Potent, Selective Inhibitors of Fibroblast Growth Factor Receptor … · 2011. 8. 26. ·...

Therapeutic Discovery

Potent, Selective Inhibitors of Fibroblast Growth FactorReceptor Define Fibroblast Growth Factor Dependencein Preclinical Cancer Models

Matthew Squires1,2, George Ward2, Gordan Saxty2, Valerio Berdini2, Anne Cleasby2, Peter King3,Patrick Angibaud3, Tim Perera3, Lynsey Fazal2, Douglas Ross2, Charlotte Griffiths Jones2, Andrew Madin2,Rajdeep K. Benning2, Emma Vickerstaffe2, Alistair O'Brien2, Martyn Frederickson2, Michael Reader2,Christopher Hamlett2, Michael A. Batey4, Sharna Rich2, Maria Carr2, Darcey Miller2, Ruth Feltell2,Abarna Thiru2, Susanne Bethell2, Lindsay A. Devine2, Brent L. Graham2, Andrew Pike2,Jose Cosme2, Edward J. Lewis2, Eddy Freyne3, John Lyons2, Julie Irving4, Christopher Murray2,David R. Newell4, and Neil T. Thompson2

AbstractWe describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor

receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over

the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was

defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or

translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile

for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an

important role. Mol Cancer Ther; 10(9); 1–11. �2011 AACR.

Introduction

The fibroblast growth factor (FGF) family and their 4receptor tyrosine kinases, FGFR1/2/3/4, mediatenumerous physiologic processes including cell migra-tion, proliferation, survival, and differentiation. Giventhe importance of FGF/FGFR, it is unsurprising thataberrant FGFR signaling is found in many tumor typesincluding multiple myeloma, gastric, endometrial, pros-tate, and breast (1, 2). Gain-of-function mutations inFGFRs are the most common kinase abnormality incancer with activation occurring via a range of mechan-isms such as point mutation, amplification, chromosomaltranslocation, and aberrant splicing (3). For example, thet(4;14)(p16;q32) chromosomal translocation found in 15%of multiple myeloma patients often results in overexpres-sion of FGFR3 (4–6). The overexpressed FGFR3 is usuallywild type and although somatic mutations are occasion-

ally found, the cells remain sensitive to FGF (7). ActivatedFGFR3 has a role in myelomagenesis and the ability ofanti-FGFR3 antibodies and kinase inhibitors, for example,PD173074 and CHIR258, to inhibit multiple myeloma cellgrowth, both in vitro and in vivo, validates FGFR3 as atherapeutic target (8–17). The FGFR2 gene is amplified insome cases of gastric cancer, resulting in a highly over-expressed and constitutively active receptor. Small mole-cule inhibitors and FGFR2 knockdown reveal a criticalrole for FGFR2 amplification in gastric cancer cell growthboth in vitro and in xenograft models (18, 19). Recentpublications have identified FGFR1 amplification inaround 20% of squamous non–small cell lung carcinoma(20, 21) and around 10% of breast cancers (22). FGFR4amplification has been observed in rhabdomyosarcomaand activating mutations characterized in 7% of cases(23). FGFs have a role in tumor angiogenesis and mediateresistance to VEGFR2 inhibitors (24). Together, thesecompelling data support the development of a specific,potent inhibitor of FGFR1–4 for cancer therapy.

The aims of this drug discovery program were first togenerate potent, selective, orally bioavailable inhibitorsof the FGFR family of tyrosine kinases, to establishmodel systems capable of defining the appropriate phar-macology, and to investigate the role of FGFR in cancer.Selectivity over closely homologous kinases, includingVEGFR2, was considered essential. Broad spectrumtyrosine kinase inhibitors exist, but FGFR is never themost potently inhibited kinase and such mixed inhibitorsfail to inhibit FGFR completely because of interveningkinase activities and their associated toxicities. Starting

Authors' Affiliations: 1Novartis Pharma AG, Basel, Switzerland; 2AstexTherapeutics Ltd., Cambridge, United Kingdom; 3Ortho Biotech OncologyR&D, Beerse, Belgium; and 4Newcastle Cancer Centre, Northern Institutefor Cancer Research, Newcastle University, Newcastle upon Tyne, UnitedKingdom

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: N.T. Thompson, Astex Therapeutics Ltd., 436Cambridge Science Park, Milton Road, Cambridge CB4 0QA, UnitedKingdom. Phone: 441223435015; Fax: 441223226201; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0426

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org OF1

on April 20, 2021. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 15, 2011; DOI: 10.1158/1535-7163.MCT-11-0426

http://mct.aacrjournals.org/

with fragment-derived hits, we used a structure-baseddesign (25–27) to optimize leadmolecules to potent FGFRinhibitors with selectivity against VEGFR2, which shares57% sequence identity with the kinase domain of FGFR1–3 and 54% with that of FGFR4. Further optimization ofpharmacokinetic properties resulted in a series of imida-zopyridine leads, the properties of which are describedhere. The profile in preclinical models presented heredescribes a paradigm for the response of cellular systemswith a defined genetic background to a specific FGFRinhibitor.

Materials and Methods

Compounds 1 and 2 are 1-{3-[7-(4-fluorophenyl)imi-dazo[1,2-a]pyridin-3-yl]phenyl}-3-(2,2,2-trifluoroethyl)urea and 1-{3-[7-(5-methyl-[1,3,4]oxadiazol-2-yl)imidazo[1,2-a]pyridin-3-yl]phenyl}-3-(2,2,2-trifluoroethyl)urea,respectively. These and additional compounds usedweresynthesized by Astex Therapeutics Ltd. or Ortho Biotech(28).

Cell linesKG-1, MFE-296, and RT112/84 cell lines were from the

European Collection of Animal Cell Cultures. Snu-1, Snu-16, Kato-III,Hec-1A, andAN3-CAcell lineswere obtainedfrom the American Type Culture Collection. RPMI-8226and wild-type Ba/F3 cell lines were obtained from Ger-man Collection ofMicroorganisms andCell Cultures. TheKMS-11 cell line was obtained from the Japanese Collec-tion of Research Bioresources. The Jim-1 cell line wasobtainedunder license fromCancerResearchTechnology.Stably transfected Ba/F3 cell lines expressing TEL-FGFR1/3/4 were generated from fusion expression con-structs of the TEL oligomerization domain linked to the 50

end of FGFR kinase domains in pcDNA3.1 (Invitrogen)and electroporation into wild-type Ba/F3 cells. Selectionof stable cell lines was conducted with Geneticin (Invitro-gen) in the absence of mouse interleukin 3. Human umbi-lical vein endothelial cells (HUVEC) were obtained fromClonetics and grown in EGM2 medium (Clonetics).

Antibodies were from Cell Signaling Technology,except total FGFR2 and total FRS2-a from Santa CruzBiotechnology.

Cloning, expression, and purification of kinasedomains for FGFR1 and VEGFR2

A construct spanning residues 455 to 763 of the wild-type human FGFR1 with L455V, C486A, and C582Smutations was expressed as a His-tagged protein in Sf9insect cells.

Cell pellets were lysed by sonication in a buffer con-taining 25 mmol/L Tris-HCl at pH 8.0, 250 mmol/Lsodium chloride, 10% glycerol, 10 mmol/L imidazole,and 5 mmol/L b-mercaptoethanol. After centrifugation,the supernatant was incubated with Ni-NTA fast flowresin and eluted with an imidazole gradient. The eluatewas incubated overnight with TEV protease to remove

the His-tag and purified on a Ni column. Ion exchangeand size exclusion chromatography were used to furtherpurify the protein.

A construct encompassing residues 805 to 1,171, butomitting 50 residues of the kinase insert domain, wasmade for the kinase domain of VEGFR2. This constructwas expressed and purified as described (29).

Crystallization, diffraction data collection, structuredetermination, and refinement

Crystals were obtained by the hanging drop method.FGFR1 crystals were obtained by seeding into a solutioncontaining 10 to 15 mg/mL of protein, 100 mmol/L Bis–Tris buffer at pH 6.5, 300 mmol/L ammonium sulfate,12% to 16% PEG10000, and 5% ethylene glycol.

VEGFR2 crystals were obtained from drops containing7 mg/mL of protein mixed with 100 mmol/L HEPES,pH 7.2, 2.0 to 2.2 mol/L ammonium sulfate, and 4%mPEG550.

Complexes of FGFR1 with compounds 1, 2, and 4 wereprepared by soaking the crystals in saturated solutions in10% dimethyl sulfoxide (DMSO) and well solution for 3,3, and 2 days, respectively. The complex of VEGFR2 withcompound 4was prepared by soaking the compound intothe crystal for 3 hours in 10% DMSO and well solution.

All data for FGFR were collected using a Jupiter CCDdetector mounted on an R200 rotating anode x-ray gen-erator. Data were processed and scaled using d*trek.X-ray data for VEGFR2 were collected at the ESRF onID23.1 and processed and scaled using MOSFLM. Allstructures were solved using molecular replacement anddifference Fourier methods and refined using REFMAC(CCCP4, United Kingdom).

In vitro kinase activitiesInhibition of kinase activity in vitro was conducted as

described in Supplementary Data.

Proliferation assaysCells were seeded into 96-well plates at 5� 103 cells per

well before addition of compound in 0.1% DMSO for 72hours. A solution of 10% v/v Alamar Blue (BiosourceInternational) was added following compound incuba-tion and cells incubated for a further 6 hours. Platefluorescence was read at lex ¼ 535 nm and lem ¼ 590 nm.

Phospho-FGFR3 and phospho-VEGFR2 ELISAPhospho-FGFR3 was measured in KMS-11 cell lysates

using a DuoSetIC ELISA (R&D Systems). KMS-11 cellswere plated out in serum-free medium (2 � 105 cells perwell) and treated with compounds for 30 minutes beforelysing in 125 mL of TG lysis buffer [20 mmol/L Tris,pH 7.6, 0.14 mol/L NaCl, 1% (v/v) Triton X-100, 10%(v/v) glycerol, 0.05 mol/L NaF, and 1 mmol/L Na3V04 þprotease inhibitor tablet from Roche (Mini Complete;used at 1 tablet/10 mL)] for 30 minutes at 4�C. Lysates(100 mL) were assayed according to the manufacturer’sprotocol.

Squires et al.

Mol Cancer Ther; 10(9) September 2011 Molecular Cancer TherapeuticsOF2




Phospho-VEGFR2 was measured using a DuoSetICELISA (R&D Systems). HUVECs (5 � 104 cells per well)were plated out in EGM2 medium (including 1% serum)and left to recover overnight. The cells were switched toserum-free medium and left for further 16 hours and thentreated with compounds for 30 minutes and stimulatedwith recombinant human VEGF165 (R&D Systems) at100 ng/mL for 5 minutes at 37�C before lysing in 125 mLof TG lysis buffer for 30 minutes at 4�C. Lysates (100 mL)were assayed according to the manufacturer’s protocol.

Western blottingCell or xenograft lysates were prepared in TG lysis

buffer (see above), cleared by centrifugation, and normal-ized for total protein by BCA assay (Thermo Scientific).Equal quantities of total protein were denatured and runon SDS-PAGE gels (Invitrogen) and transferred to nitro-cellulose blots (Invitrogen). Bound primary antibodieswere detected using IR-labeled secondary antibodies(Li-Cor) and an Odyssey imager (Li-Cor).

Xenograft modelsAll animal studies were conducted according to the

relevant national regulatory guidelines and individualexperiments approved by the appropriate institutionalanimal welfare committee. Cells from tissue culturewere implanted subcutaneously in the right flank of 8-to 10-week-old BALB/c nu/nu mice (Charles River) orBALB/c Hsd:athymic nude-Foxn1nu (Harlan) at 5 � 106

cells per animal in 50% Matrigel basement media (BDBiosciences) and 50% (v/v) RPMI 1640 media (Invitro-gen). Treatment commenced when tumors were palpable(approximately 5 mm � 5 mm, 10–14 days postimplanta-tion). Groups of tumor-bearing animals (n ¼ 8) receiveddosing vehicle (control) by oral gavage or FGFR3 inhi-bitor as indicated in the schedule in a dosing volume of 10mL/kg. Tumor volume was calculated by caliper (Mitu-toyo) measurements using the equation a2 � b/2, where ais the smallest measurement and b the largest. Data arepresented as mean relative tumor volume, where thetumor volume on the initial day of treatment (day 0) isassigned a relative tumor volume value of 1.A complete regression was defined as a decrease in

tumor volume to an undetectable size, less than 3 mm inany dimension. Tolerability was estimated bymonitoringbody weight loss, clinical signs, and survival. Statisticalsignificance between control and treatment was deter-mined by using ANOVAwith Dunnett’s post test for 3 ormore groups or Student’s t test for 2 groups.

Pharmacodynamic studiesSubcutaneous xenograft tumors were removed from

nude mice at the indicated times following a single oraladministration of FGFR inhibitor. Tumor samples wereground to a fine powder under liquid nitrogen andprotein extracted by addition of 1 mL triton lysis buffer.Western blots were conducted as outlined in Materialsand Methods.

Results

Structure-based design of compounds 1 and 2,inhibitors of FGFR, and basis for VEGFR2selectivity

Fragment screening against FGFR was conductedusing a combination of nuclear magnetic resonance spec-troscopy, thermal denaturation, and x-ray crystallogra-phy, resulting in the identification of greater than 30x-ray structures of fragments in FGFR1. One attractivestarting point for medicinal chemistry was the imidazo-pyridine fragment (compound 3). Because of the lowmolecular weight of this fragment hit, it had a relativelylow potency with an IC50 of 120 mmol/L versus FGFR3.However, when potency was normalized with respect tosize, an encouraging value of 0.38 kcal per heavy atomwas obtained as a measure of the fragment’s ligandefficiency (30). The FGFR1 crystal structure shows thatthe imidazopyridine binds in the ATP site of the kinaseforming a single hydrogen bond to the backbone NH ofAla564 (see Fig. 1B). The binding site suggests that theagent acts as an inhibitor by competing for binding withATP. The experimentally determined binding mode sug-gested how potency might be readily improved. First,replacement of the chlorine with substituted aromaticscould be used to form hydrogen bonds with the sidechain of Asp641 and in addition, access to this part of theenzyme might be used to drive selectivity through inter-actions with Ala640, which in VEGFR2 is substituted bythe larger amino acid, cystine. Second, the crystal struc-ture indicated that the ester group at position 6 of theimidazopyridine template was probably not useful foraffinity, whereas elaboration at position 7 might facilitategood surface complementarity between the ligand and aregion of the protein where affinity increases are oftenobserved in kinases.

This structure-based drug design approach led to iden-tification of the selective and potent FGFR inhibitor com-pound 1 that has an FGFR3 potency of 3 nmol/L and aselectivity of about 30-fold over VEGFR2 (Fig. 1A and Cand Table 1). Further optimization led to compound 2(Fig. 1A and C) with reduced lipophilicity relative tocompound 1. Both compounds were potent inhibitors ofall 4 FGFR isoforms with greater selectivity over VEGFR2,platelet-derivedgrowth factor receptor (PDGFR) b, and theepidermal growth factor receptor (EGFR) family than TKI-258 or brivanib (Table 1). Compound 1 in particularretained activity against VEGFR1 and VEGFR3 and Flt-3,although a screen conducted against a larger panel ofkinases indicated that only MKNK and RIPK2 were sensi-tive to the compounds at 100 nmol/L and below (Supple-mentary Table S1).More details on the fragment screening,the structure-based drug design approach, and the asso-ciated structure activity relationshipswill be reported else-where in due course.

Crystal structures of compounds 1 and 2 bound toFGFR1 were obtained (Fig. 1C). Both compounds showeda positioning of the imidazopyridine template similar to

Characterization of Small Molecule Inhibitors of FGFR

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compound 3 and the formation of the hydrogen bondwith the N–H of Ala564 on the hinge of the kinase. Asexpected, the aromatic group at position 7 of the imida-

zopyridine showed good surface complementarity withthe protein. From the structure activity relationships ofrelated compounds (data not shown), it is apparent that

Figure 1. A, chemical structuresof 1-{3-[7-(4-fluorophenyl)-imidazo[1,2-a]pyridin-3-yl]-phenyl}-3-(2,2,2-trifluoroethyl)-urea (1); 1-{3-[7-(5-methyl-[1,3,4]oxadiazol-2-yl)-imidazo[1,2-a]pyridin-3-yl]-phenyl}-3-(2,2,2-trifluoroethyl)-urea (2); 3-chloro-imidazo[1,2-a]pyridine-6-carboxylic acidmethyl ester (3). B, the initialfragment ligand (3) bound intoFGFR1 as a protein–ligandcocomplex with the van der Waalsprotein surface (orange). C, theprotein–ligand cocomplex of bothadvanced compounds (1, blue)and (2, purple). D, the chemicalstructure of compound4 (1-{3-[7-(4-acetyl-piperazin-1-yl)-imidazo[1,2-a]pyridin-3-yl]-phenyl}-3-(2,2,2-trifluoroethyl)-urea). Figure 1D shows compound4 bound to both FGFR1 (blue) andVEGFR2 (purple); Arg627, Ala640,and Tyr563 are labeled accordingto the FGFR sequence andnumbering (the correspondingVEGFR2 residues are Arg1032,Cys1045, and Phe918).The water-mediated contactbetween the ligand and Arg627 isonly present in the FGFR1structure, because in the VEGFR2structure, the correspondingarginine adopts a very differentconformation.

D

AIa640

Arg627

Tyr563

F

F

FN

N

O

F

HN

HN

Compound 1

CI

O

ON

N

Compound 3

F

FF

N

Compound 2

NN

NO

O

HNH

N

Compound 4

FF

F

N

NN

N

O

O

HNH

N

A

B C

Table 1. In vitro assays were conducted for the indicated kinases

Kinase Compound1 (IC50), nmol/L

Compound2 (IC50), nmol/L

Brivanib(IC50), nmol/L

TKI-258(IC50), nmol/L

ZD6474(IC50), nmol/L

FGFR3 3 15 52 18 2,200FGFR1 13 78 15 13 1,300FGFR2 33 66 32 21 260FGFR4 34 94 >1,000 470 9,300VEGFR1 13 440 9.0 53 75VEGFR3 68 320 ND 42 220VEGFR2 100 380 4.2 5.4 12PDGFRb 290 2,500 ND 15 1,500Flt3 82 60 ND ND NDEGFR >10,000 >10,000 ND >10,000 30ErbB2 >10,000 >10,000 ND ND 2,900

NOTE: IC50wascalculatedusingPrismsoftwareandexpressedasmeanof at least 2 independent experiments conducted in duplicate.Abbreviation: ND, not determined.

Squires et al.





the urea group is important for both potency and selec-tivity of the compounds. The 2 N–Hs of the urea formed adual hydrogen bond with the carboxylate of Asp641,whereas the carbonyl of the urea formed awater-mediated

hydrogen bond with side chain of Arg627. To our knowl-edge, this positioning for Arg627 has not been seen beforein FGFR1 crystal structures and it appeared to be inducedby the water-mediated interaction with the urea.

Compound 1

Gastric cancer

Endometrial cancer

Myeloproliferative disease

*

*

KG-1Snu-1Snu-16Kato-IIIHec-1AMFE-296AN3-CART112/84RPMI-8226KMS-11FGFR1 Ba/f3FGFR4 Ba/f3FGFR3 Ba/f3Ba/f3 WT

KG-1Snu-1Snu-16Kato-IIIHec-1AMFE-296AN3-CART112/84RPMI-8226KMS-11FGFR1 Ba/f3FGFR4 Ba/f3FGFR3 Ba/f3Ba/f3 WT

Multiple myeloma

Engineered cell lines

Bladder cancer

* -Example of FGFR-independent cell line



*

*

+

+ -FGFR4 Ba/f3 N/D for compound 1


Gastric cancer

Compound 2

1.51.00.50.0–0.5–1.0

1.51.00.50.0–0.5–1.0

log IC50-mean log IC50 (nmol/L)


1.51.00.50.0–0.5–1.0


*

Endometrial cancer

Multiple myeloma


Bladder cancer


Gastric cancer

Endometrial cancer

Multiple myeloma


Bladder cancer

*

*

Brivanib

*

*

*

*

KG-1Snu-1Snu-16Kato-IIIHec-1AMFE-296AN3-CART112/84RPMI-8226KMS-11FGFR1 Ba/f3FGFR4 Ba/f3FGFR3 Ba/f3Ba/f3 WT*

Figure 2. Inhibition of proliferation and survival in FGFR-dependent cell lines. Antiproliferative activity of compounds 1 and 2 were assessed in a panelof human tumor cell lines as described in Materials and Methods. Cell lines known not to be dependent upon FGFR signaling were included (*).Experiments were conducted in triplicate, and data presented are composed of the mean of at least 2 independent experiments and values expressedas the fold change from the mean log IC50.






The lack of potency of compounds 1 and 2 againstVEGFR2 and their low solubility in the crystallographybuffer system precluded us from obtaining a structure ofeither in complex with VEGFR2. However, we did suc-ceed in obtaining a VEGFR2 crystal structure for therelatively potent and selective compound 4 (Fig. 1A)from the same series (FGFR3 IC50 ¼ 12 nmol/L; VEGFR2IC50 ¼ 220 nmol/L). Figure 1D shows the experimentallydetermined binding mode of compound 4 in VEGFR2superimposed on the experimental binding mode of thesame compound in FGFR1. The 2 binding modes for thisligand are very similar and selectivity in the series mustbe driven by subtle energetic differences. One possibleexplanation for the selectivity is induced movement ofArg627 in the FGFR1 structure that does not occur withthe corresponding residue (Arg1032) in VEGFR2. Thislack of movement of Arg1032 is also seen in otherVEGFR2 structures from this series, suggesting a higherenergetic penalty associated with this protein movementin VEGFR2. However, it should be noted that our FGFR1protein structures are obtained with nonphosphorylated

protein, whereas the VEGFR2 crystals are obtained from amixture of mono- and diphosphorylated protein, so dif-ferences in conformational mobility associated with dif-ferent phosphorylation states cannot be discounted.Another possible driver for selectivity is that in VEGFR2,the sulfur of Cys1045 (equivalent to Ala640 in FGFR1) is

approximately 3.4 A�away from the N–H and 3.5 A

�away

from the carbon of the urea group and that these closecontacts are slightly unfavorable in VEGFR2.

Compounds 1 and 2 inhibit proliferation andsurvival of a panel of FGFR-dependent cell lines

Compounds 1 and 2 were assayed for antiproliferativeactivity against a panel of 14 cell lines, comprising Ba/F3cells engineered to express constitutively active forms ofFGFR1, 3, and 4 and lines representative of a number ofdiseases in which FGFR signaling is known to be upre-gulated. In addition, 3 control cell lines were included[Snu-1, a gastric line harboring mutant ras; HEC-1A, awild-type FGFR2 endometrial line; and RPMI-8226, amultiple myeloma line without the t(4;14) translocation].

Figure 3. Compounds 1 and 2exhibit selectivity for FGFR3 overVEGFR2 in cells. Serum-starvedHUVECs (VEGFR2) or KMS-11cells (FGFR3) were incubated withthe indicated compounds for 30minutes. HUVECs were stimulatedby the addition of 100 ng/mLVEGF165 for 5 minutes. An ELISAwas used to monitor eitherpVEGFR2 or pFGFR3. Curves arerepresentative of at least 3individual experiments in eachcase.

120pVEGFR2 - 240 nmol/L

Compound 1 Compound 2

120

20

40

60

80

100 pFGFR3 - 22 nmol/L

% C

on

tro

l%

Co

ntr

ol

% C

on

tro

l

% C

on

tro

l

20

40

60

80

100pFGFR3 - 117 nmol/L

pVEGFR2 - 1,422 nmol/L

–4 –3 –2 –1 0 1 20

log [Compound 1] µmol/L log [Compound 2] µmol/L

–3 –2 –1 0 1 20

20

TKI-258 Brivanib

20

40

60

80

100

120pVEGFR2 - 28 nmol/LpFGFR3 - 300 nmol/L

p-VEGFR2 - 27 nmol/L

p-FGFR3 - 200 nmol/L

–4 –3 –2 –1 0 1 2–20

0

20 % C

on

tro

l

20

40

60

80

100

120

–20

0

20

log [TKI-258] µmol/LZD6474

–5 –4 –3 –2 –1 0 1 2

log [Brivanib] µmol/L

40

60

80

100

120pVEGFR2 - 190 nmol/LpFGFR3 - >10,000 nmol/L

–4 –3 –2 –1 0 1 2–20

0

20

log [ZD6474] µmol/L

Squires et al.





Finally, wild-type Ba/F3 cells that are dependent uponcytokine signaling for survival were included. Bothcompounds 1 and 2 were potent inhibitors of FGFR-dependent cell survival with mean IC50 values inFGFR-dependent cells of 320 and 670 nmol/L andFGFR-independent cells of 3,500 and >6,500 nmol/L,respectively. Examples of cell lines activated by each ofthe FGFR isoforms were included in the panel, and cellproliferation was inhibited in all of these consistent withthe pan-FGFR activity of the compounds (Table 1).Figure 2 is a graphical representation of the data withlog IC50 values expressed as a fold change from themean value. Brivanib alaninate, an example of a broad-spectrum tyrosine kinase inhibitor, was significantlyless potent in the panel overall and exhibited minimalselectivity for FGFR-dependent versus independentlines with a mean IC50 of 2,500 and 6,500 nmol/L,respectively. These data clearly illustrate the selectivityof compounds 1 and 2 in FGFR-dependent systemscompared with those cell lines transformed by othermechanisms.

Compounds 1 and 2 selectively inhibit FGFR in cellsAn ELISA was used to monitor levels of phospho-

FGFR3 in KMS-11 multiple myeloma cells and phos-pho-VEGFR2 in HUVECs following incubation with a

concentration range of compound 1, compound 2, themixed VEGFR2/FGFR inhibitors TKI-258 and brivanib,or the VEGFR inhibitor ZD6474. Both compounds 1 and 2inhibited phospho-FGFR at concentrations around 10-fold lower than those required to inhibit phospho-VEGFR2 (Fig. 3). This is consistent with the selectivityof these compounds in vitro (Table 1). The less selectivecompounds TKI-258, brivanib, and ZD6474 were morepotent against VEGFR2 in HUVECs (28, 27, and 190nmol/L) than against FGFR in KMS-11 (300, 200, and>10,000 nmol/L; Fig. 3).

Inhibition of FGFR2 signaling in cells was confirmedin the FGFR2-amplified gastric cancer cell line Snu-16(Fig. 4). Snu-1 gastric lines were included as a controlline, as they express only low levels of FGFR2 andharbor a Ras mutation. Both compounds 1 and 2 inhib-ited phosphorylation of the FGFR2 receptor in Snu-16cells at concentrations above 100 nmol/L. Inhibition ofdownstream signaling in the mitogen-activated proteinkinase (MAPK) and AKT pathways was also observedat the levels of extracellular signal–regulated kinase(ERK), AKT, and S6. Incubation of Snu-1 cells withthe same concentrations had no inhibitory effect onthese signaling pathways consistent with the lowerantiproliferative activity of the compounds in this cellline (Fig. 2). Inhibition of phospho-FGFR3 and down-

10*1010.100 µmol/LCpd 1/PD173074*

Snu-16Snu-1

ACompound 1

pFGFR2

Total FGFR2

p-FRS-2

Total FRS-2

– + + + + +10*1010.100

– + + + + +FGF1:

p-ERK1/2

Total ERK1/2 p-S6Total S6

Snu-16Snu-1

BCompound 2

µmol/L Cpd 20.0 0.0

60.1

20.2

50.5 1 0.0 0.0

60.1

20.2

50.5 1

pFGFR2

Total FGFR2

p-ERK1/2

Total ERK1/2

µmol/LCpd 1/PD173074*10*1010.100– + + + + +FGF1:

p-S6

Total S6

p-AKT

Total AKT

CCompound 1

KMS-11

pFGFR3

Total FGFR3

p-FRS-2

Total FRS-2

p-ERK1/2

Total ERK1/2

Figure 4. Inhibition of FGFRsignaling in tumor cell lines. Theindicated cell lines were incubatedwith the indicated concentrationsof compounds 1 (A and C) and 2(B) or 10 mmol/L PD173074. ForKMS-11 cells, FGF [(100 ng/ml)/heparin (100 µg/ml)] was addedfor the final 5 minutes. Westernblotting was conductedon cell lysates. Data arerepresentative of at least 3independent experiments.






stream signaling was observed in KMS-11 cells at con-centrations below 100 nmol/L (Fig. 4C). Inhibition ofFGFR1 signaling in KG-1 cells, FGFR2 in AN3-CA andMFE-296, and FGFR3 in 97/7, RT-4, and RT112/84bladder cell lines was also confirmed by the samemethods (Supplementary Fig. S1).

Pharmacokinetic characterization of compounds1 and 2

Supplementary Figure S2 shows the pharmacokineticprofiles of compounds 1 and 2 following oral dosing inthe mouse. Compound 1 exhibits high oral bioavailabilityin the mouse (79%) and excellent dose linearity withrespect to Cmax (19.5 and 28.6 mg/mL) and area undercurve (AUC; 179 and 287 h mg/ml for 50 and 100 mg/kg,respectively). Compound 2 exhibits similarly favorablekinetics with an oral bioavailability of 100% and doselinearity (Cmax values of 16, 33, and 49 mg/mL andAUC values of 65, 130, and 330 h mg/ml for 12.5, 25,and 50 mg/kg, respectively).

Stability in liver microsomes suggested low clearancefor both compounds (Supplementary Table S2) and thiswas confirmed in vivo. Metabolic clearance was 2.3 and5.1 mL/min/kg, respectively, for compound 1 in mouseand rat and 5.2 and 1.1 mL/min/kg for compound 2following intravenous dosing. Low turnover in humanmicrosomes suggested that therapeutic exposures shouldbe achievable in human subjects.

Pharmacodynamic studies in human tumorxenografts

Inhibition of FGFR and downstream signaling wasinvestigated in human tumor xenograft models (Fig. 5).A single dose of either compound 1 or 2 at 50 mg/kgorally completely ablated phospho-FGFR3 in the Ba/F3-

TEL/FGFR3 model (Fig. 5A). This inhibition was shownto be dose dependent for compound 1 (SupplementaryFig. S3). In Snu-16 (Fig. 5B) and KMS-11 (Fig. 5C) xeno-grafts, phospho-FGFR2 and phospho-FGFR3 were inhib-ited for more than 8 hours following a single dose ofcompound 1. Inhibition of downstream MAPK and AKTsignaling and an increase in the levels of cleaved PARP, amarker of apoptosis, were observed at 8 hours in theSnu-16. Figure 5D shows RPMI-8226 tumors removedfrom animals 4 hours after treatment with the indicateddoses of compound 1. In this FGFR-independent model,no effect was observed on either MAPK or PARP.

Efficacy in human tumor xenograftsThe antitumor efficacy of both compounds 1 and 2 was

investigated in FGFR3-dependent multiple myelomaxenografts (Fig. 6A and C). For this purpose, we usedKMS-11 cells that express high levels of a mutant FGFR3(Fig. 4C) and Jim-1 cells that express physiologic levels ofwild-type FGFR3 (Supplementary Fig. S3). Compoundswere administered orally, once daily at doses that toler-ability studies suggested were below the maximum tol-erateddoses.Administrationof 50mg/kgcompound1 forup to 21 days resulted in minimal body weight loss andno observations of gross toxicity were made. For com-pound 2, no adverse toxicities were observed at dosesup to 100 mg/kg once daily when dosed for up to26 days. Compound 1 caused tumor growth inhibitionat 12.5mg/kg orally in the Jim-1model with regression oftumor volume observed at both 25 and 50 mg/kg. At50 mg/kg, all animals showed tumor shrinkage by day 5,which persisted to day 10. By day 16, 50% of these tumorswere still smaller than on day 1. Compound 2 was lesseffective in the Jim-1 model at 50 mg/kg consistent withthe lower potency of this compound. The higher dose of

Figure 5. Inhibition of FGFRsignaling in xenograft tumors.Mice bearing Ba/F3-TEL/FGFR3(A), Snu-16 (B), or KMS-11 (C)xenografts were treated with asingle dose of compound 1 or 2, at50 mg/kg, as indicated. Triplicatetumor samples were removed atsubsequent times and processedto determine levels of phospho-signaling by immunoblotting.Nude mice bearing RPMI-8226xenografts (D) had compound 1administered daily for 21 days atthe indicated doses. Tumors wereremoved at 4 hours following thefinal dose.

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100 mg/kg, however, caused an equivalent growthinhibition in the KMS-11 model to that caused by50 mg/kg compound 1. Both compounds were alsoefficacious in the endometrial xenograft model AN3-CA. Compound 1 was not efficacious even at the highestdose (50 mg/kg) tested in the FGFR-independent RPMI-8226 model (Fig. 6B), a dose and schedule that resultedin complete growth inhibition in the FGFR-positive Jim-1 model. Compound 2 had no efficacy in the FGFR-independent MDA-MB-231 breast cancer xenograft, amodel known to be dependent upon EGFR rather thanFGFR for survival (Fig. 6H). In contrast to compounds 1and 2, the mixed FGFR/VEGFR2 inhibitor TKI-258exhibited similar growth inhibitory activity in boththe Jim-1 and RPMI-8226 xenografts (Fig. 6A and B).This suggests that a proportion of the activity of thiscompound in xenograft models is due to inhibition ofkinases in addition to FGFR, most probably an effecton tumor angiogenesis due to VEGFR2 inhibition. Theselective inhibition of FGFR-dependent systems by

compounds 1 and 2 showed the potential to exploitthese selective compounds in a targeted fashion.

Discussion

Here, we describe the characterization of 2 fragment-derived, specific inhibitors of the FGFR family of kinases.Compounds 1 and 2 are potent inhibitors of all 4 isoformsof the FGFR family with no appreciable activity in a panelof related receptor tyrosine kinases including EGFR andPDGFRb. The compounds were around 20-fold selectiveagainst the highly homologous VEGFR2 kinase. Theywere selective both in cell-based assays and in vivowherethis apparently modest level of selectivity in vitro trans-lated to a highly specific tumor inhibitory effect in FGFR-dependent xenografts with no activity in xenografts notdependent upon FGFR signaling. These data more thanany other confirm that the compounds are acting in anFGFR-specific manner when exerting their effects in thesemodel systems.

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Figure 6. Compounds 1 and 2 are efficacious in FGFR-dependent tumor xenograft models. Compound 1 was administered orally to mice bearingFGFR-dependent (KMS-11, Jim-1, AN3-CA; A) or FGFR-independent xenografts (RPMI-8226; B) once daily at the indicated doses and time periods. For theJim-1 and RPMI-8226 studies, TKI-258 was included as a positive control. Compound 2 was administered orally to mice bearing FGFR-dependent(KMS-11, Jim-1, AN3-CA); C or FGFR-independent xenografts (MDA-MB-231); D once daily for the indicated doses and time periods. Groups were takenoff study at the end of the study period or when tumors reached greater than 1,000 mm3.






This is the first detailed pharmacologic characteriza-tion of compounds that exhibit selectivity for FGFRfamily members over VEGFR2. With the growing interestin targeting FGFR, broad studies such as those presentedhere will help define the pharmacologic profile of aspecific FGFR inhibitor across a relevant panel of pre-clinical systems. Several studies in defined tumor typeshave been conducted with PD173074 (12, 31, 32), but theypredominantly use engineered cell lines or those repre-senting a single disease type. A lack of small moleculeand biological inhibitors specific for FGFR and a lack ofwell-characterized reagents have, to date, preventedsimilar studies.

There are several indications within oncology inwhich targeting the FGFR pathway specifically maybe of benefit. These include tumor types with specificactivating mutations in FGFR (5–7, 31), those withamplification of one of the receptor family members(2) or more broadly as an antiangiogenic therapyfollowing VEGFR therapy in tumors in which FGF-2has been upregulated (24). Several molecules are orhave been investigated with the aim of potentiallyexploiting their FGFR activity. PD173074 showedselectivity for the FGFR receptor in vitro and a numberof reports described its activity in individual FGFR-dependent systems (12, 14), but it did not proceed intoclinical development. Compounds explored clinicallyinclude brivanib alaninate (BMS-582664; ref 29, 33) inhepatocellular carcinoma (NCT00355238), endometrial(NCT06888173), and colorectal cancer (NCT00207051)and TKI-258 (Novartis) in multiple myeloma(NCT01058434), renal cell carcinoma (NCT00715182),urothelial cell carcinoma (NCT00790426), and breastcancer (NCT 00958971). Isoform-specific antibodytherapies are also in preclinical development. Two

recent publications describe the potent activity ofFGFR2-IIIb–specific (34) and FGFR2-specific (35) anti-bodies in preclinical models of gastric cancer, harboringFGFR2 amplification. This further supports the impor-tant role of FGFR2 in this indication.

Data presented herein show that broad spectrumcompounds have in vivo activity against both FGFR-dependent and -independent models, most likely dri-ven by their antiangiogenic activity via VEGFR2 inhibi-tion. Thus, it is difficult to show FGFR-specific effectsattributable to these compounds either preclinically orin clinical studies. One possible limitation of theseinhibitors in FGFR-driven disease is the appearanceof intervening toxicities due to inhibition of additionalkinases, which prevents maximal inhibition of FGFR inrelevant tumor settings.

Compounds 1 and 2 described here exhibit very dis-tinct activity profiles in FGFR-dependent and -indepen-dent in vitro and in vivo systems. As such, the compoundsoffer an exciting and innovative approach to targetingcancers dependent upon FGFR signaling.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

Aspects of this work were supported by grants from Cancer Research UK andLeukaemia and Lymphoma Research.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received June 8, 2011; accepted June 21, 2011; published OnlineFirstJuly 15, 2011.

References1. Turner N, Grose R. Fibroblast growth factor signalling: from devel-

opment to cancer. Nat Rev Cancer 2010;10:116–29.2. Turner N, Pearson A, Sharpe R, Lambros M, Geyer F, Lopez-Garcia

MA, et al. Integrative molecular profiling of triple negative breastcancers identifies amplicon drivers and potential therapeutic targets.Oncogene 2010;29:2013–23.

3. Forbes S, Clements J, Dawson E, Bamford S, Webb T, Dogan A, et al.COSMIC 2005. Br J Cancer 2006;94:318–22.

4. Malgeri U, Baldini L, Perfetti V, Fabris S, Vignarelli MC, Colombo G,et al. Detection of t(4;14)(p16.3;q32) chromosomal translocation inmultiple myeloma by reverse transcription-polymerase chain reactionanalysis of IGH-MMSET fusion transcripts. Cancer Res 2000;60:4058–61.

5. Chesi M, Nardini E, Lim RS, Smith KD, Kuehl WM, Bergsagel PL. Thet(4;14) translocation inmyeloma dysregulates both FGFR3 and a novelgene, MMSET, resulting in IgH/MMSET hybrid transcripts. Blood1998;92:3025–34.

6. Chang H, Qi XY, Samiee S, Yi QL, Chen C, Trudel S, et al. Genetic riskidentifiesmultiplemyelomapatientswhodonot benefit fromautologousstem cell transplantation. Bone Marrow Transplant 2005;36:793–6.

7. Chesi M, Brents LA, Ely SA, Bais C, Robbiani DF, Mesri EA,Activated fibroblast growth factor receptor 3 is an oncogene thatcontributes to tumor progression in multiple myeloma. Blood2001;97:729–36.

8. Li Z, Zhu YX, Plowright EE, Bergsagel PL, Chesi M, Patterson B, et al.The myeloma-associated oncogene fibroblast growth factor receptor3 is transforming in hematopoietic cells. Blood 2001;97:2413–9.

9. Plowright EE, Li Z, Bergsagel PL, Chesi M, Barber DL, Branch DR,et al. Ectopic expression of fibroblast growth factor receptor 3 pro-motes myeloma cell proliferation and prevents apoptosis. Blood2000;95:992–8.

10. Pollett JB, Trudel S, Stern D, Li ZH, Stewart AK. Overexpressionof the myeloma-associated oncogene fibroblast growth factorreceptor 3 confers dexamethasone resistance. Blood 2002;100:3819–21.

11. Chen J, Lee BH, Williams IR, Kutok JL, Mitsiades CS, Duclos N, et al.FGFR3 as a therapeutic target of the small molecule inhibitor PKC412in hematopoietic malignancies. Oncogene 2005;24:8259–67.

12. Grand EK, Chase AJ, Heath C, Rahemtulla A, Cross NC. TargetingFGFR3 in multiple myeloma: inhibition of t(4;14)-positive cells bySU5402 and PD173074. Leukemia 2004;18:962–6.

13. Trudel S, Ely S, Farooqi Y, Affer M, Robbiani DF, Chesi M, Inhibition offibroblast growth factor receptor 3 induces differentiation and apop-tosis in t(4;14) myeloma. Blood 2004;103:3521–8.

14. Trudel S, Li ZH, Wei E, Wiesmann M, Chang H, Chen C, et al. CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for thepotential treatment of t(4;14) multiple myeloma. Blood 2005;105:2941–8.

Squires et al.





15. Trudel S, Stewart AK, Rom E, Wei E, Li ZH, Kotzer S, et al. Theinhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to t(4;14) multi-ple myeloma cells. Blood 2006;107:4039–46.

16. Xin X, Abrams TJ, Hollenbach PW, Rendahl KG, Tang Y, Oei YA, et al.CHIR-258 is efficacious in a newly developed fibroblast growth factorreceptor 3-expressing orthotopic multiple myeloma model in mice.Clin Cancer Res 2006;12:4908–15.

17. Qing J, Du X, Chen Y, Chan P, Li H, Wu P, et al. Antibody-basedtargeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiplemyeloma in mice. J Clin Invest 2009;119:1216–29.

18. Kunii K, Davis L, Gorenstein J, Hatch H, Yashiro M, Di Bacco A, et al.FGFR2-amplified gastric cancer cell lines require FGFR2 and Erbb3signaling for growth and survival. Cancer Res 2008;68:2340–8.

19. Takeda M, Arao T, Yokote H, Komatsu T, Yanagihara K, Sasaki H,et al. AZD2171 shows potent antitumor activity against gastric cancerover-expressing fibroblast growth factor receptor 2/keratinocytegrowth factor receptor. Clin Cancer Res 2007;13:3051–7.

20. Weiss J, Sos ML, Seidel D, Peifer M, Zander T, Heuckmann JM, et al.Frequent and focal FGFR1 amplification associates with therapeuti-cally tractable FGFR1 dependency in squamous cell lung cancer. SciTrans Med 2010;2:1–6.

21. Ramos AH, Dutt A, Mermel C, Perner S, Cho J, Lafargue CJ, et. al.Amplification of chromosomal segment 4q12 in non-small cell lungcancer. Cancer Biol Ther 2009;8:2042–50.

22. Elbauomy Elsheikh S, Green AR, Lambros MB, Turner NC, GraingeMJ, Powe D, et al. FGFR1 amplification in breast carcinomas: achromogenic in situ hybridisation analysis. Breast Cancer Res 2007;9:R23.

23. Taylor JG, Cheuk AT, Tsang PS, Chung JY, Song YK, Desai K, et al.Identification of FGFR4-activating mutations in human rhabdomyo-sarcomas that promote metastasis in xenotransplantedmodels. J ClinInvest 2009;119:3395–407.

24. Casanovas O, Hicklin DJ, Bergers G, Hanahan D. Drug resistance byevasion of antiangiogenic targeting of VEGF signaling in late-stagepancreatic islet tumors. Cancer Cell 2005;8:299–309.

25. Hartshorn MJ, Murray CW, Cleasby A, Frederickson M, Tickle IJ, JhotiH. Fragment-based lead discovery using X-ray crystallography. JMedChem 2005;48:403–13.

26. Gill A, Cleasby A, Jhoti H. The discovery of novel protein kinaseinhibitors by using fragment-based high-throughput X-ray crystal-lography. Chembiochem 2005;6:506–12

27. Murray CW, Rees DC. The rise of fragment based discovery chem-istry. Nat Chem 2009;1:87–92.

28. Berdini V, Besong GE, Callaghan O, Carr MG, Congreve MS, Gill AL,et al. Preparation of bicyclic heterocyclic compounds as FGFRinhibitors. United States patent application US20100120761 2010May 13

29. McTigue MA, Wickersham JA, Pinko C, Showalter RE, Parast CV,Tempczyk-Russell A, et al. Crystal structure of the kinase domain ofhuman vascular endothelial growth factor receptor 2: a key enzyme inangiogenesis. Structure 1999;7:319–30.

30. Hopkins AL, Groom CR, Alex A. Ligand efficiency: a useful metric forlead selection. Drug Discov Today 2004;9:430–1.

31. Byron SA, Gartside MG, Wellens CL, Mallon MA, Keenan JB, PowellMA, et al. Inhibition of activated fibroblast growth factor receptor 2 inendometrial cancer cells induces cell death despite PTEN abrogation.Cancer Res 2008;68:6902–7.

32. Pardo OE, Latigo J, Jeffery RE, Nye E, Poulsom R, Spencer-Dene B,et al. The fibroblast growth factor receptor inhibitor PD173074 blockssmall cell lung cancer growth in vitro and in vivo. Cancer Res2009;69:8645–51.

33. Cai ZW, Zhang Y, Borzilleri RM, Qian L, Barbosa S, Wei D, et. al.Discovery of brivanib alaninate ((S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)pro-pan-2-yl)2-aminopropanoate), a novel prodrug of dual vascularendothelial growth factor receptor-2 and fibroblast growth factorreceptor-1 kinase inhibitor (BMS-540215). J Med Chem 2008;51:1976–80.

34. Bai A, Meetze K, Y.Vo N, Kollipara S, Mazsa EK, Winston WM, et al.GP369, an FGFR2-IIIb-specific antibody, exhibits potent anti-tumoractivity against human cancers driven by activated FGFR2 signaling.Cancer Res 2010;70:7630–9.

35. Zhao W, Wang L, Park H, Chhim S, Tanphanich M, Yashiro M, et al.monoclonal antibodies to fibroblast growth factor receptor 2 effec-tively inhibit growth of gastric tumor xenografts. Clin Cancer Res2010;16:5750–8.






Published OnlineFirst July 15, 2011.Mol Cancer Ther Matthew Squires, George Ward, Gordan Saxty, et al. ModelsDefine Fibroblast Growth Factor Dependence in Preclinical Cancer Potent, Selective Inhibitors of Fibroblast Growth Factor Receptor

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