www.sciencetranslationalmedicine.org/cgi/content/full/2/62/62ra93/DC1

Supplementary Materials for

Frequent and Focal FGFR1 Amplification Associates with Therapeutically Tractable FGFR1 Dependency in Squamous Cell

Lung Cancer

Jonathan Weiss, Martin L. Sos,* Danila Seidel, Martin Peifer, Thomas Zander, Johannes M. Heuckmann, Roland T. Ullrich, Roopika Menon, Sebastian Maier, Alex Soltermann, Holger Moch, Patrick Wagener, Florian Fischer, Stefanie Heynck, Mirjam Koker, Jakob

Schöttle, Frauke Leenders, Franziska Gabler, Ines Dabow, Silvia Querings, Lukas C. Heukamp, Hyatt Balke-Want, Sascha Ansén, Daniel Rauh, Ingelore Baessmann, Janine

Altmüller, Zoe Wainer, Matthew Conron, Gavin Wright, Prudence Russell, Ben Solomon, Elisabeth Brambilla, Christian Brambilla, Philippe Lorimier, Steinar Sollberg,

Odd Terje Brustugun, Walburga Engel-Riedel, Corinna Ludwig, Iver Petersen, Jörg Sänger, Joachim Clement, Harry Groen, Wim Timens, Hannie Sietsma, Erik Thunnissen, Egbert Smit, Daniëlle Heideman, Federico Cappuzzo, Claudia Ligorio, Stefania Damiani,

Michael Hallek, Rameen Beroukhim, William Pao, Bert Klebl, Matthias Baumann, Reinhard Buettner, Karen Ernestus, Erich Stoelben, Jürgen Wolf, Peter Nürnberg, Sven

Perner, Roman K. Thomas*

*To whom correspondence should be addressed. E-mail: martin.sos@nf.mpg.de (M.L.S.); nini@nf.mpg.de (R.K.T.)

Published 15 December 2010, Sci. Transl. Med. 2, 62ra93 (2010)

DOI: 10.1126/scitranslmed.3001451

The PDF file includes:

Methods Fig. S1. Significant deletions are observed in squamous cell lung cancer. Fig. S2. FGFR1 amplification has no significant impact on overall survival of SQLC patients. Fig. S3. FGFR1 amplification correlates with FGFR1 protein expression. Fig. S4. Expression of FGFR ligands does not correlate with FGFR1 amplification status. Fig. S5. PD173074 binds inside the ATP-binding pocket of FGFR1. Fig. S6. Knockdown of genes adjacent to FGFR1 on 8p12 does not affect cell viability. Fig. S7. PD173074 is not active in the PDGFRA- and FGFR1-amplified cell line H1703. Fig. S8. PD173074 shows antitumor activity in vivo.

Table S1. Significant amplifications and deletions are noted in a subset of 155 SQLC samples. Table S2. Clinical features and co-occurrent mutations of FGFR1-amplified SQLC samples. Table S3. Significant amplifications and deletions are noted in a subset of 77 adenocarcinoma samples. Table S4. FGFR1 amplification is detected using FISH on tumor microarrays. Table S5. GI50 values are not associated with mutation status across the lung cancer cell line panel. Table S6. PD173074 induces apoptosis in FGFR1-amplified cell lines. Table S7. PD173074 has specific activity against two kinases. Table S8. FGFR1 and SOX2 amplification in squamous cell lung carcinoma. Table S9. Sequences of all shRNA constructs that were used in the study. References

Frequent and Focal FGFR1 Amplification Associates with Therapeutically Tractable FGFR1

Dependency in Squamous-cell Lung Cancer.

Weiss J. et al.

Correspondence:

Martin L. Sos: martin.sos@nf.mpg.de

Roman K. Thomas: nini@nf.mpg.de

Supplementary Methods

Immunohistochemical staining

Mice bearing FGFR1-amplified tumors were sacrificed at the end of treatment. Tumors were exgrafted

and embedded in paraffin. H&E and immunohistochemistry stainings were performed according to

standard protocols (1). Antibodies recognizing pAKT, pERK, AKT and ERK were obtained from Cell

signaling.

Structural model of compound binding

Amino acid side chains of gatekeeper mutant alleles of FGFR1 were modelled mith MOE (package

version 2009.10, Molecular Operating Environment) based on the crystal of FGFR1 in complex with

PD173074 (pdb code 2FGI) (2). Figures were prepared with PyMol software (DeLano Scientific

LLC).

Pharmacokinetics of PD173074

Pharmacokinetic data were created by taking blood-samples from male nude mice by puncture of the

tail-vein under isoflurane anesthesia. Blood samples of 50μl each were drawn and mixed with 5μl

heparin (5000I.U./ml), 0.5, 1, 1.5, 2, 3, 4, 5, 6, 12, 14, 16, 18, 20, 22 and 24 hours after a single dose

of either 100 or 50mg/kg PD173074. All blood samples were centrifuged; the blood plasma-

containing supernatant was extracted and stored at -20°C. Analytic samples were prepared from the

blood plasma by acetonitrile extraction with an internal standard of 1μM griseofulvin. PD173074

concentrations were determined using HPLC (Shimadzu-A20) and tandem mass spectrometry (4000

Q-Trab LC-MS/MS, AB SCIEX) with quantification limits of 63.34 ng/ml. Pharmacokinetic data was

evaluated with WinNonlin Software (Pharsight).

Immunoprecipitation

For the detection of FGFR1 phosphorylation, whole-cell lysates (0.5-1 mg) in NP40 lysis buffer was

incubated with agarose A/G Plus preconjugated with the FGFR1 antibody. Immunoprecipitates were

washed in NP40 lysis buffer, boiled in sample buffer, and subjected to SDS-PAGE followed by

immunoblotting using an anti phospho-tyrosine residues antibody to detect specific phosphorylation of

FGFR1.

Enzyme linked immunosorbent assay (ELISA)

Cells were seeded in 6-well dishes, 24h prior to the experiment. Cells were rinsed with PBS and

incubated for 24h with serum free cell culture media. Supernatants were harvested and a FGF2 ELISA

(Raybiotech) was performed according to manufactures protocol. Absorbance was measured on a

Mirthas LB940 (Berthold Technologies).

References and Notes

1. M. L. Sos, S. Fischer, R. Ullrich, M. Peifer, J. M. Heuckmann, M. Koker, S. Heynck,

I. Stuckrath, J. Weiss, F. Fischer, K. Michel, A. Goel, L. Regales, K. A. Politi, S. Perera, M. Getlik, L. C. Heukamp, S. Ansen, T. Zander, R. Beroukhim, H. Kashkar, K. M. Shokat, W. R. Sellers, D. Rauh, C. Orr, K. P. Hoeflich, L. Friedman, K. K. Wong, W. Pao, R. K. Thomas, Identifying genotype-dependent efficacy of single and combined PI3K- and MAPK-pathway inhibition in cancer. Proc Natl Acad Sci U S A 106, 18351 (2009).

2. M. Mohammadi, S. Froum, J. M. Hamby, M. C. Schroeder, R. L. Panek, G. H. Lu, A. V. Eliseenkova, D. Green, J. Schlessinger, S. R. Hubbard, Crystal structure of an angiogenesis inhibitor bound to the FGF receptor tyrosine kinase domain. EMBO J 17, 5896 (1998).

3. R. Beroukhim, C. H. Mermel, D. Porter, G. Wei, S. Raychaudhuri, J. Donovan, J. Barretina, J. S. Boehm, J. Dobson, M. Urashima, K. T. Mc Henry, R. M. Pinchback, A. H. Ligon, Y. J. Cho, L. Haery, H. Greulich, M. Reich, W. Winckler, M. S. Lawrence, B. A. Weir, K. E. Tanaka, D. Y. Chiang, A. J. Bass, A. Loo, C. Hoffman, J. Prensner, T. Liefeld, Q. Gao, D. Yecies, S. Signoretti, E. Maher, F. J. Kaye, H. Sasaki, J. E. Tepper, J. A. Fletcher, J. Tabernero, J. Baselga, M. S. Tsao, F. Demichelis, M. A. Rubin, P. A. Janne, M. J. Daly, C. Nucera, R. L. Levine, B. L. Ebert, S. Gabriel, A. K. Rustgi, C. R. Antonescu, M. Ladanyi, A. Letai, L. A. Garraway, M. Loda, D. G. Beer, L. D. True, A. Okamoto, S. L. Pomeroy, S. Singer, T. R. Golub, E. S. Lander, G. Getz, W. R. Sellers, M. Meyerson, The landscape of somatic copy-number alteration across human cancers. Nature 463, 899 (2009).

4. R. Beroukhim, G. Getz, L. Nghiemphu, J. Barretina, T. Hsueh, D. Linhart, I. Vivanco, J. C. Lee, J. H. Huang, S. Alexander, J. Du, T. Kau, R. K. Thomas, K. Shah, H. Soto, S. Perner, J. Prensner, R. M. Debiasi, F. Demichelis, C. Hatton, M. A. Rubin, L. A. Garraway, S. F. Nelson, L. Liau, P. S. Mischel, T. F. Cloughesy, M. Meyerson, T. A. Golub, E. S. Lander, I. K. Mellinghoff, W. R. Sellers, Assessing the significance of chromosomal aberrations in cancer: methodology and application to glioma. Proc Natl Acad Sci U S A 104, 20007 (2007).

5. http://www.kinase-screen.mrc.ac.uk. (2010).

Supplementary Figures

Supplementary Figure 1: Significant deletions are observed in squamous cell lung cancer.

Depicted are the significant (3, 4) (FDR-value; x-axis) deletions across all chromosomes (y-axis) in

squamous-cell lung cancer (n=155) as determined by GISTIC.

Supplementary Figure 2: FGFR1 amplification has no significant impact on overall survival of

SQLC patients.

Plotted are the Kaplan-Meier survival curves (y-axis: probability of overall survival; x-axis: time in

month) for FGFR1-amplified and FGFR1 wildtype SQLC patients. FGFR1 amplification was

assessed by FISH analysis as described in the method section of the manuscript. Only samples

showing high amplification of FGFR1 (copy number >9) were used for the analysis of the overall

survival of FGFR1 amplified samples.

Supplementary Figure 3: FGFR1 amplification correlates with FGFR1 protein expression.

Whole cell lysates from two FGFR1-amplified (indicated by “+”) and four FGFR1-wildtype

(indicated by “-“) cells were subjected to immunoblotting and total protein of FGFR1 was detected.

Actin was used as loading control.

Supplementary Figure 4: Expression of ligands does not correlate with FGFR1

amplification status.

(A) Cells were seeded into 6-well plates and after 24h of incubation in serum free media, supernatant

was harvested and used for detection of FGF2 via ELISA. Shown are the levels of FGF2 (y-axis,

pg/ml) across a panel of two FGFR1-amplified cells (H520, H1581) and four FGFR1 wildtype

(H2009, HOP92, H1299, H1650) NSCLC cell lines (x-axis).

(B) Gene expression arrays were utilized to measure expression of FGFR ligands in primary SQLC

samples harboring FGFR1-amplification, SOX2-amplification and in samples that were wildtype for

both genes. Hierarchical clustering analysis (Genepattern) was performed using the normalized

average signal intensity of the indicated FGFR ligands.

(C) The FGFR1-amplified cell line H1581 was plated in 6cm dishes containing either FCS containing

media (first lane) or serum free media and stimulated (20 minutes before harvesting) with 50ng/ml of

the indicated FGFR ligands (FGF2, lane 3 and 5; FGF9, lane 4) and treated with 1μM of the FGFR

inhibitor PD173074 (lane 5).

FGFR

Supplementary Table 1: Significant amplifications and deletions are noted in a subset of 155 SQLC samples

cytoband

q value

wide peak boundaries

Frequency (%)

amplification/ deletion

# of genes in peak

genes in w

ide peak

1p34.2 0.010253 chr1:39823091-40594085 0.02 A 15 BMP8B COL9A2 MYCL1 PPT1 RLF ZMPSTE24 PPIE CAP1 HEYL HPCAL4 TRIT1 OXCT2 NT5C1A MFSD2 TMCO2 _ _ _ _ _

1p12 0.23967 chr1:119749239-121057404 0.01 A 11 FCGR1B HMGCS2 HSD3B1 HSD3B2 NOTCH2 ADAM30 PHGDH REG4 ZNF697 NBPF7 FAM72B _ _ _ _ _ _ _ _ _

2p15 1.59E-05 chr2:58248048-61514993 0.02 A 9 PEX13 REL USP34 BCL11A FANCL PAPOLG KIAA1841 AHSA2 PUS10 _ _ _ _ _ _ _ _ _ _ _

2q14.2 0.1993 chr2:120994001-121753902 0.00 A 2 GLI2 TFCP2L1 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

3q11.2 6.13E-06 chr3:95012938-95644451 0.01 A 5 PROS1 NSUN3 ARL13B DHFRL1 STX19 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

3q26.2 7.41E-57 chr3:170704346-171880521 0.05 A 14 MDS1 CLDN11 PRKCI SKIL TLOC1 GPR160 MYNN SLC7A14 LRRC31 PHC3 ARPM1 LRRC34 LOC344657 SAMD7 _ _ _ _ _ _

3q26.33 7.65E-83 chr3:183084517-183194172 14.19 A 1* [SOX2] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

3q29 3.77E-30 chr3:198339265-199501827 1.94 A 8 hsa-mir-922 BDH1 DLG1 RPL35A KIAA0226 IQCG FYTTD1 LRCH3 LMLN _ _ _ _ _ _ _ _ _ _ _

4q13.1 0.016482 chr4:58688620-60118436 0.65 A 1* [IGFBP7] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

5p15.1 6.38E-18 chr5:17414909-18218383 1.94 A 1* [BASP1] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

6p12.1 0.11312 chr6:53947191-54536418 0.00 A 2 TINAG C6orf142 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

7p11.2 1.25E-06 chr7:54354013-54878875 2.58 A 2 SEC61G VSTM2A _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

8p12 8.82E-28 chr8:38436349-38569287 10.32 A 2 FGFR1 FLJ43582 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

8q24.21 1.29E-16 chr8:128316332-128614800 2.58 A 1* [MYC] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

9p13.3 0.0004157 chr9:35227774-35833079 3.87 A 20 CA9 CD72 NPR2 TESK1 TLN1 TPM2 RGP1 RUSC2 CREB3 UNC13B SPAG8 SIT1 C9orf127 GBA2 HINT2 C9orf100 CCDC107 C9orf128 PC-3 LOC730112

11q13.2 1.07E-15 chr11:68864575-69292157 5.81 A 3 CCND1 FGF19 ORAOV1 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

12p12.1 1.31E-08 chr12:24544505-25115257 2.58 A 5 BCAT1 LRMP SOX5 FLJ32894 LOC196415 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

12q15 0.061719 chr12:66060452-66573584 1.29 A 1 DYRK2 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

14q21.1 0.016482 chr14:37146900-37686962 1.94 A 1 TTC6 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

18q11.2 0.04975 chr18:17261675-17780549 1.29 A 4 hsa-mir-1-2 SNRPD1 MIB1 ESCO1 ABHD3 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

18q12.3 0.061719 chr18:36964003-39698813 0.65 A 3 PIK3C3 RIT2 SYT4 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

19q12 8.47E-06 chr19:34875012-35098226 2.58 A 2 CCNE1 C19orf12 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

19q13.2 0.00024071 chr19:45708413-45923005 2.58 A 6 LTBP4 NUMBL SPTBN4 ADCK4 ITPKC SHKBP1 _ _ _ _ _ _ _ _ _ _ _ _ _ _

20p12.2 0.04975 chr20:10745071-11329827 0.65 A 1* [JAG1] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

22q12.2 0.076247 chr22:28529591-28883850 1.94 A 3 MTMR3 ASCC2 HORMAD2 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

1p36.22 0.21959 chr1:10159766-10982661 1.29 D 9 CORT DFFA PEX14 PGD UBE4B KIF1B CASZ1 C1orf127 APITD1 _ _ _ _ _ _ _ _ _ _ _

1q25.1 0.21959 chr1:173063030-173068495 2.58 D 1 RABGAP1L _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

2q22.1 0.21959 chr2:141565515-141968318 1.94 D 1 LRP1B _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

3p14.1 0.21959 chr3:65163494-65191783 3.87 D 1* [MAGI1] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

3p12.3 0.21959 chr3:74857827-84356945 0.65 D 3 GBE1 ROBO1 ROBO2 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

3q12.1 2.84E-12 chr3:100427724-100432004 21.29 D 1* [DCBLD2] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

3q26.1 0.21959 chr3:163606062-163626942 2.58 D 1* [LOC131149] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

3q26.1 0.21959 chr3:166541968-166568123 1.29 D 1* [SLITRK3] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

4p15.1 0.21959 chr4:27960115-29350568 1.94 D 1* [PCDH7] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

4q21.3 0.21959 chr4:87194249-87198968 1.29 D 1 MAPK10 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

4q27 0.21959 chr4:122501424-122510971 3.23 D 1 GPR103 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

4q27 0.21959 chr4:138311872-138312388 1.29 D 1* [PCDH18] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

4q32.1 0.00061535 chr4:161272225-161303320 1.94 D 1* [RAPGEF2] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

4q32.2 3.64E-16 chr4:162093452-162104050 0.65 D 1* [FSTL5] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

7p11.2 0.0035601 chr7:54354001-54360982 0.00 D 1* [VSTM2A] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

7q21.3 0.21959 chr7:97233400-97242176 1.29 D 1* [TAC1] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

8q21.2 0.21959 chr8:85420546-85433237 0.65 D 1 RALYL _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

9p21.3 7.24E-14 chr9:21958713-23000072 7.10 D 3 CDKN2A CDKN2B DMRTA1 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

9p21.3 3.64E-16 chr9:24493818-24509684 6.45 D 1* [ELAVL2] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

10p12.33 0.21959 chr10:18115029-18270264 2.58 D 4 hsa-mir-511-2MRC1 FAM23B MRC1L1 FAM23A _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

10q23.2 0.21959 chr10:89500286-89690845 2.58 D 2 PTEN ATAD1 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

14q21.1 7.78E-06 chr14:40678110-40746090 1.94 D 1* [LRFN5] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

16p13.11 0.21959 chr16:15711158-16049858 1.94 D 4 ABCC1 MYH11 NDE1 C16orf63 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

17p11.2 1.87E-21 chr17:18175193-18409922 0.65 D 2 SHMT1 LOC654346 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

18q22.1 4.08E-05 chr18:62057848-62063490 2.58 D 1* [CDH19] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

18q22.1 2.45E-07 chr18:64894327-64908676 1.94 D 1 [CCDC102B] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

[] = genes in the proximity of the peak

* = # of genes in the proxmimity of the peak

Significant copy number alterations across all primary SQLC lung cancer samples (n=155) used in this study as defined by GISTIC (4 ) are shown.

.

sample overall survival smoking history age gender UICC TP53 mut

1 34.4 former 72 m Ib 1

2 9.5 current 76 m Ib 1

3 12.2 current 76 m IIb 0

4 59.6 former 76 m IIb 0

5 22.8 NA 62 m IIIb 1

6 31.9 former 68 m IIb 1

7 9.4 NA 72 m IIIb 1

8 11.8 current 75 f Ia 1

9 NA NA 58 m IIIa 1

10 NA NA 76 m Ib 1

11 53 current 60 m Ib 0

12 152 current 64 m IIb 1

13 90 current 70 m Ib 1

14 34.5 current 56 m Ib 0

15 56.1 current 76 f Ia 0

NA= not available

For all FGFR1 amplified primary squamous-cell lung cancer samples overall survival, smoking history,

age, gender, UICC status and TP53 mutation status is shown.

Supplementary Table 2: Clinical features and co-occurrent mutations of FGFR1-amplified SQLC samples.

Supplementary Table 3: Significant amplifications and deletions are noted in a subset of 77 adenocarcinoma samples.

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1q21.3 0.000015 chr1:149932264-151165868 A 38 FLG IVL SMCP RORC S100A10 S100A11 TCHH TDRKH TNRC4 LCE2B CRNN OAZ3 CRCT1 MRPL9 SNX27 LCE3D THEM4 TCHHL1 LCE4A LCE5A THEM5 LINGO4 LCE1A LCE1B LCE1C LCE1D LCE1E LCE1F LCE2A LCE2C LCE2D LCE3A LCE3B LCE3C LCE3E HRNR FLG2 KPRP

1q23.1 0.0018713 chr1:156607966-157135125 A 14 MNDA SPTA1 OR6N2 OR6K2 OR10T2 OR10X1 OR10Z1 OR6K6 OR6N1 OR10R2 OR10K2 OR10K1 OR6Y1 OR6K3

1q31.1 4.61E-02 chr1:184043312-185462178 A 8 PDC PLA2G4A PTGS2 TPR PRG4 OCLM C1orf27 HMCN1

2p11.2 0.013394 chr2:88907420-89329960 A 1 [RPIA]

3q26.1 3.51E-05 chr3:167433768-168291810 A 1 [ZBBX]

5p15.33 5.23E-11 chr5:2409990-6805384 A 10 SRD5A1 POLS KIAA0947 NSUN2 IRX1 MED10 C5orf38 IRX2 ADAMTS16 FLJ33360

5p14.3 8.33E-13 chr5:22696141-26975680 A 4 CDH9 CDH10 CDH12 PRDM9

6p12.3 2.10E-01 chr6:45511753-47037993 A 11 RUNX2 MEP1A PLA2G7 SLC25A27 RCAN2 ENPP4 CYP39A1 CLIC5 ENPP5 GPR116 TDRD6

7p21.3 0.16594 chr7:9039631-12409566 A 4 NDUFA4 PHF14 TMEM106B THSD7A

7q31.1 1.10E-01 chr7:107289401-120776914 A 39 CAPZA2 CAV1 CAV2 CFTR DLD IFRD1 KCND2 LAMB1 DNAJB9 MET NRCAM PPP1R3A WNT2 ST7 DOCK4 FAM3C ZNF277 TFEC LAMB4 TSPAN12 TES MDFIC PNPLA8 WNT16 LSM8 GPR85 ING3 LRRN3 ANKRD7 TMEM168 C7orf58 IMMP2L CTTNBP2 FOXP2 ASZ1 FLJ31818 LOC154907 THAP5 C7orf53

8p12 0.0044363 chr8:36899368-38225167 A 15 ADRB3 EIF4EBP1 STAR ASH2L BAG4 ERLIN2 PROSC DDHD2 GPR124 LSM1 BRF2 ZNF703 RAB11FIP1 GOT1L1 KCNU1

8p11.21 5.35E-03 chr8:41556180-42718136 A 13 hsa-mir-486 ANK1 CHRNB3 IKBKB PLAT POLB SLC20A2 VDAC3 MYST3 AP3M2 DKK4 C8orf40 AGPAT6 NKX6-3

8q21.3 1.12E-04 chr8:87194072-88419621 A 7 CPNE3 WWP1 FAM82B CNGB3 SLC7A13 CNBD1 ATP6V0D2

8q22.1 1.95E-04 chr8:96855958-97879928 A 6 SDC2 UQCRB PTDSS1 PGCP MTERFD1 GDF6

8q23.3 0.0000186 chr8:111552576-113449721 A 1 CSMD3

8q24.21 1.76E-09 chr8:127683117-129001544 A 1 MYC

8q24.21 6.00E-07 chr8:130358408-130841164 A 1 MLZE

11q13.3 0.025142 chr11:68218662-70141465 A 18 CCND1 CPT1A CTTN FGF3 FGF4 IGHMBP2 PPFIA1 FADD MTL5 FGF19 SHANK2 MYEOV TMEM16A MRGPRD MRPL21 MRGPRF TPCN2 ORAOV1

12p13.33 0.16594 chr12:1-1076721 A 11 NINJ2 RAD52 JARID1A SLC6A12 SLC6A13 ERC1 WNK1 CCDC77 B4GALNT3 HSN2 IQSEC3

12p12.1 0.0018713 chr12:25087734-25524186 A 5 KRAS LRMP CASC1 LYRM5 IFLTD1

12q15 0.23745 chr12:68072888-72990072 A 18 CNOT2 PTPRB PTPRR TSPAN8 LGR5 CCT2 FRS2 RAB21 KCNMB4 TRHDE TMEM19 TBC1D15 THAP2 RAB3IP TPH2 BEST3 CCDC131 LRRC10

13q32.1 1.49E-01 chr13:90853977-96511094 A 13 DCT GPC5 DNAJC3 CLDN10 GPC6 ABCC4 SOX21 DZIP1 TGDS OXGR1 UGCGL2 GPR180 HS6ST3

14q13.3 0.00083895 chr14:35734580-36524615 A 6 PAX9 NKX2-1 NKX2-8 MBIP SLC25A21 SFTPH

18p11.32 0.12741 chr18:1-1348036 A 10 ADCYAP1 CETN1 TYMS YES1 USP14 THOC1 CLUL1 ENOSF1 COLEC12 C18orf56

18q11.2 0.021688 chr18:22225775-22361927 A 1 KCTD1

20p11.21 0.11023 chr20:22034169-23431679 A 10 FOXA2 SSTR4 THBD CST8 CD93 NXT1 NAPB GZF1 CSTL1 CST11

7p11.2 1.61E-13 chr7:54354001-54360982 D 1 [VSTM2A]

9p21.3 9.01E-08 chr9:19861077-22402963 D 23 hsa-mir-31 hsa-mir-491 CDKN2A CDKN2B IFNA1 IFNA2 IFNA4 IFNA5 IFNA6 IFNA7 IFNA8 IFNA10 IFNA13 IFNA14 IFNA16 IFNA17 IFNA21 IFNB1 IFNW1 MLLT3 MTAP KIAA1797 KLHL9 IFNE1 PTPLAD2

11q11 0.018755 chr11:51421005-54530469 D 1 [OR4C46]

[] = genes in the proximity of the peak

* = # of genes in the proxmimity of the peak

Significant copy number alterations across all primary adenocarinoma lung cancer samples used in this study (n=77) as defined by GISTIC (4 ) are shown.

Supplementary Table 4: FGFR1 amplification is detected using FI SH on tumor microarrays.

SQLCplate FGFR1-HA FGFR1-LA FGFR1-N sumZTMA88 15 15 32 62ZTMA86 16 15 39 70CTMA4 3 4 14 21all 34 34 85 153frequency(%) 22.2222222 22.2222222 55.5555556 100

FGFR1-N: FGFR1 copy number=2FGFR1-LA: FGFR1 copy number<9FGFR1-HA: FGFR1 copy number>9

Displayed are the numbers of samples with FGFR1 amplification in squamous cell lung cancer, based on the given thresholdsResulting frequencies are given in percent (%).

Supplementary  Table  5:  GI50  values  are  not  associated  with  mutation  status  across  the  lung  cancer  cell  line  panel.

Cell  line

Histology

GI50  PD173074

NRASm

ut

EGFRmut

BRAFmut

PIK3CAm

ut

EML4-­‐ALK

A427 Carcinoma.  Not  specified 0.531 0 0 0 0 0A549 Carcinoma.  Not  specified 20.89 0 0 0 0 0Calu1 Epidermoid  carcinoma 15.31 0 0 0 0 0Calu3 Adenocarcinoma 7.763 0 0 0 0 0Calu6 Anaplastic  carcinoma 15.85 0 0 0 0 0Colo699 Adenocarcinoma 12.16 0 0 0 0 0Dms-­‐114 SCLC 0.006 0 0 0 0 0DV90 Adenocarcinoma 19.05 0 0 0 0 0EKVX Adenocarcinoma 19.95 0 0 0 0 0H1299 Carcinoma.  Not  specified 17.38 1 0 0 0 0H1355 Adenocarcinoma 19.95 0 0 0 0 0H1395 Adenocarcinoma 21.38 0 0 1 0 0H1437 Adenocarcinoma 40 0 0 0 0 0H1563 Adenocarcinoma 15.14 0 0 0 1 0H1568 Adenocarcinoma 20.89 0 0 0 0 0H157 Squamous  cell  carcinoma 13.49 0 0 0 0 0H1581 carcinoid 0.026 0 0 0 0 0H1648 Adenocarcinoma 15.85 0 0 0 0 0H1650 Adenocarcinoma.  Bronchoalveolar  carcinoma 22.65 0 1 0 0 0H1666 Adenocarcinoma.  Bronchoalveolar  carcinoma 10.23 0 0 1 0 0H1734 Adenocarcinoma 8.222 0 0 0 0 0H1755 Adenocarcinoma 6.166 0 0 1 0 0H1770 Neuroendocrine  carcinoma 14.79 0 0 0 0 0H1781 Adenocarcinoma.  Bronchioalveolar  carcinoma 16.79 0 0 0 0 0H1792 Adenocarcinoma 16.6 0 0 0 0 0H1819 Adenocarcinoma 15.14 0 0 0 0 0H1838 Adenocarcinoma 13.8 0 0 0 0 0H1915 Poorly  differentiated  carcinoma 14.79 0 0 0 0 0H1944 Adenocarcinoma 17.58 0 0 0 0 0H1975 Adenocarcinoma 19.72 0 0 0 0 0H1993 Adenocarcinoma 22.91 0 0 0 0 0H2009 Adenocarcinoma 14.13 0 0 0 0 0H2030 Adenocarcinoma 13.03 0 0 0 0 0H2052 Mesothelioma 9.333 0 0 0 0 0H2087 Adenocarcinoma 3.589 1 0 1 0 0H2110 Non-­‐small  cell  lung  cancer.  Not  specified 10.72 0 0 0 0 0H2122 Adenocarcinoma 19.95 0 0 0 0 0H2126 Adenocarcinoma 40 0 0 0 0 0H2172 Non-­‐small  cell  lung  cancer.  Not  specified 20.42 0 0 0 0 0H2228 Adenocarcinoma 14.29 0 0 0 0 1H23 Adenocarcinoma 16.6 0 0 0 0 0H2347 Adenocarcinoma 17.38 1 0 0 0 0H2444 Non-­‐small  cell  lung  cancer.  Not  specified 18.62 0 0 0 0 0H28 Mesothelioma 13.65 0 0 0 0 0H2882 Squamous  cell  carcinoma 18.2 0 0 0 0 0H3122 Adenocarcinoma 18.41 0 0 0 0 1H322 Adenocarcinoma 15.31 0 0 0 0 0H322M Adenocarcinoma 17.18 0 0 0 0 0H3255 Adenocarcinoma 13.8 0 1 0 0 0H358 Bronchioalveolar  carcinoma 12.59 0 0 0 0 0H441 Papillary  Adenocarcinoma 17.18 0 0 0 0 0H460 Large  cell  lung  cancer 40 0 0 0 1 0H520 Squamous  cell  carcinoma 5.06 0 0 0 0 0H522 Adenocarcinoma 20.18 0 0 0 0 0H596 Adenosquamous  carcinoma 16.22 0 0 0 1 0H647 Adenosquamous  carcinoma 19.5 0 0 0 0 0H661 Large  cell  lung  cancer 14.96 0 0 0 0 0H820 Papillary  Adenocarcinoma 13.03 0 0 0 0 0HCC1171 Adenocarcinoma 14.29 0 0 0 0 0HCC1195 Adenosquamous  carcinoma 9.661 1 0 0 0 0HCC1359 Spindle-­‐giant  cell 14.79 0 0 0 0 0HCC15 Squamous  cell  carcinoma 20.42 1 0 0 0 0HCC1833 Adenocarcinoma 20.65 0 0 0 0 0HCC193 Adenocarcinoma 4.266 0 0 0 0 0HCC2279 Adenosquamous  carcinoma 17.58 0 1 0 0 0HCC2429 Non-­‐small  cell  lung  cancer.  Not  specified 16.22 0 0 0 0 0HCC2450 Adenocarcinoma 17.99 0 0 0 1 0HCC2935 Adenocarcinoma 19.5 0 1 0 0 0HCC364 Adenocarcinoma 17.99 0 0 1 0 0HCC366 Adenosquamous  carcinoma 10.72 0 0 0 0 0HCC4006 Adenocarcinoma 16.6 0 1 0 0 0HCC44 Adenocarcinoma 17.99 0 0 0 0 0HCC461 Adenocarcinoma 14.13 0 0 0 0 0HCC515 Adenocarcinoma 15.49 0 0 0 0 0HCC78 Adenocarcinoma 16.22 0 0 0 0 0HCC827 Adenocarcinoma 30.55 0 1 0 0 0HCC95 Squamous  cell  carcinoma 7.244 0 0 0 0 0HOP62 Adenocarcinoma 18.84 0 0 0 0 0HOP92 Large  cell  lung  cancer 15.14 0 0 0 0 0LCLC97TM1 Large  cell  lung  cancer 8.318 0 0 0 0 0LouNH91 Squamous  cell  carcinoma 17.99 0 1 0 0 0PC9 Adenocarcinoma 12.74 0 1 0 0 0SKLU1 Adenocarcinoma 6.531 0 0 0 0 0

Summary  of  the  raw  GI50  values  [μM]  derived  from  high-­‐throughput  cell  line-­‐based  screening.  GI50  values  were  determined  using  the  statistical  data  analysis  software  “R”  with  the  “ic50”  package.  Mutations  in  EGFR,  NRAS,  BRAF,  PIK3CA  and  EML4/ALK  are  annotated  as  “1”  for  presence  or  “0”  for  absence.  If  known,  the  histotype  of  the  respective  cell  line  is  given.

ce

ll l

ine

ind

uc

tio

n o

f a

po

pto

sis

sta

nd

ar

d d

ev

iati

on

FG

FR

1-a

mp

lifi

ca

tio

nH520 26.4 1.98 1

DMS114 20 2.69 1

H1581 17.5 2.12 1

A427 12.6 0.42 0

HCC15 7.65 2.19 0

HCC827 4.9 1.70 0

A549 2.55 0.07 0

H2052 2.45 0.21 0

EKVX 2.15 8.41 0

H1355 1.65 0.64 0

H2444 1.25 2.33 1

H1395 1 0.99 0

H2087 1 2.12 0

Calu3 0 3.39 0

H2110 0 2.69 0

H2122 0 4.10 0

H358 0 0.35 0

H1650 0 0.00 0

H1666 0 2.62 0

H1755 0 2.05 0

H1792 0 0.71 0

H1975 0 0.92 0

H2172 0 0.07 0

LCLC97TM1 0 5.52 0

for FGFR1-amplification: "1" copy number > 4; "0" copy number < 4

Cells were seeded in six-well plates and treated with either DMSO (control) or 0.5 µM PD173074

for 72 h. Apoptosis was detected by measuring the annexinV and propidium iodide (PI) content.

The relative induction of apoptosis across 24 NSCLC cells is displayed.

Supplementary Table : P 73074 induces apoptosis in FGFR1-amplified cell lines. D16

Supplementary Table : PD173074 has specific activity against two kinases.

kin

ase

ac

tiv

ity

[%

]

sta

nd

ard

de

via

tio

n

kin

ase

ac

tiv

ity

[%

]

sta

nd

ard

de

via

tio

n

FGF-R1 8 0 MARK1 99 3

VEG-FR 10 3 TAK1 100 8

Lck 26 3 MST2 100 4

YES1 40 12 MAPKAP-K3 100 8

MST4 49 5 CK2 101 0

p38a MAPK 51 11 PAK5 101 4

p38b MAPK 76 1 GCK 101 1

IKKb 79 8 EPH-B4 101 25

RIPK2 81 3 PKCz 101 17

MLK3 81 3 PAK4 101 0

IRAK4 81 11 SmMLCK 102 24

HER4 83 9 BTK 103 6

CK1 85 4 MSK1 103 11

CSK 85 5 PKA 103 4

GSK3b 85 6 DYRK1A 104 2

ERK8 86 14 JNK1 105 4

PIM1 87 5 EPH-B2 105 8

SRPK1 87 6 PKCa 105 6

MARK4 87 8 BRSK1 105 7

MKK1 87 2 JNK3 105 2

JAK2 88 2 IGF-1R 106 17

Src 88 4 S6K1 106 5

SYK 88 4 MARK2 106 10

PKBa 88 18 LKB1 106 27

NUAK1 89 9 MNK1 106 9

IR 89 4 HIPK1 106 2

MLK1 89 9 CHK1 107 0

p38d MAPK 90 2 PDK1 107 5

ROCK 2 90 1 NEK6 107 10

IRR 90 7 MELK 107 14

PKD1 90 10 PLK1 107 7

CDK2-Cyclin A 91 1 MINK1 108 5

PKBb 92 8 PRK2 108 16

SGK1 92 3 CAMKKb 109 11

MNK2 93 3 PHK 109 2

PAK2 93 6 Aurora A 109 0

EPH-A4 94 13 ERK2 109 7

EPH-B3 95 4 PIM3 110 5

DYRK2 95 1 Aurora B 110 8

AMPK 95 12 BRSK2 111 19

MARK3 95 2 ERK1 112 13

EF2K 95 11 CHK2 112 15

PIM2 96 1 MAPKAP-K2 113 12

TrkA 96 7 HIPK3 117 1

CAMK1 97 2 TTK 118 27

p38g MAPK 97 4 HIPK2 118 17

DYRK3 97 15 PRAK 120 1

RSK1 97 11 PAK6 121 6

NEK2a 98 14 EPH-A2 122 33

JNK2 98 2 RSK2 122 47

CLK2 99 4 TBK1 123 10

DAPK1 99 4 IKKe 130 29

MEKK1 137 2

Biochemical measurement of the remaining activity (in %) of 105 kinases in the presence of PD173074 is displayed. Each measurement was performed in

triplicate and standard deviations are displayed. Screening was performed at the protein phosphorylation unit at the University of Dundee (5)

7

sam

ple

3q

26

.33

8p

12

sam

ple

3q

26

.33

8p

12

sam

ple

3q

26

.33

8p

12

sam

ple

3q

26

.33

8p

12

1 0 0 47 2 0 93 0 0 139 1 0

2 1 0 48 0 0 94 1 0 140 1 0

3 0 0 49 2 0 95 1 0 141 0 0

4 2 0 50 0 2 96 2 0 142 0 0

5 0 0 51 2 0 97 0 0 143 1 0

6 1 1 52 0 1 98 2 0 144 0 0

7 0 0 53 1 0 99 1 0 145 0 0

8 0 0 54 2 0 100 2 0 146 0 0

9 2 0 55 1 0 101 0 2 147 0 0

10 0 0 56 2 0 102 0 0 148 1 0

11 0 0 57 2 0 103 0 2 149 0 0

12 0 0 58 2 0 104 1 0 150 0 0

13 1 0 59 0 0 105 1 1 151 0 0

14 2 0 60 0 1 106 0 0 152 0 0

15 0 0 61 0 1 107 1 1 153 2 1

16 0 0 62 2 0 108 1 2 154 0 0

17 1 2 63 0 0 109 0 0 155 0 2

18 2 0 64 1 0 110 0 1

19 0 0 65 0 0 111 0 0

20 2 0 66 0 0 112 0 0

21 0 0 67 1 2 113 1 1

22 0 0 68 0 0 114 1 0

23 0 0 69 0 0 115 2 0

24 0 0 70 1 0 116 1 0

25 0 0 71 1 0 117 0 0

26 0 0 72 0 0 118 0 0

27 0 0 73 0 0 119 0 1

28 0 0 74 0 2 120 0 0

29 0 0 75 0 0 121 1 0

30 0 0 76 0 0 122 1 0

31 1 0 77 2 0 123 1 0

32 2 0 78 2 2 124 0 0

33 1 0 79 2 2 125 2 0

34 1 0 80 0 2 126 2 0

35 2 0 81 0 0 127 0 1

36 0 0 82 0 0 128 0 0

37 2 0 83 0 0 129 0 2

38 0 0 84 0 0 130 0 1

39 1 0 85 1 0 131 0 0

40 0 2 86 1 2 132 2 0

41 0 0 87 1 0 133 1 0

42 1 0 88 0 0 134 1 2

43 1 0 89 0 0 135 1 1

44 1 0 90 0 0 136 2 0

45 2 0 91 2 0 137 0 0

46 0 2 92 0 2 138 2 0

0: copy number t<2.8; 1: 0.5<copy number t< 0.9; 2: copy number t>0.9

Genetic copy number changes in 8p12 and 3q26 were identified in primary squamous-cell lung cancer samples. Two different thresholds (t) for calling a copy

number change were chosen. All samples with copy number < 2.8 change are assigned with “0”. All samples with copy number > 2.8 but < 3.7 are assigned as “1”.

The remaining samples with copy number > 3.7 at the given chromosomal position are denoted with “2”.3.7

Supplementary Table : FGFR1 and SOx2 amplification in squamous cell lung carcinoma.8

TRC number Gene Sequence

TRCN0000121106 FGFR1 CCGGGAGATGGAGGTGCTTCACTTACTCGAGTAAGTGAAGCACCTCCATCTCTTTTTG

TRCN0000121186 FGFR1 CCGGTCTTGAAGACTGCTGGAGTTACTCGAGTAACTCCAGCAGTCTTCAAGATTTTTG

TRCN0000015613 WHSC1L1 CCGGCGAGAGTATAAAGGTCATAAACTCGAGTTTATGACCTTTATACTCTCGTTTTT

TRCN0000015614 WHSC1L1 CCGGCCATCATCAATCAGTGTGTATCTCGAGATACACACTGATTGATGATGGTTTTT

TRCN0000015615 WHSC1L1 CCGGCGAGAATATCATGTCCAGTTTCTCGAGAAACTGGACATGATATTCTCGTTTTT

TRCN0000015616 WHSC1L1 CCGGGCTTCCATTACGATGCACAAACTCGAGTTTGTGCATCGTAATGGAAGCTTTTT

TRCN0000015617 WHSC1L1 CCGGGCAGGGAATTGTTTGAGTCTTCTCGAGAAGACTCAAACAATTCCCTGCTTTTT

TRCN0000137790 FLJ43582 CCGGGACTCGCATCTGTGGTTTCTTCTCGAGAAGAAACCACAGATGCGAGTCTTTTTTG

TRCN0000138235 FLJ43582 CCGGCAAGTTTGGGACAGAAGGGAACTCGAGTTCCCTTCTGTCCCAAACTTGTTTTTTG

TRCN0000138014 FLJ43582 CCGGGACTCCTTTCTTGTCCTGGAACTCGAGTTCCAGGACAAGAAAGGAGTCTTTTTTG

TRCN0000138332 FLJ43582 CCGGCGCAGTAACACACGTAGGTTTCTCGAGAAACCTACGTGTGTTACTGCGTTTTTTG

TRCN0000138861 FLJ43582 CCGGGCAGTTCCAAAGAGCCAAGAACTCGAGTTCTTGGCTCTTTGGAACTGCTTTTTTG

For all shRNA constructs used in the study, the according TRC library number and sequence are given.

Supplementary Table S9. Sequences of all shRNA constructs that were used in the study.