptosis

mediators were predominantly affected by FGFR inhibition

(PD173074, 1 μmol/L; Supplementary Fig. S12A); loss of the

mitochondrial membrane potential as well as cytochrome C

release occurred robustly after 72 hours in FGFR-dependent

cell lines (Supplementary Fig. S12B).

Further analysis of RNAseq data revealed that tumor sam-

ples, in which the amplicon centered on FGFR1 , expressed

higher levels of MYC ( P = 0.002) compared with other 8p12-

amplifi ed samples (Supplementary Fig. S8B). However, we

were not able to detect a statistically signifi cant co-occurrence

of amplifi ed 8p12 and MYC (Supplementary Fig. S3). There-

fore, we analyzed the transcription levels of MYC in our cell

line panel ( n = 14 ). Levels of MYC gene expression predicted

FGFR inhibitor sensitivity in individual 8p12-amplifi ed cell

lines ( P = 0.02; Fig. 4C ) as well as in groups of sensitive versus

insensitive cell lines ( Fig. 4D ).

Altogether, we used cell culture and xenograft experi-

ments ( Fig. 3B and D ) to study the interplay of FGFR1 with

MYC. In all independent approaches, we observed that MYC






Figure 5. MYC expression in primary FGFR1 -amplifi ed squamous cell lung carcino-mas. A, enrichment of FGFR1 phosphorylation, independence of MYC expression in a cohort of 86 FGFR1 -amplifi ed lung cancer patients. Tumor biopsies were analyzed by FGFR1 FISH and stained for MYC expression as well as FGFR1 phosphorylation. Frequencies of positive stains were compared by the Fisher exact test. B, pathologic examination of a squamous cell tumor biopsy of the BGJ398 responder [BGJ398 trial ( 10 )]. The sample was scored (degrees 0–3) by FGFR1 dual-color FISH (top, normalized copy-number ratio) as well as MYC IHC (bottom, nuclear staining intensity). C, fused scans of positron emission tomography (PET) and compu-ter tomography (CT) before (top left, baseline) and after begin of BGJ389 therapy (top right, 4 weeks). Baseline CT scan (bottom left); CT after 8 weeks (bottom right) of BGJ398 therapy, showing tumor regression. Target lesions for evaluation of tumor response are highlighted by red arrows. IHC, immunohistochemistry.

A B

P = 0.0008

pFGFR1 IHC-positive

c-MYC IHC-positive

Patient samples (n = 86)

P = 0.76

C

FGFR1 FISH:

2.6 Ratio

88% 5 < gene copies

MYC IHC score:

3 of 3

Baseline

After 8 weeks, BGJ398

After 4 weeks, BGJ398

Baseline

Fre

quency (

%)

FISH

positive

FGFR1 FISH

negative

FGFR1

1

0.75

0.5

0.25

0

modulates oncogenic transformation, cell-autonomous sig-

naling, and FGFR inhibitor response in FGFR1 -amplifi ed or

overexpressing cells ( Fig. 4 ).

Prevalence of MYC Expression in Primary FGFR1 -Amplifi ed Lung Tumors

To extrapolate our fi nding that MYC expression levels

dictate FGFR inhibitor sensitivity of FGFR1 -amplifi ed lung

cancer to a larger panel of primary tumors, we screened a

cohort of 306 squamous cell lung cancer biopsies for the

presence of FGFR1 amplifi cation by FISH ( 37 ). In this cohort

8p12 amplifi cation occurred at a frequency of approximately

20%. A subcohort ( n = 86) enriched for FGFR1 amplifi cation

(78%) was further analyzed for p-FGFR1 and MYC expression

by immunohistochemistry using a 4-tier scale by three inde-

pendent observers ( Fig. 5A and Supplementary Fig. S13). We

found strong membranous p-FGFR1 staining in this cohort

(Supplementary Fig. S13). Only 26% of the amplifi ed samples

exhibited low scores of FGFR1 phosphorylation ( Fig. 5A and

Supplementary Fig. S13A). In contrast, high levels of nuclear

MYC staining did not segregate with amplifi cation status of

FGFR1 (frequency 40% in FGFR1 amp vs. 46% in FGFR1 non-amp ;

P = 0.76; Fig. 5A and Supplementary Fig. S13A). Thus,

whereas most FGFR1 -amplifi ed squamous cell lung cancers

exhibited FGFR1 phosphorylation, only a fraction of these

cases also showed nuclear MYC expression. The fi nding that

only a minority of FGFR1 -amplifi ed lung tumors are likely to

respond to FGFR inhibition ( 10, 11 ) is consistent with the

possibility that MYC expression predicts FGFR dependency

in this cohort.

We identifi ed a 65-year-old caucasian man with a 70-pack-

per-year smoking history. The patient was diagnosed with

stage IV squamous cell lung cancer and had been initially

treated with two chemotherapy lines (a combination of car-

boplatinum and paclitaxel and docetaxel monotherapy). We

observed amplifi cation of FGFR1 (2.6 ratio-signals per cell on






average, plus 88% of the cells harbored 5 or more gene cop-

ies) in the patient’s tumor ( Fig. 5B ). Immunohistochemical

assessment revealed elevated expression levels of MYC with a

score of 3 ( Fig. 5B and Supplementary Fig. S13B). The patient

agreed to treatment with BGJ398, a highly specifi c FGFR

inhibitor ( 38 ), which was being evaluated in a fi rst-in-humans

trial at our center. After cardiac assessment and baseline

thoracic computed tomography (CT; ref. 10 ), treatment with

100 mg BGJ398 was started. We observed a regression with-

out cavitation of the tumor [CT scans after 4 and 8 weeks,

partial response (PR) according to RECIST 1.1 criteria] and

the patient experienced improvement of symptoms ( Fig. 5C ).

After 10 months of therapy, progressive disease (PD) was

diagnosed in the kidney (PD as to RECIST1.1 criteria), so that

BGJ398 treatment was stopped.

Another patient was diagnosed with metastatic squamous

cell lung cancer and high-level amplifi cation of FGFR1 (10.1

signals per cells on average) and high expression of MYC (Sup-

plementary Fig. S14). The FGFR1 amplifi cation was highly

focal, as determined by hybrid-capture–based massively parallel

sequencing of 302 genes, enriched for the chromosomal region

covering the 8p12 amplicon (Supplementary Fig. S14A). The

patient refused chemotherapy, but consented to off-label use of

pazopanib, a multikinase inhibitor with weak activity against

FGFR ( 39, 40 ). After cardiac assessment and baseline thoracic

CT (Supplementary Fig. S14B), treatment with pazopanib 400

mg b.i.d. was started. Four weeks and eight weeks after the start

of pazopanib, CT showed tumor regression with cavitation

(Fig. S14b). Because of grade 2 fatigue, stomatitis, and gastroin-

testinal side effects, the patient decided to stop pazopanib after

6 months. At that time, no clinical or radiologic signs of tumor

progression were present. We note that the inhibitory profi le

of pazopanib and the pseudocavernous response are also com-

patible with a predominant antiangiogenic effect. However, in

light of our preclinical fi ndings, we speculate that the patient’s

response might also be attributable to FGFR inhibition in the

context of an MYC-expressing, FGFR1 -amplifi ed lung cancer.

DISCUSSION Preliminary results from an early FGFR inhibitor trial as

well as our conclusions from cell culture experiments suggest

that not all patients with FGFR1 amplifi cation will benefi t

from FGFR inhibition ( 6 , 10 , 11 , 41 ). Here, we show marked

heterogeneity of the 8p12 amplifi cation event in squamous

cell lung cancer. This heterogeneity was also evident when

assessing mechanisms of receptor activation and signaling

modulation. We report that the 8p12 amplifi cation results

from broad genomic rearrangements, such that FGFR1 is

located in the epicenter of the amplicon in only 28% of all

8p12-amplifi ed cases. Furthermore, although FGFR1 phos-

phorylation was present in 74% of FGFR1 -amplifi ed primary

lung tumors and is therefore unlikely to be predictive of the

much more infrequent sensitivity to inhibition alone, high

expression levels of MYC associated with FGFR dependency

across different cell line models. Supporting an oncogenic

role for amplifi ed FGFR1 , we show that the mesenchymal

splice variants that we found to be expressed by amplifi ed

tumors are transforming in vitro and in vivo . This effect was

strongly enhanced by coexpression of MYC. The role of high-

level expression of MYC in mediating FGFR dependency was

further strengthened by clinical observations of two patients

with amplifi ed FGFR1 and high MYC-expressing squamous

cell lung cancer who responded to FGFR inhibition. Even

though an FGFR-independent mechanism of response could

not be ruled out, these clinical observations were in line with

our preclinical observations. Beyond these cell-autonomous

mechanisms of activation and modulation of drug response,

we provide support for a role of autocrine and/or paracrine

ligand-dependent receptor activation in modulating sensitiv-

ity to FGFR inhibition, which may be of potential therapeutic

relevance.

In conclusion, we reveal that high expression of MYC

as well as FGF ligand concentrations modulate oncogenic

transformation and response to FGFR inhibition in FGFR1 -

amplifi ed lung cancer. We hope that our fi ndings may help

in refi ning the selection of patients who are most likely to

benefi t from treatment with FGFR inhibitors.

METHODS Cell Lines

Cancer cell lines, HEK293T and NIH3T3 cells were purchased

from American Type Culture Collection and German Resource Cen-

tre for Biological Material (DSMZ) and cultured using either RPMI

or Dulbecco’s Modifi ed Eagle Medium (DMEM) high-glucose media,

supplemented with 10% fetal calf serum (FCS). Adherent cells were

routinely passaged by washing with PBS buffer and subsequent

incubation in Trypsin/EDTA. Trypsin was inactivated by the addi-

tion of culture medium and cells were plated or diluted accordingly.

Suspension cell lines were passaged by suitable dilution of the cell

suspension. All cells were cultured at 37°C and 5% CO 2 . The identity

of all cell lines included in this study was authenticated by genotyp-

ing (SNP 6.0 arrays, Affymetrix) and all cell lines are tested for infec-

tion with mycoplasma (MycoAlert, Lonza). Furthermore, the identity

of the H1581 cell line was ensured by short tandem repeat profi ling

(DNA fi ngerprinting).

Cell Line Stimulation Cell lines were starved from bovine serum for 24 hours and stimu-

lated by a collection of 6 FGF ligands (1 ng/mL) and heparin (10 μg/

mL) for 20 minutes. In addition, the FGFR inhibitor PD173074

(1 μmol/L) was added 40 minutes before stimulation by FGF-1 and

FGF-2. Phosphorylation of FGFR, ERK, AKT, and the FGFR1 signal-

ing adapter protein FRS2α as well as total expression of ERK and

FGFR1 were assessed by immunoblotting.

Whole Transcriptome Sequencing (RNAseq) Total RNA was extracted from fresh-frozen lung tumor tissue

containing at least 60% tumor cells. Depending on the tissue size,

15–30 slides were cut using a cryostat (Leica) at −20°C. Material for

RNA extraction was disrupted and homogenized for 2 minutes at 20

Hz by Tissue Lyser (Qiagen). RNA was extracted using the Qiagen

RNeasy Mini Kit. RNA quality was assessed by a Bioanalyzer; sam-

ples showing an RNA integrity number (RIN) > 8 were retained for

transcriptome sequencing. We cloned cDNA strands of 250 bp into

a sequencing library, allowing us to sequence 95-bp paired-end reads

without overlap. All RNAseq libraries were analyzed on the Illumina

Genome Analyzer IIx.

Gene coverage was used to differentiate splice variants of FGFR1 .

Mesenchymal splice variants of FGFR1 were differentiated by cover-

age of exon 2, whereas coverage of tissue-specifi c exons 8 (IIIb/IIIc)

distinguished epithelial (IIIb) from mesenchymal (IIIc) forms.






Xenograft Mouse Models All animal procedures were approved by the local animal protec-

tion committee and the local authorities. Transduced NIH3T3 and

tumor cells were resuspended in RPMI or DMEM medium and

injected (5 × 10 6 cells per tumor) subcutaneously into the fl anks

of 8- to 15-week-old male nude mice [Rj:NMRI-nu (nu/nu), Janvier

Europe] under 2.5% isofl urane anesthesia ( 42 ).

To assess the effect of FGFR inhibitors in vivo , NVP-BGJ 398

(Novartis) was dissolved in a vehicle solution (33% PEG300, 5% glu-

cose) for xenograft application ( 6 , 38 ). Tumor size was monitored

every second day by measurement of perpendicular diameters by an

external caliper ( 42 ) and calculated by use of the modifi ed ellipsoid

formula [V = 1/2 (Length × Width 2 )]. Oral therapy was started when

tumors reached a volume of 100 mm 3 . Mice received daily either

BGJ398 (15 mg/kg) or vehicle solution. After 14 (NIH3T3 FGFR1β

+ MYC), 16 (NIH3T3 EML4–ALK, KRAS G12V), or 25 (NIH3T3 e.V.,

FGFR1α/β) days of therapy, respectively, mice were sacrifi ced by intra-

peritoneal injection of ketamine/xylazine (300/60 mg/kg).

To examine ligand dependency in vivo , AdCMV-null virus (Vector

Biolabs) and AdsFGFR virus (titer: 1 × 10 10 , contributed as a kind gift

by Gerhard Christofori, University of Basel) were mixed with tumor

cells in DMEM for subcutaneous injection. Tumor formation was

monitored twice a week by careful visual inspection and palpation of

the skin. As soon as tumors became palpable, diameters were meas-

ured by an external caliper to determine tumor volumes. In addition,

animal weights were documented weekly. Eight weeks after injection

of H1581 and A549 tumor cells, animals were sacrifi ced.

Subcutaneous tumors as well as livers were resected and fi xed in

4% formaldehyde for immunohistochemical staining and virus detec-

tion, respectively.

ELISA Assay Cell culture supernatants were collected, centrifuged (200 rcf, 5

minutes), concentrated by ultracentrifugation units (Satorius AG)

and analyzed for FGF concentrations by ELISA (Abcam). In addi-

tion, protein was extracted from cells, collected in equal amounts of

lysis buffer (Cell Signaling Technology), and measured by Bradford

assay (Pierce). Normalized FGF concentrations (c Norm ) were derived as

ratios of FGF and lysate protein concentrations.

For further details, refer to Supplementary Methods.

Disclosure of Potential Confl icts of Interest F. Malchers is a consultant/advisory board member of Blackfi eld

AG. L. Nogova has received honoraria from the speakers’ bureaus

of Novartis and Roche and is a consultant/advisory board member

of Amgen, Lilly, and Bayer. J.M. Heuckmann has ownership interest

(including patents) and is a co-founder and shareholder in Black-

fi eld AG. J. Diebold is a consultant/advisory board member of Roche

and Pfi zer. M. Scheffl er is a consultant/advisory board member of

Boehringer Ingelheim. M. Peifer has ownership interest (including

patents) and is a founder and shareholder of Blackfi eld AG, and is

a consultant/advisory board member of the same. T. Zander has

received honoraria from the speakers’ bureaus of Amgen, Roche,

Novartis, and Boehringer Ingelheim and is a consultant/advisory

board member of Amgen. F. Ringeisen is employed as a Senior Medi-

cal Director, Clinical Research, in Novartis Pharma and has owner-

ship interest (including patents) in the same. J. Wolf has received

honoraria from the speakers’ bureau of Novartis and is a consult-

ant/advisory board member of the same. R.K. Thomas has received

commercial research grants from AstraZeneca, Merck, and EOS, has

ownership interest (including patents) in Blackfi eld AG, and is a

consultant/advisory board member of JNJ, Blackfi eld, AstraZeneca,

Roche, Lilly, Sanofi , and Merck. No potential confl icts of interest

were disclosed by the other authors.

Authors’ Contributions Conception and design: F. Malchers, F. Dietlein, P.S. Mainkar,

S. Chandrasekhar, D. Rauh, R. Buttner, J. Wolf, R.K. Thomas

Development of methodology: F. Malchers, F. Dietlein, J.M.

Heuckmann, M. Peifer, A. Florin, N. Karre, D. Rauh, R. Buttner, L.C.

Heukamp, R.K. Thomas

Acquisition of data (provided animals, acquired and managed

patients, provided facilities, etc.): F. Malchers, F. Dietlein, J. Schöttle,

L. Nogova, K. Albus, J.M. Heuckmann, O. Gautschi, J. Diebold,

D. Plenker, M. Gardizi, M. Scheffl er, M. Bos, D. Seidel, S. Chandrasekhar,

J. George, S. Silling, T. Zander, R. Ullrich, H.C. Reinhardt, R. Buttner,

L.C. Heukamp, J. Wolf, R.K. Thomas

Analysis and interpretation of data (e.g., statistical analysis,

biostatistics, computational analysis): F. Malchers, F. Dietlein,

J. Schöttle, X. Lu, L. Nogova, J.M. Heuckmann, M. Bos, F. Leenders,

M. Peifer, P.S. Mainkar, N. Karre, S. Chandrasekhar, D. Rauh, R. Ullrich,

R. Buttner, J. Wolf, R.K. Thomas

Writing, review, and/or revision of the manuscript: F. Malchers,

F. Dietlein, L. Nogova, O. Gautschi, J. Diebold, M. Scheffl er, M. Bos,

J. George, R. Ullrich, H.C. Reinhardt, F. Ringeisen, R. Buttner,

L.C. Heukamp, J. Wolf, R.K. Thomas

Administrative, technical, or material support (i.e., reporting or

organizing data, constructing databases): F. Dietlein, L. Fernandez-

Cuesta, D. Seidel, A. Richters, A. Florin, S. Chandrasekhar, S. Silling,

H.C. Reinhardt, R. Buttner, R.K. Thomas

Study supervision: F. Malchers, F. Dietlein, S. Chandrasekhar,

D. Rauh, J. Wolf, R.K. Thomas

Acknowledgments The authors thank Will Morrel for invaluable technical help and

Gerhard Christofori for providing the AdsFGFR virus. They also

thank Dr. Klaus Strobel and Dr. Peter Hofman for CT images and

photograpy.

Grant Support This work was co-funded by the German federal state North Rhine

Westphalia (NRW) and the European Union (European Regional Devel-

opment Fund: Investing In Your Future) as part of the PerMed NRW

initiative (grant 005-1111-0025; to R.K. Thomas and J. Wolf). This

work was also supported by the EU-Framework Program CURELUNG

(HEALTH-F2-2010-258677; to R.K. Thomas and J. Wolf), by the Deut-

sche Forschungsgemeinschaft through TH1386/3-1 (to R.K. Thomas)

and through SFB832 (TP6 to R.K. Thomas; TP5 to L.C. Heukamp),

by the German Ministry of Science and Education (BMBF) as part

of the NGFNplus program (grant 01GS08100; to R.K. Thomas and

J. Wolf), by the Deutsche Krebshilfe as part of the Oncology Centers

of Excellence funding program (to R. Buttner and R.K. Thomas) and

through the Mildred-Scheel-Doktorandenprogramm (grant 110770;

to F. Dietlein and R.K. Thomas), by the Max Planck Society, by the

Behrens-Weise Foundation (M.I.F.A.NEUR8061; to R.K. Thomas), and

by an anonymous foundation (to R.K. Thomas). R.K. Thomas is sup-

ported by a Stand Up To Cancer Innovative Research Grant, a Program

of the Entertainment Industry Foundation (SU2C-AACR-IR60109).

Received June 27, 2013; revised November 22, 2013; accepted

November 25, 2013; published OnlineFirst December 3, 2013.

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2014;4:246-257. Published OnlineFirst December 3, 2013.Cancer Discovery Florian Malchers, Felix Dietlein, Jakob Schöttle, et al.

in Lung CancerFGFR1Transformation by Amplified Cell-Autonomous Mechanisms of−Cell-Autonomous and Non

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