Assessing the Activity of Cediranib, a VEGFR-2/-3 …mct.aacrjournals.org/content/molcanther/early/2011/03/24/1535-7163... · 4 Abstract Cediranib is a potent inhibitor of the vascular

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Assessing the Activity of Cediranib, a VEGFR-2/-3 tyrosine kinase inhibitor, against

VEGFR-1 and members of the structurally related PDGFR-family

Sandra R. Brave1, Kirsty Ratcliffe1, Zena Wilson1, Neil H. James1, Sue Ashton1, Anna

Wainwright1, Jane Kendrew1, Philippa Dudley1, Nicola Broadbent1, Graham Sproat1, Sian

Taylor1, Claire Barnes1, Jeffrey C. Silva2, Charles L. Farnsworth2, Laurent Hennequin3,

Donald J. Ogilvie1,5, Juliane M. Jürgensmeier1, Masabumi Shibuya4, Stephen R. Wedge1,and

Simon T. Barry1

Authors Affiliations: 1Cancer Bioscience, AstraZeneca, Alderley Park, Macclesfield, Cheshire,

SK10 4TG, United Kingdom; 2 Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923,

United Sates of America; 3AstraZeneca Pharma, Centre de Recherches, Z.I. La Pompelle,

Chemin de Vrilly, Reims, France and 4Department of Molecular Oncology, Tokyo Medical and

Dental University, Tokyo 113-8519, Japan. 5Current address: Paterson Institute for Cancer

Research Wilmslow Rd., Manchester, M20 4BX, United Kingdom

Running Title: Assessment of cediranib selectivity

Key Words: Cediranib, AZD2171, VEGF, VEGF receptor-1, c-Kit, PDGFR,

angiogenesis, tyrosine kinase,

Abbreviations

AML, acute myeloid leukemia

ATP, adenosine triphosphate

BrdU, bromodeoxuridine

BSA, bovine serum albumin

CST, Cell Signaling Technology

DMEM, Dulbecco's modified Eagle's medium

DMSO, dimethyl sulphoxide

ELISA, enzyme-linked immunosorbent assay

FGFR, fibroblast growth factor receptor

on September 16, 2018. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 25, 2011; DOI: 10.1158/1535-7163.MCT-10-0976

http://mct.aacrjournals.org/

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GIST, gastrointestinal stromal tumor

HCl, hydrochloric acid

IC50, half maximal inhibitory concentration

LTQ, linear trap quadrupole

MAPK, mitogen-activated protein kinase

MSD, Meso Scale Discovery

NaF, sodium fluoride

PDGFR, platelet-derived growth factor receptor

PlGF, placental growth factor

SCF, stem cell factor

SCLC, small cell lung cancer

SDS, sodium dodecylsulfate

SEM, standard error of the mean

VEGFR, vascular endothelial growth factor receptor

VSMC, vascular smooth muscle cells

XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium

hydroxide

Financial Support:

Sandra R. Brave, Kirsty Ratcliffe, Zena Wilson, Neil H. James, Sue Ashton, Anna Wainwright,

Jane Kendrew, Philippa Dudley, Nicola Broadbent, Graham Sproat, Sian Taylor, Claire

Barnes, Laurent Hennequin, Donald J. Ogilvie, Juliane M. Jürgensmeier, Stephen R. Wedge,

and Simon T. Barry are current or former employees and shareholders of AstraZeneca.

Jeffrey C. Silva, Charles L. Farnsworth, are employees of Cell Signaling Technology.

The analysis was performed by Cell Signaling Technology funded by AstraZeneca.

Corresponding Authors:

Simon Barry Cancer Bioscience, AstraZeneca, Alderley Park, Macclesfield, Cheshire, SK10

4TG, United Kingdom (+44 1625 513350) e-mail: [email protected]




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Steve Wedge Cancer Bioscience, AstraZeneca, Alderley Park, Macclesfield, Cheshire, SK10

4TG, United Kingdom (+441625 523236) e-mail: [email protected]




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Abstract

Cediranib is a potent inhibitor of the vascular endothelial growth factor receptor (VEGFR)-2

and -3 tyrosine kinases. This study assessed the activity of cediranib against the VEGFR-1

tyrosine kinase and the platelet-derived growth factor receptor (PDGFR)-associated kinases

c-Kit, PDGFR-α and -β. Cediranib inhibited VEGF-A-stimulated VEGFR-1 activation in

AG1-G1-Flt1 cells (IC50, 1.2nM). VEGF-A induced greatest phosphorylation of VEGFR-1 at

tyrosine residues Y1048 and Y1053; this was reversed by cediranib. Potency against

VEGFR-1 was comparable to that previously observed versus VEGFR-2 and R-3. Cediranib

also demonstrated significant activity against wild-type c-Kit in cellular phosphorylation assays

(IC50, 1−3nM) and in a stem cell factor-induced proliferation assay (IC50, 13nM). Furthermore,

phosphorylation of wild-type c-Kit in NCI-H526 tumor xenografts was reduced markedly

following oral administration of cediranib (≥1.5 mg/kg/day) to tumor-bearing nude mice. The

activity of cediranib against PDGFR-β and -α was studied in tumor cell lines, vascular smooth

muscle cells (VSMC) and a fibroblast line using PDGF-AA and PDGF-BB ligands. Both

receptor phosphorylation (IC50, 12–32nM) and PDGF-BB-stimulated cellular proliferation (IC50,

32nM in human VSMC; 64nM in osteosarcoma cells) were inhibited. In vivo, ligand induced

PDGFR-β phosphorylation in murine lung tissue was inhibited by 55% following treatment

with cediranib at 6 mg/kg, but not ≤3 mg/kg. In contrast, in C6 rat glial tumor xenografts in

mice, ligand induced phosphorylation of both PDGFR-α and -β was reduced by 46−61% by

cediranib 0.75 mg/kg. Additional selectivity was demonstrated versus Flt-3, CSF1R, EGFR,

FGFR1 and FGFR4. Collectively, these data indicate that cediranib is a potent pan-VEGFR

kinase inhibitor with similar activity against c-Kit, but is significantly less potent versus

PDGFR-α and –β.




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Introduction

Inhibition of vascular endothelial growth factor-A (VEGF) mediated signalling has been found

to give therapeutic benefit in oncology, as monotherapy in the treatment of renal cell cancer

(1, 2), and when combined with certain cytotoxic therapies in other disease settings (3).

Approaches used to inhibit VEGF receptor (VEGFR) signalling include small molecule

inhibitors that bind into the adenosine triphosphate (ATP)-binding pocket within the kinase

domain of VEGFR-2, to prevent ATP catalysis and propagation of receptor signalling. This

receptor predominantly transduces the angiogenic and permeability activity of VEGF-A. Two

other VEGF receptors are also described with differential binding of VEGF family ligands.

Each small molecule differs in its potency against the VEGF receptors, selectivity versus

other kinases, physicochemical properties and pharmacokinetic profile (4, 5). Some VEGFR

tyrosine kinase inhibitors have additional activity against one or more members of the platelet-

derived growth factor receptor (PDGFR) family of kinases (class III), which comprises

PDGFR-α, PDGFR-β, c-Kit, colony-stimulating factor receptor-1 and Flt-3; these receptors

having some structural homology to the VEGFR family members in that each harbors a

kinase-insert sequence in its intracellular domain (6). However, VEGFR kinase inhibitors have

also been described with activity against tyrosine kinases outside of the PDGFR class, and in

some case against serine/threonine kinases. Inhibiting multiple kinases simultaneously may

provide additional therapeutic opportunities in defined disease settings, but may impact

adversely on tolerability, particularly if chronic administration or usage in combination with

concurrent cytotoxic treatment, is required.

Cediranib has been previously shown to be a potent inhibitor of VEGFR-2 and -3 signalling in

cellular assays and to inhibit the growth of both angiogenic blood vessels and lymph-

angiogenic vessels in vivo (7−9). In recombinant kinase assays, cediranib also inhibits

VEGFR-1 kinase activity (IC50 of 5 nM) within a similar concentration range to VEGFR-2 and -

3 (IC50 values of <0.1 nM and ≤3nM respectively) (7). However, a more quantitative

assessment of inhibitor activity against endogenous VEGFR-1 kinase has proven technically

challenging in endothelial cells because of the low intrinsic activity of this receptor. Cediranib

demonstrates selectivity versus other kinases (7). Cediranib has similar potency against c-Kit




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when compared to VEGFR-2 in phosphorylation assays, but less potency against PDGFR-α

and -β, particularly in a PDGF-AA/PDGFR-α driven tumor cell proliferation assay. However,

the activity of cediranib against a c-Kit driven phenotypic endpoint, or PDGFR-α and -β

signalling in normal cell types (which may also influence therapeutic response), or a

comparative inhibition of these targets in vivo, was not previously examined.

This study primarily aimed to further describe the pharmacology of cediranib by (i)

determining activity against VEGFR-1 in cells, (ii) using a wider complement of cell lines and

assays to examine activity against c-Kit and PDGFR-α/-β signalling in vitro, and (iii) to

examine pharmacodynamic inhibition of c-Kit, PDGFR-α and PDGFR-β in vivo over a dose-

range where cediranib has previously shown activity in tumor models. The data confirm that

cediranib is primarily a pan-VEGFR inhibitor that can inhibit wild-type c-Kit. The data also

suggest that cediranib may have some partial pharmacodynamic activity against PDGFR-α

and -β receptor activation in tumors, although this inhibition may be of limited functional

relevance, but does inhibit FGFR1 and FGFR4.




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Materials and Methods

Reagents

Cediranib [(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)

quinazoline (AZD2171) (Fig.1) was synthesized according to the processes described in

WO00/47212, in particular those described in Example 240 of WO/47212. The free base of

cediranib was used in these preclinical studies, with a molecular weight of 450.51. For all in

vitro assays, cediranib was prepared initially as a 10 mM stock solution in dimethyl sulphoxide

(DMSO) and diluted in the relevant assay media, such that the final concentration of DMSO

did not exceed 0.01%, with the exception of studies examining direct effects on tumor cells

where 1% DMSO was required to examine higher concentrations of cediranib. All in vivo

studies were conducted by once-daily oral gavage. For studies in mice, cediranib was

suspended in 1% (weight/volume) aqueous polysorbate 80 (polyoxyethylene (20) sorbitan

mono-oleate in deionized water) and dosed at 0.1 ml/10 g of body weight.

Cell culture

NCI-H526 (a human small cell lung cancer [SCLC] line), U118MG (a human glioblastoma

line), MG63 (a human osteosarcoma line) and C6 (a rat glial line) cells were purchased from

the American Type Culture Collection (Manassas, Virginia, USA), no further authentication

was performed on these lines. The M07e (a human acute myelogenous leukemia [AML] line)

cells were purchased from the German Collection of Microorganisms and Cell Cultures

(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of

Microorganisms and Cell Cultures); Braunschweig, Germany), no further authentication was

performed. NIH 3T3 (mouse fibroblast) cells were obtained from A. Wong, Jefferson Cancer

Institute, no further authentication was performed. Human aortic and coronary vascular

smooth muscle cells (VSMC) were purchased from PromoCell GmbH (Heidelberg, Germany).

All cell lines were routinely passaged less than 10 times with the exception of the primary

vascular cells which were passaged no more than 4 times. NCI-H526, U118MG, MG63, C6,

M07e, NIH 3T3, human aortic and coronary VSMC were maintained in culture as per

providers recommendation. M07e cells were maintained in culture in the presence of

interleukin-3 (5 ng/ml) and granulocyte macrophage colony stimulating factor (5 ng/ml).




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Inhibition of growth factor stimulated receptor phosphorylation in vitro

The ability of cediranib to inhibit receptor phosphorylation in cells was determined using

Western blotting. Cells were serum starved overnight in the presence (M07e and NCI-H526)

or absence (aortic and coronary VSMC) of 0.1% bovine serum albumin (BSA) or in the

presence of 1% charcoal stripped serum (MG63, U118MG, C6, NIH 3T3 cells). Cells were

then incubated with cediranib for 60−120 min and stimulated with the relevant ligand: stem

cell factor (SCF, 50 ng/ml), PDGF-AA or PDGF-BB (50 ng/ml) for 5−10 min. SCF was

obtained from R&D Systems Inc. (Abingdon, Oxfordshire, UK) and PDGF-AA and PDGF-BB

from Sigma-Aldrich (Poole, Dorset, UK). Cell lysates of NCI-H526, M07e, aortic and coronary

VSMC were made in lysis buffer I (50 mM Tris-HCl pH 7.6; 137 mM sodium chloride; 10%

glycerol; 0.1% Igepal; 0.1% sodium dodecylsulfate [SDS]; 50 mM sodium fluoride; 1mM

sodium orthovanadate and cocktail protease inhibitor tablets [Roche Diagnostics Ltd., Lewes,

UK)). Cell lysates of MG63, U118MG, C6 and NIH 3T3 cells were made in lysis buffer 2 (10%

glycerol, 2% SDS, 50 mM Tris-HCL, 200mM 2-mercaptoethanol).

The protein concentration in the lysates was determined using a bicinchoninic acid (BCA)

assay kit (Pierce, IL, USA) and Western blotting was performed on whole cell lysates (50–75

μg of protein loaded per lane) using standard SDS-PAGE methods with detection by

enhanced chemiluminescence. Total and phosphorylated proteins were measured using

antibodies to c-Kit (CST 3391; Cell Signaling Technology, Inc. [CST], Beverley,

Massachusetts, USA), and phosphorylated c-Kit (SC5535; Santa Cruz Biotechnology Inc,

Santa Cruz, California, USA); PDGFR-α (AF-307, R&D Systems Inc.), PDGFR-α (CST 3164),

and phosphorylated PDGFR-α (SC12910, Santa Cruz); PDGFR-β (AF-385, R&D Systems

Inc.), PDGFR-β (1469-1, Epitomics, Suffolk, UK), phosphorylated PDGFR-β (CST 3161);

mitogen-activated protein kinase (MAPK) (CST 9102) and phosphorylated MAPK (CST 9101).

Phosphorylation was quantitated using the ChemiGenius imaging system for

Chemiluminescence (Syngene, USA) with the exception of the human coronary VSMC which




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were quantified by enzyme-linked immunosorbent assay (ELISA) (Goat Anti-PDGFR Alpha

R&D systems AF-307-NA capture, R&D Systems Inc. Duoset detection).

AG1-G1-Flt-1 cells were established with the permission of the Ethics Committee for scientific

research at the Institute of Medical Science, University of Tokyo, Tokyo, Japan. Briefly, a

human adult benign angioma was excised surgically and plated with F12-nutrient mixture

(Ham) medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS)

(JRH Biosciences, Leneza, Kansas) and 40 μg/ml kanamycin (Wako, Osaka, Japan). A

pEF1α-SV40 large T antigen plasmid was introduced into the cells using dimethyl sulphoxide

(DMSO) and polybrene (SIGMA-Aldrich, St Louis, Missouri). An SV40 large T-positive clone

AG1-G1 cell was isolated, and then pBCMGS-Neo-Flt-1 carrying the full length of Flt-1 cDNA

(10), or the empty vector pBCMGS-Neo plasmid was transfected into AG1-G1 cell by

Effectene Transfection Reagent (QIAGEN, Valencia, CA). Clone selection and culture were

performed with HAMS F-12 medium containing 10% FBS, 40 μg/ml kanamycin and 400 μg/ml

geneticin G418 (Wako). G418 was decreased to 200 μg/ml in regular culture. To examine

inhibition of VEGFR-1 phosphorylation, cells were placed in serum-free media overnight and

then incubated with cediranib for 90 min and stimulated with VEGF 50 ng/ml (R & D Systems

Inc., Abingdon, UK) for the last 5 min of incubation. Cell lysates were made in lysis buffer 1

and phosphorylated VEGFR-1 evaluated using Meso Scale methodology (Meso Scale

Discovery [MSD], Maryland, USA). The phosphorylated VEGFR-1 was analyzed by MSD

ELISA. Total VEGFR-1 antibody (MAB321, R & D Systems Inc., Abingdon, UK) was spotted

onto high-bind MSD plates and incubated for 2 h at room temperature after which time plates

were blocked and then washed. Cell lysates were added and incubated overnight at 4°C.

Plates were washed, the sulphotagged-pY20 detection antibody added and left to incubate for

1 h at room temperature. Following washing, read buffer was added and plates were read

immediately on the SECTOR™ Imager 6000 (MSD, Maryland, USA). To visualize

phosphorylated VEGFR-1 by Western blot, VEGFR1 was immune-precipitated with an

antibody to total VEGFR-1 (SC316, Santa Cruz), and immunoblotted with the anti-

phosphotyrosine antibody PY20 (SC508, Santa Cruz). Levels of total VEGFR-1 were

confirmed by immunoblotting with SC316.




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Sample preparation and mass spectrometry for identification of phosphotyrosine

modification of VEGFR-1

Analysis of VEGFR-1 phosphorylated epitope changes was performed using Cell Signaling

Technology’s proprietary PhosphoScan™ methodology. AG1-G1-Flt-1 cells were placed in

serum-free media overnight and stimulated with VEGF (50 ng/ml) or placenta growth factor

(PlGF) (100 ng/ml, R&D Systems) for 5 min, the former also with and without cediranib (100

nM; 85 min pre-incubation and 5 min co-incubation with ligand). Protein extracts from

AG1-G1-Flt1 cells were prepared by suspending cells in Urea Lysis Buffer (20 mM HEPES

[pH 8.0], 9.0 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-

glycerol phosphate). Lysates generated from approximately 2 x 108 cells were prepared for

each sample condition (control, VEGF-treated, PlGF-treated and VEGF- + cediranib-treated.

The resulting protein extracts (40 mg total protein each) were then reduced with dithiothreitol

(4.5 mM), carboxamidomethylated using iodoacetamide (10.0 mM), and subsequently

digested with trypsin (1:100 weight, trypsin to total protein). Peptides were separated from

non-peptide material by solid-phase extraction with Sep-Pak C18 cartridges. Lyophilized

peptides were re-dissolved, and phosphorylated peptides were isolated using a slurry of

immobilized phosphorylated tyrosine antibody (CST #9411) conjugated to protein-G agarose

beads (Roche, Indianapolis, Indiana, USA). Peptides were eluted from antibody-resin into a

total volume of 100 µL in 0.15% trifluoroacetic acid. Eluted peptides were concentrated with

PerfectPure C18 tips immediately prior to liquid chromatography−mass spectrometry (MS)

analysis. Peptides were loaded directly onto a 10 cm x 75 µm PicoFrit capillary column

packed with Magic C18 AQ reversed-phase resin. The column was developed with a 45 min

linear gradient of acetonitrile in 0.125% formic acid delivered at 280 nL/min. Tandem mass

spectra were collected with an linear trap quadrupole (LTQ)-Orbitrap hybrid mass

spectrometer, using a top-ten method, a dynamic exclusion repeat count of 1 and a repeat

duration of 30 s. MS spectra were collected in the Orbitrap component of the mass

spectrometer, and MS/MS spectra were collected in the LTQ. MS/MS spectra were evaluated

using TurboSequest in the Proteomics Browser package (v. 27, rev. 13) The ratios of the

integrated peak height intensities for phosphopeptide quantification were obtained using the




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XCalibur software 2.0.7 (ThermoFinnigan, San Jose, CA). A reduction in peak intensity in

VEGF-treated cells compared to control, or PlGF-treated cells compared with control or VEGF

plus cediranib-treated cells compared to control was expressed as a negative fold change

value. All integrated peak intensity calculations were manually reviewed to ensure proper

integration of consistently shaped, co-eluting peaks. Changes in phosphorylated peptide

levels were measured by taking the ratio of raw intensities between control and treated cells,

with the untreated sample as the reference (denominator) in each case. Raw intensity ratios

were normalized using a median adjustment technique whereby the log2 ratios comprising

each binary comparison (inhibitor treated versus control) was independently and globally

adjusted such that the normalized median log2 ratio is zero. The normalized log2 ratios were

then converted to their corresponding normalized fold-changes.

Inhibition of growth factor mediated cellular proliferation

NCI-H526 cells were used to determine the effect of cediranib on SCF-stimulated

proliferation. Cells were seeded at a density of 1x105 per ml in 96-well microtiter plates in

phenol-red free low-serum containing media (0.2% FBS) overnight. The following day cells

were pre-treated with cediranib (0.1–100 nM) for 30 minutes before stimulation with 50 ng/ml

SCF and then incubation for 72 h at 37°C. Cell proliferation was determined using an XTT

endpoint (Roche Diagnostics Ltd., Lewes, UK). All assays were performed in triplicate and the

mean ± standard error of the mean (SEM) was calculated from six independent experiments.

Human aortic VSMC were used to determine the effect of cediranib on PDGF-BB-stimulated

proliferation. Cells were seeded at 10,000 cells/well in black-walled 96-well plates in smooth

muscle cell growth medium 2 (Promocell GmbH Heidelberg, Germany) and incubated

overnight at 37°C. The following day the medium was replaced with Dulbecco's modified

Eagle's medium (DMEM; Gibco, Abingdon, UK) containing 0.1% FBS, PDGF-BB (50 ng/ml)

and cediranib. After 24-h incubation a bromodeoxuridine (BrdU) reagent (Amersham, UK)

was added and cells incubated for a further 24 h at 37oC. Cells were fixed in formalin for 15

min and proliferation assessed by staining for BrdU using the Cell Proliferation Fluorescence

kit (Amersham, UK). Cells were imaged on the ArrayScan (Cellomics, USA). All assays were

performed in triplicate and mean ± SEM was calculated from three independent experiments.




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MG63 cells were used to determine the effect of cediranib on PDGF-AA- and PDGF-BB-

stimulated proliferation. Cells were seeded at 1500 cells/well in 96-well plates in phenol red

DMEM containing 1% charcoal stripped serum for 24 h at 37°C. The following day the

medium was replaced with DMEM (Sigma-Aldrich) containing PDGF-AA or PDGF-BB (50

ng/ml) and cediranib for a further 72 h. Cell proliferation was determined as described above.

All assays were performed in triplicate and a mean ± SEM calculated from three independent

experiments.

Inhibition of receptor phosphorylation in vivo

The activity of cediranib was evaluated in a NCI-H526 human SCLC tumor xenograft model.

Tumors were implanted subcutaneously in the hind flank of female nude (nu/nu genotype)

mice of at least 8 weeks of age. When tumors reached a volume 0.36 ± 0.02 cm3, mice were

randomized (8−10 per group) and dosed with cediranib (0.75–6 mg/kg/d) or vehicle

administered once daily by oral gavage. Tumor volumes were assessed by bilateral Vernier

caliper measurement at least twice weekly and calculated using the formula (length x width) x

√ (length x width) x (π/6), where length was taken to be the longest diameter across the tumor

and width the corresponding perpendicular. To remove any size dependency prior to

statistical evaluation (the variance in mean tumor volume data increases proportionally with

volume and is therefore disproportionate between groups), data was log-transformed prior to

statistical evaluation using a one-tailed two-sample t test. NCI-H526 xenograft tumors were

evaluated for c-Kit receptor phosphorylation ex-vivo using immunoprecipitation, following

acute (2 days; eight animals per group) or chronic (17 days; three animals per group)

treatment with cediranib. Tumors were homogenized in lysis buffer I and following a protein

assay, 5 mg of protein from each sample was immunoprecipated overnight at 4°C with

anti-c-Kit conjugated agarose beads. The immune complexes were washed and proteins

eluted by boiling in SDS sample buffer. Standard SDS-Page methods were performed to

enable detection of total and phosphorylated c-Kit using antibodies as previously described.

Protein phosphorylation was quantitated using the ChemiGenius as described above.




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The activity of cediranib was also evaluated in a C6 rat glial tumor xenograft model in mice.

Cells were cultured in 199 media (Life Technologies, Abingdon, UK) supplemented with 10%

FCS and 2 mM glutamine and maintained in 7.5% CO2. For all tumor studies, C6 glial cells

were resuspended in sterile phosphate-buffered saline and inoculated subcutaneously

(1 x 105 in 100 μl) in the hind flank of male athymic mice. Tumor volumes were assessed as

described above. Approximately 21 days post implant when the C6 glial tumor volume was

between 0.5−0.8 cm3, mice were randomized into groups of 10 prior to treatment. Mice

received a single bolus oral dose of either cediranib or vehicle control. Animals were

sacrificed 4 h post dose. Five minutes prior to sacrifice, VEGF-A (20 μg per mouse:

AstraZeneca) and PDGF-BB (75 μg per mouse: Biosource, Paisley UK) were co-administered

intravenously (0.3 ml/mouse in PBS) to all animals. Terminal blood samples were taken from

the vena cava into lithium heparin tubes to collect plasma samples for pharmacokinetic

analysis. Tumors and lungs were excised and halved, each tissue half weighed and snap

frozen immediately in liquid nitrogen. Tissue samples were stored at –80°C until processed for

Western blot analysis for total and phosphorylated VEGFR-2 and PDGFR-β. Lungs were

homogenized in lysis buffer I and tumors were homogenized in lysis buffer III (60mM Tris,

150mM sodium chloride, 1mM ethylenediaminetetraacetic acid, 1% Igepal, 0.25% sodium

deoxycholate, 40% glycerol, 8% SDS, 200mM Tris-HCl, Phosphatase Inhibitor Cocktail 1 and

2 [Sigma-Aldrich]).




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Results

Cediranib is a potent inhibitor of VEGFR-1

To determine the potency of cediranib against VEGFR-1 in cells, a cell line stably transfected

with full length VEGFR-1 (AG1-AG-Flt-1) was used. Cediranib inhibited VEGF-A driven

VEGFR-1 phosphorylation with an IC50 value of 1.2 nM (Fig. 2A). This is comparable with the

cellular potency versus VEGFR-2 (0.5 nM) (7) and VEGFR−3 (<1 nM) (8), and consistent with

the primary pharmacology of the compound being that of a potent pan-inhibitor of VEGFR-1,

-2 and -3 tyrosine kinase activity.

To identify the tyrosine phosphorylation sites on VEGFR-1 modulated by ligand-induced

autophosphorylation and inhibition by cediranib, PhosphoscanTM was performed on VEGFR-1

isolated from the AG1-G1 cells treated with VEGF-A, and VEGF-A in the presence of 100 nM

cediranib. Phosphorylated receptor was enriched via a total phospho-tyrosine

immune-precipitation. The residues phosphorylated on VEGFR-1 in each treated lysate were

examined by specifically identifying phosphorylated peptides corresponding to VEGFR-1 (Fig.

2A, B; Supplementary Table 1). Upon stimulation with VEGF-A or PlGF, significant induction

of phosphorylation of peptides incorporating tyrosine residues Y1053, Y1048/Y1053, Y1048

was observed. Modest induction of phosphorylation was also detected at residues 794, 1242

but the magnitude of change was lower (Fig. 2A). The pattern of ligand induced

phosphorylation by both VEGF-A and PlGF was similar although the magnitude of induction

was higher with VEGF-A than with PlGF. Serine phosphorylated peptides were also detected,

although the significance of these modifications is unclear (Supplementary Table 1). This

shows that under these conditions the phosphorylation status of VEGFR-1 is dynamically

regulated on a restricted number of residues upon engaging VEGF-A or PlGF, with Y1048

and Y1053 showing the greatest fold changes.

To determine which residues were dynamically regulated by cediranib we compared protein

extracts from cells stimulated with VEGF-A with those from cells stimulated with VEGF in the

presence of 100 nM cediranib (Fig. 2C). There was a marked reduction in the relative

abundance of peptides corresponding to Y794, Y1053, Y1053/48, and Y1048 in cediranib-




15

treated samples, with a 37-fold reduction in the presence of the peptide corresponding to

pY1053/48 in the cediranib-treated samples (Fig. 2C). The total tyrosine phosphorylation

status of VEGFR-1 in the lysates used for this specific analysis was also assessed by ELISA.

VEGFR-1 from each lysate was captured and the level of tyrosine phosphorylation detected

using an anti-phosphotyrosine antibody (Fig. 2D). Both VEGF-A and PlGF induced significant

phosphorylation of VEGFR-1 in the lysates. Cediranib inhibited the VEGF-A induced

phosphorylation of VEGFR-1.

Cediranib inhibits c-Kit phosphorylation and SCF-induced proliferation

Cediranib inhibits c-Kit with a similar potency to that with which it inhibits the tyrosine kinase

activity of the VEGF receptors (7). The activity of cediranib against c-Kit was tested in two cell

lines, M07e and NCI-H526. SCF-stimulated c-Kit phosphorylation was inhibited with IC50

values of 3 and 1 nM respectively (Fig. 3 A and B). MAPK as a downstream signalling marker

was also inhibited with an IC50 value similar to that for inhibition of receptor phosphorylation

(Fig. 3A and B). The relationship between inhibition of acute ligand-induced phosphorylation

and SCF/c-Kit-dependent proliferation was determined using NCI-H526 cells. Cediranib

inhibited SCF-stimulated proliferation of NCI-H526 cells after 72 h, with an IC50 value of 0.013

nM (Fig. 3C), and full inhibition being achieved at concentrations of between 20 nM and 50

nM. From these experiments, it appears that about 10-fold higher concentrations were

required to inhibit functional consequences of c-Kit signalling (i.e. c-Kit-dependent cellular

proliferation) than for inhibition of receptor phosphorylation.

Mutations in c-Kit are associated with certain tumors such as gastrointestinal stromal tumors

(GIST) and AML where they drive tumor growth. The activity of cediranib against a range of

common c-Kit mutations was also determined using a panel of cell lines that either expressed

mutated c-Kit endogenously, or were transiently transfected with mutated receptors. The c-Kit

mutations assessed were V560G, V559D, W557R, Del 557-558, V654A, T670I, D816V,

D816Y and N822K. To assess the potential of the compound to inhibit phosphorylation of

these receptors, cells were incubated in the presence and absence of 20 nM of cediranib.

Cediranib inhibited phosphorylation of c-Kit mutants V560G, V559D, W557R and Del




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557-558, V654A and N822K markedly, but it did not inhibit constitutive phosphorylation of

c-Kit mutants T670I D816V and D816Y (Supplementary Table 2).

Inhibition of c-Kit phosphorylation by cediranib in vivo

Inhibition of c-Kit phosphorylation was examined in vivo in established NCI-H526 tumor

xenografts, following chronic once-daily dosing of cediranib (17 doses of either 6, 3, 1.5 or

0.75 mg/kg) to tumor-bearing mice (Fig. 3D). The dose-range examined has been previously

determined to result in dose-dependent inhibition of a wide range of human tumor xenograft

models that do not express or have a dependency upon c-Kit. C-Kit was immunoprecipated

and the samples analyzed for phosphorylated and total receptors. In NCI-H526 tumors,

cediranib reduced phosphorylation of the receptor by greater than 80% at doses as low as

0.75 mg/kg. Although NCI-H526 tumor growth has been suggested to be dependent on c-Kit

(11), despite the tumors expressing constitutively phosphorylated c-Kit, no enhanced effects

on growth or survival of the tumor cells was observed in these experiments (Supplementary

Fig. 1) compared to other xenografts not expressing c-Kit.

Cediranib inhibits PDGFR-mediated autophosphorylation and PDGF-driven

proliferation at higher concentrations

In recombinant kinase assays, cediranib has been previously shown to demonstrate lower

potency, 10- and 36-fold for inhibition of PDGFR-α and -β than for VEGF receptors or 2.5-

and 19-fold for c-Kit (7). The activity against PDGFR-α and -β signalling was further explored

using a range of cell types including other tumor cells, VSMC, and fibroblasts (Fig. 4 A-F).

PDGF-AA and PDGF-BB ligands were used in stimulation assays, the former inducing

homodimerization of PDGFR-α and the latter homodimerization of PDGFR-β and

heterodimerization of PDGFR-α and -β. U118MG human glioma cells express both human

PDGFR-α and -β. Cediranib inhibited PDGF-AA-induced phosphorylation of PDGFR-α and

PDGF-BB-induced phosphorylation of PDGFR-β with mean IC50 values of 20 nM and 32 nM,

respectively. In C6 rat glioma cells, a similar IC50 value of 24 nM was observed versus PDGF-

AA stimulation of PDGFR-α (Fig. 4C). In NIH3T3 cells (mouse fibroblast line) cediranib was

slightly more potent, inhibiting PDGF-BB mediated phosphorylation of PDGFR-β with an IC50




17

value of 12 nM (Fig. 4D). Comparable activity was found in smooth muscle cell types. In

cultured human coronary VSMC the primary PDGFR receptor is PDGFR-α (data not shown).

Cediranib inhibited PDGF-AA stimulated receptor phosphorylation with an IC50 value of 15 nM

(Fig. 4E). In contrast, in human aortic VSMC the primary PDGFR receptor is PDGFR-β (data

not shown). In these cells cediranib inhibits PDGF-BB-induced phosphorylation of PDGFR-β

with an IC50 value of 23 nM (Fig. 4F). To determine how effectively cediranib inhibits the

functional consequences of PDGFR activation, its potency was assessed in both PDGF-AA

and PDGF-BB driven proliferation assays. In human aortic VSMC cediranib inhibited PDGF-

BB-stimulated proliferation after 48 h with an IC50 value of 36 nM (Fig. 4G) similar to the

potency versus PDGFR-β phosphorylation in the same cells. In MG63 cells, cediranib

inhibited PDGF-BB stimulated proliferation with an IC50 value of 63.5 nM (Fig. 4H), similar to

the previously reported IC50 value of 40 nM versus PDGF-AA-induced proliferation in the

same cell line (7).

Cediranib gives differential inhibition of PDGFR signalling in C6 tumors and murine

lung tissue in ligand-induced acute pharmacodynamic assays

We have previously demonstrated time and dose dependent inhibition of VEGFR-2 in murine

lung tissue using a ligand induced pharmacodynamic assay (12). This approach was taken

because the inter-animal variability in pVEGFR-2 levels was found to be high, making

accurate assessment of inhibitor dose responses extremely difficult. Addition of exogenous

ligand to stimulate receptor phosphorylation results overcame this issue. Here we used a

similar approach to assess the inhibition of PDGFR activation relative to VEGFR-2 to gain

greater insight in to the effects of cediranib on these receptors in vivo. The relative potency of

cediranib versus VEGFR-2 and PDGFR-β was compared directly in vivo in the same animal.

To normalize levels of phosphorylated VEGFR-2 and stimulate PDGFR-α and -β

phosphorylation, animals were injected with both VEGF-A and PDGF-BB immediately prior to

sacrifice. Lungs from animals bearing C6 tumors receiving cediranib 6, 3, 1.5 or 0.75 mg/kg

for 4 h were assessed for levels of phosphorylated VEGFR-2 (Fig. 5A) and phosphorylated

PDGFR-β (Fig. 5B) 4 hours after dosing. This timepoint was chosen as we established the

maximal exposure of cediranib occurs between 2-3 hours in mice (data not shown).




18

Consistent with previous data (12), VEGFR-2 phosphorylation in lung was significantly

reduced over a range of doses from 6 mg/kg down to 0.75 mg/kg (Fig. 5A). In contrast,

phosphorylation of PDGFR-β was only significantly inhibited in lung at 6 mg/kg with no

significant inhibition achieved at lower doses (Fig. 5B).

To further explore the relative activity against activity PDGFR-α and PDGFR-β expressed in

tumour cells we examined C6 tumour xenografts. C6 cells express both PDGFR-α and

PDGFR-β. Whilst PDGFR-α is constitutively phosphorylated in C6 cells in vivo, the injection of

PDGF-BB results in the additional phosphorylation of PDGFR-β, enabling inhibition of both

receptors to be studied in the same tumor. In separate studies (acute and chronic) we have

assessed the activity of cediranib against PDGFR-α in C6 tumour xenografts (at 6 hours) and

established that cediranib gives up to 60% inhibition of PDGFR-α phosphorylation. Again due

to variability in phosphorylation between tumours these data were variable (data not shown).

Adopting an acute ligand stimulation approach allowed us to further examine inhibition of both

PDGFRs in tumor and lung within the individual animals. In contrast to the modulation of

receptor phosphorylation in lung tissue, phosphorylation of both PDGFR-α (Fig. 5C) and -β

(Fig. 5D) in C6 tumors, in the same animals, was partially inhibited by all doses of cediranib

examined.

Selectivity against other tyrosine kinase receptors

Some VEGFR tyrosine kinase inhibitors also have activity against other kinases. Selectivity of

cediranib against mutFlt-3 and CSF-1R members of the PDGFR family have been shown

previously (Wedge et al, 2005). In addition to determining the relative activity against

VEGFR-1, c-Kit and PDGFRs, we also tested its activity against wild-type Flt-3 and fibroblast

growth factor receptor FGFR1 and FGFR4 (Supplementary Table 3). The activity against Flt-

3 was determined using OCI-AML-5 cells stimulated with Flt-3 ligand (Flt-3L). The activity

against FGFR1 and FGFR4 was determined by transiently overexpressing the receptor in

Cos-1 cells which resulted in constitutive phosphorylation of the receptor. Cediranib was

inactive against wild-type Flt-3 (IC50 value >1 μM) and had marginal activity versus FGFR-1

and -4 (IC50 values of 0.35 and 2.17 μM respectively). These data indicate that cediranib




19

has is significantly less active against these receptors than against the VEGF receptors, or c-

Kit.




20

Discussion

We have previously shown that cediranib is a potent inhibitor of VEGFR-2, and -3, and that it

reduces growth of a wide range of tumor models by targeting tumor vasculature (7, 8, 12).

Cediranib has selectivity for VEGFR-2 against a wide range of kinases, including the PDGFR-

family members CSF-1R and Flt-3 in cellular phosphorylation assays (420- and >20,000-fold

selectivity, respectively, versus VEGFR-2) (7). Here we explore the pharmacology of

cediranib in more depth, examining its activity against VEGFR-1 in cells and the PDGFR-

family members c-Kit, PDGFR-α and PDGFR-β in vitro and in vivo within a dose-range of

0.75–6 mg/kg which has been routinely examined within preclinical tumor xenograft

experiments.

Establishing the potency of small molecule inhibitors against VEGFR-1 signalling in ligand-

induced endothelial cell assays has proven challenging due to the low intrinsic kinase activity

associated with this receptor. Despite this low kinase activity there is some evidence to

implicate VEGFR-1 signalling in pathological angiogenesis (13−15), and in the recruitment of

macrophages and myeloid prescursor cell recruitment to tumors (16−18); a process which

has been linked with resistance to VEGF signalling inhibitors (19−21). The role of the

VEGFR1 kinase domain in recruitment of BM derived cells into tumours has been confirmed

using VEGFR1 TK-/- transgenic mice (22). Consequently, concurrent inhibition of VEGFR-1

and -2 signalling, may afford added therapeutic benefit. To examine inhibition of VEGFR-1,

we utilized a cell line derived from a human benign angioma into which the full-length receptor

was over-expressed by stable-transfection. This cell line does not express VEGFR-2 and

therefore enables a more accurate assessment of activity against VEGFR-1, by avoiding any

confounds that could result from VEGFR heterodimerization. The inhibition of VEGF-induced

VEGFR-1 phosphorylation by cediranib, as determined by Western blotting, was evident at a

potency that is comparable to that determined against VEGFR-2 and -3 activation in cellular

assays, thereby confirming that cediranib is a pan-VEGFR kinase inhibitor. Whilst it is clear

that VEGFR-1 does induce specific signalling (10, 23, 24), there is limited information on the

residues involved. We therefore also used a MS-based method to examine the residues

activated on VEGFR-1 by VEGF or PlGF in AG1-G1-Flt-1 cells, and inhibition of the VEGF-




21

induced response by cediranib. VEGF-A and PlGF were found to induce a broadly similar

pattern of change in VEGFR-1 increasing the phosphorylation of tyrosine residues Y794,

Y1048, Y1053 and Y1242. The greatest fold-change evident was at Y1048 and Y1053 which

are within the tyrosine kinase domain of the receptor. Cediranib treatment abolished all

VEGF-stimulated phosphorylation on the four residues described, but had greatest effect

against Y1048/1053, the signal from this peptide sequence being reduced by nearly 37 fold

when compared to the untreated control, suggesting that phosphorylation at this site was the

most labile. These data contrast with previous studies that have described a number of

VEGFR-1 residues in the C-terminal tail as being modulated by VEGF-A treatment, in

particular Y1213 (25−27), but also Y1327 and Y1333 (27), and Y1309 in response to PlGF

stimulation (24). Although peptides indicating phosphorylation at Y1213 were detected in our

study, these were not modulated by ligand activation. These differences may be attributable

to the cell line examined or technical approach used. We were unable to develop a

pharmacodynamic assay to measure inhibition of VEGFR-1 activity in vivo, due to both the

low level of receptor phosphorylation and the inability to identify selective phosphorylation-

specific antibodies to VEGFR-1. However, given the similar potency of cediranib against each

VEGF receptor in cells, it would be reasonable to assume that it has the ability to inhibit

VEGFR-1 driven signalling responses in vivo.

In addition to having activity against the VEGF receptors, cediranib also inhibits the kinase

activity of c-Kit. Inhibition of wild-type c-Kit signalling in M07e and NCI-H526 cells prevented

downstream MAPK phosphorylation. Cediranib inhibited the SCF-induced proliferation of NCI-

H526 cells and reduced an associated increase in AKT phosphorylation (data not shown).

However, a decrease in potency (of approximately 10-fold) was observed for inhibition of

SCF-induced proliferation, suggesting that up to 90% of SCF signalling through c-Kit and

MAPK needs to be suppressed to deliver a cytostatic effect in NCI-H526 cells. In vivo,

inhibition (~85%) of the constitutive phosphorylation of c-Kit in established NCI-H526

xenograft tumors was observed after 17 days of chronically dosing cediranib at 0.75−6 mg/kg.

This suggests that cediranib may elicit a pharmacodynamic affect great enough to influence




22

the phenotypic consequences of c-Kit signalling in vivo, although an enhanced anti-tumor

effect was not observed in xenografts derived from this particular tumor line.

Mutation or aberrant activation of c-Kit and its ligand SCF are associated with the progression

of numerous solid and hematological malignancies, including GIST (28), SCLC (29), and AML

(30). Approximately 95% of GIST cases are c-Kit positive, with 60−70% positive for the c-Kit

exon 11 mutations (V560G, V559D, W557R Del557-558) against which the c-Kit/PDGFR/Abl

kinase inhibitor imatinib shows activity (31). More recently secondary mutations of c-Kit have

been identified that confer acquired resistance to imatininb (32, 33). Cediranib was found to

inhibit phosphorylation of all of the imatinib sensitive c-Kit mutant forms found in GIST, and in

addition inhibited two of the secondary point mutations which confer acquired resistance to

imatinib (V654A, N822K) (34−37). However, cediranib was not active against the T670I gate

keeper mutation in c-Kit (38) or the D816V/D816Y c-Kit mutations (35−37).

Previous data generated in cellular phosphorylation assays showed that cediranib was 10-to

16-fold less active against PDGFR-α and -β, than VEGFR, and this margin increased to 100-

fold when a comparative assessment of ligand-induced proliferation was performed. The in

vitro data generated with cediranib in this study against receptor phosphorylation in multiple

cell types is consistent with our previous data, with IC50 values in the range of 15–32 nM

being slightly higher than existing data in MG63 cells (cediranib IC50 of 5 and 8 nM against

PDGFR-α and -β respectively). A previously observed drop-off in potency of approximately 7-

fold between phosphorylation and proliferation endpoints for PDGF-AA/PDGFR-α signalling in

MG63 cells was also apparent in the present studies when these cells were stimulated with

PDGF-BB. The activity of cediranib against PDGF-BB-induced PDGFR-β phosphorylation and

cellular proliferation was more comparable in the primary VSMCs, suggesting that PDGFR-

mediated signalling responses may be cell-type dependent. In vivo, cediranib inhibited

PDGFR-β signalling in C6 rat tumor xenografts across the dose range examined, in contrast

to an effect only being evident at a dose of 6 mg/kg in normal lung tissue. This apparent

discrepancy is unlikely to be due to species-specificity differences given the high degree of

receptor homology between mouse and rat. Whilst a distribution effect cannot be ruled out,




23

the tissue concentrations of cediranib in C6 tumors did not exceed those in normal lung tissue

(data not shown). Neither is the inhibition of PDGFR phosphorylation in C6 tumors due to a

bystander effect that is secondary to the anti-vascular effects of cediranib, since compound

treatment does not affect the phosphorylation of other receptor tyrosine kinases, such as

EGFR in Lovo human colorectal tumor xenografts, at doses that significantly inhibit tumor

growth (data not shown). An alternative explanation for the divergent effect observed in lung

and C6 tumors could relate to the differential regulation or function of PDGFR-β in these

tissue compartments, the receptor driving significant cellular proliferation in C6 tumors but not

in normal lung tissue.

Although cediranib inhibited PDGFR-α and -β phosphorylation in C6 tumors, this model did

not appear to have increased sensitivity to the antitumor effects of the compound. A dose of 3

mg/kg cediranib, which inhibited PDGFR-α and -β phosphorylation by 73 and 76%,

respectively, 4 h after an acute dose, inhibited tumor growth by 52% (± 4% SEM) after 10 to

14 days of continuous once-daily dosing (data not shown): an effect not dissimilar to that

observed in non-PDGFR-dependent tumor models as a consequence of inhibiting VEGF

signalling (5). This finding re-enforces the fact that very significant inhibition of PDGFR

signalling may be required to prevent phenotypic signalling responses. The activity of

cediranib against PDGFR-α and -β would therefore not be expected to contribute significantly

to an effect on tumor growth or survival, unless a tumor has a particularly high dependency on

signalling from these receptors.

This work highlights the significant challenge to accurately describe the relative activity of an

ATP-competitive inhibitor potent against more than one kinase. This requires consideration of

activity at the recombinant kinase level, within multiple cellular phosphorylation and

proliferation assays, and then in vivo potency against the pertinent kinase in target tissues.

These data are all required to fully interpret observations made in pre-clinical models. The in

vivo PD data that show across the dose range cediranib is primarily a VEGF signalling

inhibitor with activity against c-Kit. That a significant drop off in potency is observed between

ligand-induced receptor phosphorylation and cellular proliferation for c-Kit, PDGFR-α and -β,




24

but not for VEGFR-2 in endothelial cell assays (7), combined with the relative order of

potency against these targets within a number of in vitro assays suggest cediranib is primarily

a VEGFR inhibitor.




25

Acknowledgements:

We would like to acknowledge the support of the staff in CDMG for xenograft studies, Susan

Lovick for statistical analysis, Jonathon Orme for FGFR-1 and -4 assays and analyses,

Jonathan Dry for mutation analysis, James Sherwood and John Smith for c-Kit sequencing

and mutation analyses.




26

References

1. Escudier B, Pluzanska A, Koralewski P, Ravaud A, Bracarda S, Szczylik C, et al.

Bevacizumab plus interferon alfa-2a for treatment of metastatic renal cell carcinoma:

a randomised, double-blind phase III trial. Lancet 2007;370:2103-11.

2. Motzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Oudard S, et

al. Overall survival and updated results for sunitinib compared with interferon alfa in

patients with metastatic renal cell carcinoma. J Clin Oncol 2009; 27:3584-90.

3. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, et al.

Bevacizumab plus irinotecan, fluorouracil and leucovorin for metastatic colorectal

cancer. N Engl J Med 2004; 350:2335-42.

4. Ivy SP, Wick JY, Kaufman BM. An overview of small-molecule inhibitors of VEGFR

signaling Nat Rev Clin Oncol 2009;6:569-79.

5. Wedge SR and Jürgensmeier JM. VEGF receptor tyrosine kinase inhibitors for the

treatment of cancer, In: Tumour angiogenesis - basic mechanisms and cancer

therapy, Springer-Verlag Heidelberg (Eds. Marme, D. and Fusenig, N., ISBN 978-3-

540-33176-6), 2008: Chapter 23: p.395-424.

6. McTigue MA, Wickersham JA, Pinko C, Showalter RE, Parast CV, Tempczyk-Russell

A , et al. Crystal structure of the kinase domain of human vascular endothelial growth

factor receptor 2: a key enzyme in angiogenesis. Structure 1999;7:319-30.

7. Wedge SR, Kendrew J, Hennequin LF, Valentine PJ, Barry ST, Brave SR, et al.

AZD2171: a highly potent, orally bioavailable, vascular endothelial growth factor

receptor-2 tyrosine kinase inhibitor for the treatment of cancer. Cancer Res

2005;65:4389-400.

8. Heckman CA, Holopainen T, Wirzenius M, Keskitalo S, Jeltsch M, Ylä-Herttuala S, et

al. The tyrosine kinase inhibitor cediranib blocks ligand-induced vascular endothelial

growth factor receptor-3 activity and lymphangiogenesis. Cancer Res 2008;68:4754-

62.

9. Padera TP, Kuo AH, Hoshida T, Liao S, Lobo J, Kozak KR, et al. Differential

response of primary tumor versus lymphatic metastasis to VEGFR-2 and VEGFR-3

kinase inhibitors cediranib and vandetanib. Mol Cancer Ther 2008;7:2272-79.




27

10. Seetharam L, Gotoh N, Maru Y, Neufeld G, Yamaguchi S, Shibuya M. A unique

signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial

growth factor VEGF. Oncogene 1995;10:135-47.

11. Abrams TJ, Lee LB, Murray LJ, Pryer NK, Cherrington JM. SU11248 inhibits KIT

and platelet-derived growth factor receptor beta in preclinical models of human small

cell lung cancer. Mol Cancer Ther 2003;2:471-78.

12. Smith NR, James NH, Oakley I, Wainwright A, Copley C, Kendrew J, et al. Acute

pharmacodynamic and antivascular effects of the vascular endothelial growth factor

signaling inhibitor AZD2171 in Calu-6 human lung tumor xenografts. Mol Cancer Ther

2007;6:2198-208.

13. Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M et al.

Synergism between vascular endothelial growth factor and placental growth factor

contributes to angiogenesis and plasma extravasation in pathological conditions. Nat

Med. 2001; 7:575-83.

14. Fischer C, Jonckx B, Mazzone M, Zacchigna S, Loges S, Pattarini L, et al. Anti-

PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy

vessels. Cell 2007;131:463-75.

15. Wu Y, Zhong Z, Huber J, Bassi R, Finnerty B, Corcoran E, et al. Anti-vascular

endothelial growth factor receptor-1 antagonist antibody as a therapeutic agent for

cancer. Clin Cancer Res 2006;12:6573-84.

16. Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F, et al.

Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor

angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med 2002;8:831-40.

17. Kaplan RN, Riba RD, Zacharoulis S, Bramley AH, Vincent L, Costa C, et al.

VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic

niche. Nature 2005;438:820-7

18. Murakami M, Zheng Y, Hirashima M, Suda T, Morita Y, Ooehara J, et al. VEGFR1

tyrosine kinase signaling promotes lymphangiogenesis as well as angiogenesis

indirectly via macrophage recruitment. Arterioscler Thromb Vasc Biol 2008;28:658-

64.




28

19. Shojaei F, Wu X, Malik AK, Zhong C, Baldwin ME, Schanz S, et al. Tumor

refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells. Nat

Biotechnol 2007:25:911-920.

20. Shojaei F, Wu X, Zhong C, Yu L, Liang XH, Yao J, et al. Bv8 regulates myeloid-cell-

dependent tumour angiogenesis. Nature 2007;450:825-31.

21. Shojaei F, Wu X, Qu X, Kowanetz M, Yu L, Tan M, et al. G-CSF-initiated myeloid

cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy

in mouse models. Proc Natl Acad Sci U S A 2009;106:6742-7.

22. Muramatsu M, Yamamoto S, Osawa T, Shibuya M. Vascular endothelial growth factor

receptor-1 signaling promotes mobilization of macrophage lineage cells from bone

marrow and stimulates solid tumor growth. Cancer Res. 2010 70, 8211-21.

23. Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH. Different

signal transduction properties of KDR and Flt1, two receptors for vascular endothelial

growth factor. J Biol Chem 1994;269:26988-95.

24. Autiero M, Waltenberger J, Communi D, Kranz A, Moons L, Lambrechts D, et al. Role

of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1

and Flk1 Nat Med 2003;9:936-43.

25. Igarashi K, Isohara T, Kato T, Shigeta K, Yamano T, Uno I. Tyrosine 1213 of Flt-1 is a

major binding site of Nck and SHP-2. Biochem Biophys Res Commun 1998;246:95-9

26. Yu Y, Hulmes JD, Herley MT, Whitney RG, Crabb JW, Sato JD. Direct identification

of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-

1 that mediates phosphatidylinositol 3'-kinase binding. Biochem J 2001;358:465-72.

27. Ito N, Wernstedt C, Engström U, Claesson-Welsh L. Identification of vascular

endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of

SH2 domain-containing molecules. J Biol Chem 1998;273:23410-8.

28. Rubin BP, Singer S, Tsao C, Duensing A, Lux ML, Ruiz R, et al. KIT activation is a

ubiquitous feature of gastrointestinal stromal tumors. Cancer Res 2001;61:8118-21.

29. Camps C, Sirera R, Bremnes RM, Garde J, Safont MJ, Blasco A, et al. Analysis of

c-Kit expression in small cell lung cancer: prevalence and prognostic implications.

Lung Cancer 2006;52:343-7.




29

30. Advani AS. C-Kit as a target in the treatment of acute myelogenous leukemia. Curr

Hematol Rep 2005;4:51-8.

31. Heinrich MC, Corless CL, Demetri GD, Blanke CD, von Mehren M, Joensuu H,et al.

Kinase mutations and imatinib response in patients with metastatic gastrointestinal

stromal tumor. J Clin Oncol 2003;21:4342-9.

32. Wardelmann E, Merkelbach-Bruse S, Pauls K, Thomas N, Schildhaus HU, Heinicke

T, et al. Polyclonal evolution of multiple secondary KIT mutations in gastrointestinal

stromal tumors under treatment with imatinib mesylate. Clin Cancer Res

2006;12:1743-9.

33. Desai J, Shankar S, Heinrich MC, Fletcher JA, Fletcher CD, Manola J, et al. Clonal

evolution of resistance to imatinib in patients with metastatic gastrointestinal stromal

tumors. Clin Cancer Res 2007;13:5398-405.

34. Frost MJ, Ferrao PT, Hughes TP, Ashman LK. Juxtamembrane mutant V560GKit is

more sensitive to Imatinib (STI571) compared with wild-type c-Kit whereas the kinase

domain mutant D816VKit is resistant. Mol Cancer Ther 2002;1:1115-24.

35. Antonescu CR, Besmer P, Guo T, Arkun K, Hom G, Koryotowski B, et al. Acquired

resistance to imatinib in gastrointestinal stromal tumor occurs through secondary

gene mutation. Clin Cancer Res 2005;11:4182-90.

36. Shinomura Y, Kinoshita K, Tsutsui S, Hirota S. Pathophysiology, diagnosis, and

treatment of gastrointestinal stromal tumors. J Gastroenterol 2005;40:775-80.

37. Heinrich MC, Corless CL, Blanke CD, Demetri GD, Joensuu H, Roberts PJ, et al.

Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J Clin

Oncol 2006;24:4764-74.

38. Tamborini E, Bonadiman L, Negri T, Greco A, Staurengo S, Bidoli P, et al. Detection

of over-expressed and phosphorylated wild-type kit receptor in surgical specimens of

small cell lung cancer. Clin Cancer Res. 2004; 10:8214-19.




30

Figure Legends

Figure 1. The structure of cediranib

Figure 2. Cediranib inhibits activation of VEGFR-1 and modulates phosphorylation of specific

sites on VEGFR-1. The activity of cediranib against VEGFR-1 was investigated using AG1-

G1-Flt-1 cells. (A) Serum-starved cells were stimulated with 50 ng/ml VEGF-A for 5 min in the

absence or presence of a range of concentrations of cediranib. Phosphorylation of VEGFR-1

was determined in by Western blotting of VEGFR-1 immunoprecipitated with a total VEGFR-1

antibody and then blotted with a phosphotyrosine antibody. Equal loading was determined by

Western blotting using a total VEGFR-1 antibody. The IC50 was determined using MSD

ELISA; the mean IC50 ± SEM observed over a number of experiments is shown. (B) To

determine the residues on VEGFR-1 that show dynamic changes in phosphorylation upon

activation of VEGFR-1, AG1-G1-Flt1 cells were stimulated with 50 ng/ml VEGF-A and 100

ng/ml PlGF. Lysates were immunoprecipitated with an anti-phosphotyrosine antibody and the

associated proteins analyzed by MS. Fold change in phosphorylation of individual sites

versus unstimulated control samples is represented. (C) To determine the residues on

VEGFR-1 that are phosphorylated upon activation of VEGFR-1 AG1-G1-Flt1 cells were

stimulated with 50 ng/ml VEGF-A in the presence of 100 nM cediranib. Fold change in

phosphorylation versus VEGF-A treated sample on individual sites is represented. (D)

Changes in phosphorylation within the lysates subjected to MS analysis were confirmed using

MSD ELISA. Changes are consistent with those seen in multiple previous experiments.

Figure 3. Cediranib inhibits SCF driven c-Kit phosphorylation and proliferation in tumor cells.

To assess phosphorylated c-Kit inhibition NCI-H526 (A) and M07e (B) cells, serum-starved

cells were incubated in the presence or absence of cediranib as indicated for 90−120 min,

then stimulated with 50 ng/ml of SCF for 5−10 min. Representative Western blots

demonstrating the inhibition of phosphorylated c-Kit and phosphorylated MAPK in (A) NCI-

H526 cells and (B) M07e cells are shown. All data are representative of at least three similar

experiments. The mean IC50 ± SEM observed over a number of experiments is shown. (C) To

determine the ability of cediranib to inhibit SCF-stimulated proliferation NCIH526 cells were




31

stimulated with 50 ng/ml cediranib in the presence and absence of cediranib for 72 h and

proliferation assessed by BrdU incorporation. The mean IC50 ± SEM observed over a number

of experiments is shown. (D) To demonstrate cediranib inhibits phosphorylation of c-Kit in

chronically dosed NCI-H526 tumor xenografts, mice bearing NCI-H526 tumors were treated

once-daily orally with 6, 3, 1.5 and 0.75 mg/kg/day of cediranib and the levels of

phosphorylated c-Kit were determined by ex vivo analysis. c-Kit was immunoprecipitated from

tumor lysates using a total c-Kit antibody. c-Kit and phosphorylated c-Kit were detected by

Western blotting and chemiluminescence, a representative Western blot is shown.

Phosphorylated c-Kit was quantified and the mean levels in each group ± SEM are shown.

Figure 4. Cediranib inhibits PDGF-ligand stimulated phosphorylation of PDGFR-α and -β and

inhibits ligand dependent proliferation. To determine the activity of cediranib against PDGFRs

a range of cell lines U118MG (A, B), C6 (C), NIH 3T3 (D), human coronary VSMC (E) and

human aortic VSMC (F) were stimulated with PDGF-AA (50 ng/ml) or PDGF-BB (50 ng/ml) for

5−10 min and then lysed. Inhibition of phosphorylated PDGFR-β in (D) human aortic VSMC

and inhibition of phosphorylated PDGFR-α in (E) human coronary VMSC and C6 cells.

Representative Western blots showing phosphorylated PDGFR-α and -β, as well as levels of

total receptors are shown as indicated. The mean IC50 ± SEM observed over a number of

experiments is shown. To determine the effect of cediranib on PDGF-stimulated proliferation

human aortic VSMC (G) and MG63 (H) cells were stimulated with 50 ng/ml PDGF-BB for 72 h

in the presence or absence of cediranib. The mean IC50 ± SEM observed over a number of

experiments is shown.

Figure 5. Cediranib inhibits phosphorylation of PDGFR-β in lung and tumor tissue and

phosphorylation of VEGFR-2 in lung in established human C6 tumor xenograft model

following a single dose. Acute changes in phosphorylation of VEGFR-2 and PDGFR-α and -β

were determined following a single dose of cediranib at the doses 6, 3, 1.5 and 0.75 mg/kg.

Phosphorylation of PDGFR-α and -β and VEGFR-2 was stimulated by injection of PDGF-BB

and VEGF-A 5 min prior to sacrifice of the animals. (A) Levels of phosphorylated VEGFR-2 in

lung lysates were quantitated and the mean levels ± SEM are represented. A representative




32

Western blot showing phosphorylated VEGFR-2 and total VEGFR-2 is inset. (B) Levels of

phosphorylated PDGFR-β in lung were quantitated and the mean levels ± SEM are

represented. A representative Western blot showing phosphorylated PDGFR-β and total

PDGFR-β is inset. (B) Levels of phosphorylated PDGFR-α (C) and phosphorylated PDGFR-β

(D) in tumor tissue lysates were quantitated and the mean levels ± SEM are represented.

Representative Western blots showing phosphorylated PDGFR-α and -β and total

phosphorylated PDGFR-α and -β are inset.




Figure 1

O

N

N

O

O

F

N

N




(A) AG1-G1-Flt-1

pVEGFR1

MwkDa

-250

IC50 (µM) 0.0012 ± 0.0008

Figure 2

VEGFR1

pVEGFR1

cont

rol 0

0.1

0.3

0.8

2.5 7 22 66

-250

VEGF-A + cediranib (nM)

(B)25

PlGFol

-5

0

5

10

15

20PlGFVEGFA

Fold

cha

nge

vs c

ontr

o

(C)

0

e cont

rol

-40

-30

-20

-10

Fold

cha

nge

vs V

EGF-

A s

tim.

(D)

800

1200

1600

2000

pho-

VEG

FR1

coun

ts)

0

400Phos

p (c




Figure 3

(A) NCI-H526

MwkDIC50 (µM) 0 001 ± 0 0003

(B) M07e

IC50 (µM) 0 003 ± 0 0020MwkDa

-160

-160

-35

-35

pc-Kit

pMAPK

MAPK

c-Kit

kDaIC50 (µM) 0.001 ± 0.0003 IC50 (µM) 0.003 ± 0.0020

-160

-160

-35

-35

pc-Kit

pMAPK

MAPK

c-Kit

kDa

SCF + [cediranib] (nM)

cont

rol 0

0.00

1

0.01 0.

1 1 10 100

SCF + [cediranib] (nM)

cont

rol 0

0.00

1

0.01 0.

1 1 10 100

(C) NCI-H526 proliferation (D) NCI-H526 tumor PD

0 15

0.20

0.25

e (O

D)

IC50 (µM) 0.013 ± 0.005

150000

200000

250000

300000

sign

al

con 6 3 1.5 0.75

cediranib (mg/kg)MWkDa

pc-Kit(pY719)

c-Kit

160-

160-

0.00

0.05

0.10

0.15

Res

pons

e

[cediranib] (nM)Control 6 3 1.5 0.75

0

50000

100000

150000

p-cK

it s

Dose of cediranib mg/kg

*** ** ****

[cediranib] (nM) Dose of cediranib mg/kg




Figure 4

MwkDa

co

ntr

ol 0

0.1

50

.46

1.4 4.1 12

37

10

03

00

-250

-250PDGFRα

pPDGFRα

IC50 (µM) 0.020 ± 0.008

(A) U118MG

MwkDa

on

tro

l 00

.15

0.4

61

.44

.1 12

37

10

0

30

0

-250

-250PDGFRβ

pPDGFRβ

IC50 (µM) 0.032 ± 0.010

(B) U118MG

PDGF-AA + cediranib (nM)

c

MwkDa

co

ntr

ol 0

0.1

50

.46

1.4 4.1 12 37

10

03

00

-250

-250PDGFRα

pPDGFRαIC50 (µM) 0.024 ± 0.004

(C) C6MwkDa

co

ntr

ol 0

0.1

50

.46

1.4 4.1 12 37

10

03

00

-250

-250PDGFRβ

pPDGFRβIC50 (µM) 0.012 ± 0.0027

(D) NIH 3T3

PDGF-BB + cediranib (nM)

co


c


c

MwkDa

-250pPDGFRα

(E) Human coronary VSMC

IC50 (µM) 0.015 ± 0.003MwkDa

-250pPDGFRβ

IC50 (µM) 0.023 ± 0.007

(F) Human aortic VSMC


con

tro

l 0

0.3 0.8

2.5 7 22

66

20

0

-250PDGFRα


con

tro

l 0

-250PDGFRβ

0.3 0.8 2.5 7 22

66

20

0

(G) Human aortic VSMC (PDGF-BB) (H) MG63 (PDGF-BB)

5

10

15

20

Res

pons

e (B

rDu

Uni

ts)

IC50 (µM) 0.032 ± 0.004 IC50 (µM) 0.0635 ± 0.0009

2

4

6

8

10

12

Res

pons

e (O

D x

100)

0

[cediranib] (nM)

0

2

[cediranib] nM




(A) Lung phosphorylated VEGFR-2

p-VEGFR2 (pY1175)

250-

MWkDa Con 6 3 1.5 0.75

cediranib (mg/kg)

(B) Lung phosphorylated PDGFR-β

250-

MWkDa Con 6 3 1.5 0.75

cediranib (mg/kg)

p-PDGFRβ(pY751)

Figure 5

300000

350000

400000

450000

500000

550000

600000

650000

EG

FR-2

sig

nal

VEGFR2250-

20000

25000

30000

35000

40000

DG

FR-β

sign

al

PDGFRβ250-

Control 6 3 1.5 0.750

50000

100000

150000

200000

250000

300000

p-V

E


*** *** ***

***

Control 6 3 1.5 0.750

5000

10000

15000p-P

D


**

g g

( C) C6 tumor phosphorylated PDGFR-α

g g

(D) C6 tumor phosphorylated PDGFR-β

25000

30000

35000

sign

al

p-PDGFRα (pY720)

PDGFRα

250-

250-

MWkDa

Con 6 3 1.5 0.75

cediranib (mg/kg)

35000

40000

45000

50000

igna

l

PDGFRβ

250-

250-

MWkDa

Con 6 3 1.5 0.75

cediranib (mg/kg)

p-PDGFRβ (pY751)

5000

10000

15000

20000

p-P

DG

FR-α

s

***

***

***

***

5000

10000

15000

20000

25000

30000

35000

p-P

DG

FR-β

s

***

***

*** ***

Control 6 3 1.5 0.750

Dose of cediranib mg/kgControl 6 3 1.5 0.75

0





Published OnlineFirst March 25, 2011.Mol Cancer Ther Sandra R Brave, Kirsty Ratcliffe, Zena Wilson, et al. structurally related PDGFR-familykinase inhibitor, against VEGFR-1 and members of the Assessing the Activity of Cediranib, a VEGFR-2/-3 tyrosine

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Assessing the Activity of Cediranib, a VEGFR-2/-3 …mct.aacrjournals.org/content/molcanther/early/2011/03/24/1535-7163... · 4 Abstract Cediranib is a potent inhibitor of the vascular