Assessing the Activity of Cediranib, a VEGFR-2/3 Tyrosine Kinase Inhibitor, Against VEGFR-1 and Members of the Structurally Related PDGFR Family

Published OnlineFirst March 25, 2011; DOI: 10.1158/1535-7163.MCT-10-0976

Molecular Cancer Preclinical Development Therapeutics

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, Juliane M. Jurgensmeier€ 1, Masabumi Shibuya4, Stephen R. Wedge1, and Simon T. Barry1

Abstract Cediranib is a potent inhibitor of the VEGF receptor (VEGFR)-2 and VEGFR-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-a, and PDGFR-b. Cediranib inhibited VEGF-A–stimu- ¼ lated VEGFR-1 activation in AG1-G1-Flt1 cells (IC50 1.2 nmol/L). 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 with that previously observed versus VEGFR-2 and VEGFR-3. Cediranib also ¼ showed significant activity against wild-type c-Kit in cellular phosphorylation assays (IC50 1–3 nmol/L) and ¼ in a stem cell factor–induced proliferation assay (IC50 13 nmol/L). Furthermore, phosphorylation of wild- type c-Kit in NCI-H526 tumor xenografts was reduced markedly following oral administration of cediranib (1.5 mg/kg/d) to tumor-bearing nude mice. The activity of cediranib against PDGFR-b and PDGFR-a was studied in tumor cell lines, vascular smooth muscle cells (VSMC), and a fibroblast line using PDGF-AA and ¼ PDGF-BB ligands. Both receptor phosphorylation (IC50 12–32 nmol/L) and PDGF-BB–stimulated cellular ¼ In vivo proliferation (IC50 32 nmol/L in human VSMCs; 64 nmol/L in osteosarcoma cells) were inhibited. , ligand-induced PDGFR-b phosphorylation in murine lung tissue was inhibited by 55% following treatment with cediranib at 6 mg/kg but not at 3 mg/kg or less. In contrast, in C6 rat glial tumor xenografts in mice, ligand-induced phosphorylation of both PDGFR-a and PDGFR-b was reduced by 46% to 61% with 0.75 mg/kg cediranib. Additional selectivity was showed versus Flt-3, CSF-1R, 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 than PDGFR-a and PDGFR-b. Mol Cancer Ther; 10(5); 861–73. 2011 AACR.

Introduction treatment of renal cell cancer (1, 2) and, when combined with certain cytotoxic therapies, in other disease settings Inhibition of VEGF-A–mediated signaling provides (3). Approaches used to inhibit VEGF receptor (VEGFR) therapeutic benefit in oncology as monotherapy in the signaling include small molecule inhibitors that bind into the ATP-binding pocket within the kinase domain of VEGFR-2 to prevent ATP catalysis and propagation of Authors' Affiliations: 1Oncology iMED, AstraZeneca, Alderley Park, Mac- 2 receptor signaling. This receptor predominantly trans- clesfield, Cheshire, United Kingdom; Cell Signaling Technology, Danvers, Machigan; 3AstraZeneca Pharma, Centre de Recherches, Z.I. La Pom- duces the angiogenic and permeability activity of VEGF- pelle, Chemin de Vrilly, Reims, France; and 4Department of Molecular A. Two other VEGF receptors are also described with Oncology, Tokyo Medical and Dental University, Tokyo, Japan differential binding of VEGF family ligands. Each small Note: Supplementary data for this article are available at Molecular Cancer molecule differs in its potency against the VEGF recep- Therapeutics Online (http://mct.aacrjournals.org/). tors, selectivity versus other kinases, physicochemical Current address for D.J. Ogilvie: Paterson Institute for Cancer Research, Wilmslow Rd., Manchester M20 4BX, United Kingdom. properties, and pharmacokinetic profile (4, 5). Some VEGFR tyrosine kinase inhibitors have additional activity Corresponding Author: Simon Barry, Oncology iMED, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom. Phone: against 1 or more members of the platelet-derived growth 44-1625-513350; Fax: 44-1625-513350. factor receptor (PDGFR) family of kinases (class III), E-mail: Simon.T.Barry@astrazeneca.com or Steve Wedge, Oncology a b iMED, AstraZeneca, Alderley Park, Macclesfield, Cheshire, SK10 4TG, which comprises PDGFR- , PDGFR- , c-Kit, colony-sti- United Kingdom. Phone: 44-1625-523236; Fax: 44-1625-513350. mulating factor receptor-1, and Flt-3; these receptors have E-mail: [email protected] some structural homology with the VEGFR family mem- doi: 10.1158/1535-7163.MCT-10-0976 bers in that each harbors a kinase-insert sequence in 2011 American Association for Cancer Research. its intracellular domain (6). However, VEGFR kinase

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inhibitors have also been described with activity against tyrosine kinases outside the PDGFR class and, in some N cases, against serine/threonine kinases. Inhibiting multiple kinases simultaneously may provide additional ther- O O F apeutic opportunities in defined disease settings but may N impact adversely on tolerability, particularly if chronic administration or usage in combination with concurrent N O N cytotoxic treatment is required. Cediranib has been previously shown to be a potent inhibitor of VEGFR-2 and VEGFR-3 signaling in cellular Figure 1. The structure of cediranib. assays and to inhibit the growth of both angiogenic blood vessels and lymphangiogenic vessels in vivo (7–9). In recombinant kinase assays, cediranib also inhibits ¼ VEGFR-1 kinase activity (IC50 5 nmol/L) within a DMSO did not exceed 0.01%, with the exception of similar concentration range to VEGFR-2 and VEGFR-3 studies examining direct effects on tumor cells in which < (IC50 values of 0.1 and 3 nmol/L, respectively; ref. 7). 1% DMSO was required to examine higher concentra- However, a more quantitative assessment of inhibitor tions of cediranib. All in vivo studies were conducted by activity against endogenous VEGFR-1 kinase has proven once-daily oral gavage. For studies in mice, cediranib technically challenging in endothelial cells because of the was suspended in 1% (w/v) aqueous polysorbate 80 low intrinsic activity of this receptor. Cediranib shows [polyoxyethylene (20) sorbitan monooleate in deionized selectivity against other kinases (7). Cediranib has similar water] and dosed at 0.1 mL/10 g of body weight. potency against c-Kit when compared with VEGFR-2 in phosphorylation assays but less potency against PDGFR- Cell culture a and PDGFR-b, particularly in a PDGF-AA/PDGFR- NCI-H526 [a human small cell lung cancer (SCLC) a–driven tumor cell proliferation assay. However, the line], U118MG (a human glioblastoma line), MG63 (a activity of cediranib against a c-Kit–driven phenotypic human osteosarcoma line), and C6 (a rat glial line) cells endpoint, PDGFR-a and -b signaling in normal cell types were purchased from the American Type Culture Collec- (which may also influence therapeutic response), or a tion); no further authentication was done on these lines. comparative inhibition of these targets in vivo was not The M07e [a human acute myelogenous leukemia (AML) previously examined. line] cells were purchased from the German Collection of This study primarily aimed to further describe the Microorganisms and Cell Cultures (Deutsche Sammlung pharmacology of cediranib by (i) determining activity von Mikroorganismen und Zellkulturen GmbH (German against VEGFR-1 in cells, (ii) using a wider complement Collection of Microorganisms and Cell Cultures); no of cell lines and assays to examine activity against c-Kit further authentication was done. NIH 3T3 (mouse fibro- and PDGFR-a/b signaling in vitro, and (iii) to examine blast) cells were obtained from A. Wong, Jefferson Cancer pharmacodynamic inhibition of c-Kit, PDGFR-a, and Institute; no further authentication was done. Human PDGFR-b in vivo over a dose range in which cediranib aortic and coronary vascular smooth muscle cells (VSMC) has previously shown activity in tumor models. The data were purchased from PromoCell GmbH. All cell lines confirm that cediranib is primarily a pan-VEGFR inhibi- were routinely passaged less than 10 times with the tor that can inhibit wild-type c-Kit. The data also suggest exception of the primary vascular cells, which were that cediranib may have some partial pharmacodynamic passaged no more than 4 times. NCI-H526, U118MG, activity against PDGFR-a and PDGFR-b receptor activa- MG63, C6, M07e, NIH 3T3, human aortic, and coronary tion in tumors, although this inhibition may be of limited VSMCs were maintained in culture as per providers’ functional relevance but does inhibit FGFR1 and FGFR4. recommendation. M07e cells were maintained in culture in the presence of interleukin-3 (5 ng/mL) and granulo- Materials and Methods cyte macrophage colony stimulating factor (5 ng/mL).

Reagents Inhibition of growth factor–stimulated receptor Cediranib [(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-me- phosphorylation in vitro thoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; The ability of cediranib to inhibit receptor phosphor- ref. Fig. 1) was synthesized according to the processes ylation in cells was determined using Western blotting. described in WO 00/47212, in particular those described Cells were serum starved overnight in the presence in example 240 of WO/47212. The free base of cediranib (M07e and NCI-H526) or absence (aortic and coronary was used in these preclinical studies, with a molecular VSMC) of 0.1% bovine serum albumin or in the presence weight of 450.51. For all in vitro assays, cediranib was of 1% charcoal-stripped serum (MG63, U118MG, C6, and prepared initially as a 10 mmol/L stock solution in NIH 3T3 cells). Cells were then incubated with cediranib dimethyl sulphoxide (DMSO) and diluted in the rele- for 60 to 120 minutes and stimulated with the relevant vant assay media, such that the final concentration of ligand: stem cell factor (SCF; 50 ng/mL) and PDGF-AA or

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PDGF-BB (50 ng/mL) for 5 to 10 minutes. SCF was pVEGFR-1 was analyzed by MSD ELISA. Total VEGFR-1 obtained from R&D Systems and PDGF-AA and antibody (MAB321; R&D Systems Inc.) was spotted onto PDGF-BB from Sigma-Aldrich. Cell lysates of NCI- high-binding MSD plates and incubated for 2 hours at H526, M07e, and aortic and coronary VSMCs were pre- room temperature, after which time plates were blocked pared in lysis buffer I (50 mmol/L Tris-HCl, pH 7.6; 137 and then washed. Cell lysates were added and incubated mmol/L sodium chloride; 10% glycerol; 0.1% Igepal; overnight at 4C. Plates were washed and then the sul- 0.1% SDS; 50 mmol/L sodium fluoride; 1 mmol/L photagged-pY20 detection antibody was added and left sodium orthovanadate and cocktail protease inhibitor to incubate for 1 hour at room temperature. Following tablets (Roche Diagnostics Ltd.). Cell lysates of MG63, washing, read buffer was added and plates were read U118MG, C6, and NIH 3T3 cells were prepared in lysis immediately on the SECTOR Imager 6000 (MSD). To buffer 2 (10% glycerol, 2% SDS, 50 mmol/L Tris-HCl, 200 visualize pVEGFR-1 by Western blotting, VEGFR-1 was mmol/L 2-mercaptoethanol). immunoprecipitated with an antibody to total VEGFR-1 The protein concentration in the lysates was deter- (SC316; Santa Cruz Biotechnology) and immunoblotted mined using a bicinchoninic acid assay kit (Pierce) and with the anti-phosphotyrosine antibody PY20 (SC508; Western blotting was done on whole cell lysates (50–75 mg Santa Cruz Biotechnology). Levels of total VEGFR-1 were of protein loaded per lane), using standard SDS-PAGE confirmed by immunoblotting with SC316. methods with detection by enhanced chemiluminescence. Total and phosphorylated proteins were measured Sample preparation and mass spectrometry for using antibodies to c-Kit [Cell Signaling Technology identification of phosphotyrosine modification of (CST) #3391], and phosphorylated c-Kit [(pc-Kit) VEGFR-1 SC5535; Santa Cruz Biotechnology]; PDGFR-a (AF-307; Analysis of VEGFR-1–phosphorylated epitope changes R&D Systems), PDGFR-a (CST #3164), and phosphory- was done using Cell Signaling Technology’s proprietary lated PDGFR-a [(pPDGFR-a) SC12910; Santa Cruz PhosphoScan methodology. AG1-G1-Flt-1 cells were Biotechnology]; PDGFR-b (AF-385; R&D Systems), placed in serum-free media overnight and stimulated PDGFR-b (1469-1; Epitomics), phosphorylated PDGFR- with VEGF (50 ng/mL) or placenta growth factor (PlGF; b [(pPDGFR-b) CST #3161]; mitogen-activated protein 100 ng/mL; R&D Systems) for 5 minutes [the former also kinase [(MAPK) CST #9102) and phosphorylated MAPK with and without cediranib (100 nmol/L; 85 minutes [(pMAPK) CST #9101]. Phosphorylation was quantitated preincubation and 5 minutes coincubation with ligand)]. using the ChemiGenius Imaging System for Chemilumi- Protein extracts from AG1-G1-Flt1 cells were prepared by nescence (Syngene) with the exception of the human suspending cells in Urea Lysis Buffer (20 mmol/L coronary VSMCs, which were quantified by ELISA (goat HEPES, pH 8.0, 9.0 mol/L urea, 1 mmol/L sodium anti-PDGFR Alpha R&D Systems AF-307-NA Capture, orthovanadate, 2.5 mmol/L sodium pyrophosphate, 1 DuoSet detection; R&D Systems). mmol/L b-glycerol phosphate). Lysates generated from AG1-G1-Flt-1 cells were established with the permis- approximately 2 108 cells were prepared for each sion of the Ethics Committee for Scientific Research at the sample condition (control, VEGF-treated, PlGF-treated, Institute of Medical Science, University of Tokyo, Tokyo, and VEGF þ cediranib-treated). The resulting protein Japan. Briefly, a human adult benign angioma was extracts (40 mg total protein each) were then reduced excised surgically and plated with Ham’s F-12 nutrient with dithiothreitol (4.5 mmol/L), carboxamidomethy- mixture medium (Invitrogen) supplemented with 10% lated using iodoacetamide (10.0 mmol/L), and subse- FBS (JRH Biosciences) and 40 mg/mL kanamycin (Wako). quently digested with trypsin (1:100 weight, trypsin to A pEF1a-SV40 large T antigen plasmid was introduced total protein). Peptides were separated from nonpeptide into the cells, using DMSO and polybrene (SIGMA- material by solid-phase extraction with Sep-Pak C18 Aldrich). An SV40 large T-positive clone AG1-G1 cell cartridges. Lyophilized peptides were redissolved, and was isolated and then pBCMGS-Neo-Flt-1 carrying the phosphorylated peptides were isolated using a slurry of full length of Flt-1 cDNA (10), or the empty vector immobilized phosphorylated tyrosine antibody (CST pBCMGS-Neo plasmid, was transfected into AG1-G1 cell #9411) conjugated to protein G agarose beads (Roche). by the Effectene Transfection Reagent (Qiagen). Clone Peptides were eluted from antibody resin into a total selection and culture were done with Ham’s F-12 med- volume of 100 mL in 0.15% trifluoroacetic acid. Eluted ium containing 10% FBS, 40 mg/mL kanamycin, and 400 peptides were concentrated with PerfectPure C18 tips mg/mL geneticin G418 (Wako). G418 was decreased to immediately before liquid chromatography/mass spec- 200 mg/mL in regular culture. To examine inhibition of trometry (LC/MS) analysis. Peptides were loaded VEGFR-1 phosphorylation, cells were placed in serum- directly onto a 10 cm 75 mm PicoFrit capillary column free media overnight and then incubated with cediranib packed with Magic C18 AQ reversed-phase resin. The for 90 minutes and stimulated with VEGF 50 ng/mL column was developed with a 45-minute linear gradient (R&D Systems) for the last 5 minutes of incubation. Cell of acetonitrile in 0.125% formic acid delivered at lysates were prepared in lysis buffer 1 and phosphory- 280 nL/min. Tandem mass spectra were collected with lated VEGFR-1 (pVEGFR-1) was evaluated using Meso a linear trap quadrupole (LTQ)-orbitrap hybrid mass Scale methodology [Meso Scale Discovery (MSD)]. The spectrometer, using a top-ten method, a dynamic

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exclusion repeat count of 1, and a repeat duration of 30 in phenol red DMEM containing 1% charcoal-stripped seconds. MS spectra were collected in the orbitrap com- serum for 24 hours at 37C. The following day, the ponent of the mass spectrometer, and MS/MS spectra medium was replaced with DMEM (Sigma-Aldrich) con- were collected in the LTQ. MS/MS spectra were evalu- taining PDGF-AA or PDGF-BB (50 ng/mL) and cediranib ated using TurboSequest in the Proteomics Browser for a further 72 hours. Cell proliferation was determined package (v. 27, rev. 13). The ratios of the integrated peak as described earlier. All assays were done in triplicate, height intensities for phosphopeptide quantification and the mean SEM was calculated from 3 independent were obtained using the XCalibur software 2.0.7 (Ther- experiments. moFinnigan). A reduction in peak intensity in VEGF- treated cells compared with control, PlGF-treated cells Inhibition of receptor phosphorylation in vivo compared with control, or VEGF plus cediranib-treated The activity of cediranib was evaluated in an NCI- cells compared with control was expressed as a negative H526 human SCLC tumor xenograft model. Tumors fold change value. All integrated peak intensity calcula- were implanted subcutaneously in the hind flank of tions were manually reviewed to ensure proper integra- female nude (nu/nu genotype) mice of at least 8 weeks tion of consistently shaped, coeluting peaks. Changes in of age. When tumors reached a volume of 0.36 0.02 phosphorylated peptide levels were measured by taking cm3, mice were randomized (8–10 per group) and dosed the ratio of raw intensities between control and treated with cediranib (0.75–6 mg/kg/d) or vehicle adminis- cells, with the untreated sample as the reference (denomi- tered once daily by oral gavage. Tumor volumes were nator) in each case. Raw intensity ratios were normalized assessed by bilateral Vernier caliper measurement at using a median adjustment technique whereby the log2 least twice weekly and calculated using the formula ratios comprising each binary comparison (inhibitor trea- (length width) H (length width) (p/6), where ted versus control) was independently and globally length was taken to be the longest diameter across the adjusted such that the normalized median log2 ratio is tumor and width the corresponding perpendicular. To zero. The normalized log2 ratios were then converted to remove any size dependency before statistical evalua- their corresponding normalized fold changes. tion (the variation in mean tumor volume data increases proportionally with volume and is therefore dispropor- Inhibition of growth factor–mediated cellular tionate between groups), data were log-transformed proliferation before statistical evaluation by using a 1-tailed 2-sample NCI-H526 cells were used to determine the effect of t test. NCI-H526 xenograft tumors were evaluated cediranib on SCF-stimulated proliferation. Cells were for c-Kit receptor phosphorylation ex vivo by using seeded at a density of 1 105 per mL in 96-well microtiter immunoprecipitation, following acute (2 days; 8 ani- plates in phenol red–free low-serum containing media mals per group) or chronic (17 days; 3 animals per (0.2% FBS) overnight. The following day cells were pre- group) treatment with cediranib. Tumors were homo- treated with cediranib (0.1–100 nmol/L) for 30 minutes genized in lysis buffer I, and following a protein assay, before stimulation with 50 ng/mL SCF and then incuba- 5 mg of protein from each sample was immunopreci- tion for 72 hours at 37C. Cell proliferation was deter- pated overnight at 4C with anti-c-Kit–conjugated agar- mined using an XTT endpoint (Roche Diagnostics Ltd.). ose beads. The immune complexes were washed, and All assays were done in triplicate, and the mean SEM proteins were eluted by boiling in SDS sample buffer. was calculated from 6 independent experiments. Human Standard SDS-PAGE methods were done to enable aortic VSMCs were used to determine the effect of cedir- detection of total and pc-Kit, using antibodies as pre- anib on PDGF-BB–stimulated proliferation. Cells were viously described. Protein phosphorylation was quan- seeded at 10,000 cells per well in black-walled 96-well titated using the ChemiGenius as described earlier. plates in smooth muscle cell growth medium 2 (Promo- The activity of cediranib was also evaluated in a C6 rat cell GmbH) and incubated overnight at 37C. The follow- glial tumor xenograft model in mice. Cells were cultured in ing day, the medium was replaced with Dulbecco’s 199 media (Life Technologies) supplemented with 10% Modiﬁed Eagle’s Medium (DMEM; Gibco) containing fetal calf serum and 2 mmol/L glutamine and maintained 0.1% FBS, PDGF-BB (50 ng/mL), and cediranib. After in 7.5% CO2. For all tumor studies, C6 glial cells were 24-hour incubation, a bromodeoxyuridine (BrdU) reagent resuspended in sterile PBS and inoculated subcutaneously (Amersham) was added and cells were incubated for a (1 105 in 100 mL) in the hind flank of male athymic mice. further 24 hours at 37C. Cells were fixed in formalin for Tumor volumes were assessed as described earlier. 15 minutes, and proliferation was assessed by staining for Approximately 21 days postimplantation when the C6 glial BrdU by using the Cell Proliferation Fluorescence Kit tumor volume was between 0.5 and 0.8 cm3, mice were (Amersham). Cells were imaged on the ArrayScan (Cel- randomized into groups of 10 before treatment. Mice lomics). All assays were done in triplicate, and the mean received a single-bolus oral dose of either cediranib or SEM was calculated from 3 independent experiments. vehicle control. Animals were sacrificed 4 hours post–dose MG63 cells were used to determine the effect of cediranib administration. Five minutes before sacrifice, VEGF-A (20 on PDGF-AA- and PDGF-BB–stimulated proliferation. mg per mouse; AstraZeneca) and PDGF-BB (75 mgper Cells were seeded at 1,500 cells per well in 96-well plates mouse; Biosource) were coadministered intravenously

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(0.3 mL/mouse in PBS) to all animals. Terminal blood cediranib (Fig. 2C). There was a marked reduction in the samples were taken from the vena cava into lithium relative abundance of peptides corresponding to Y794, heparin tubes to collect plasma samples for pharmacoki- Y1053, Y1053/Y1048, and Y1048 in cediranib-treated netic analysis. Tumors and lungs were excised and halved, samples, with a 37-fold reduction in the presence of and each tissue was half weighed and snap frozen imme- the peptide corresponding to pY1053/48 in the cedira- diately in liquid nitrogen. Tissue samples were stored at nib-treated samples (Fig. 2C). The total tyrosine phos- 80C until processed for Western blot analysis for total phorylation status of VEGFR-1 in the lysates used for this and phosphorylated VEGFR-2 (pVEGFR-2) and PDGFR-b. specific analysis was also assessed by ELISA. VEGFR-1 Lungs were homogenized in lysis buffer I, and tumors were from each lysate was captured, and the level of tyrosine homogenized in lysis buffer III [60 mmol/L Tris, 150 phosphorylation was detected using an antiphosphotyr- mmol/L sodium chloride, 1 mmol/L ethylenediaminete- osine antibody (Fig. 2D). Both VEGF-A and PlGF induced traacetic acid, 1% Igepal, 0.25% sodium deoxycholate, 40% significant phosphorylation of VEGFR-1 in the lysates. glycerol, 8% SDS, 200 mmol/L Tris-HCl, phosphatase Cediranib inhibited the VEGF-A–induced phosphoryla- inhibitor cocktails 1 and 2 (Sigma-Aldrich)]. tion of VEGFR-1.

Results Cediranib inhibits c-Kit phosphorylation and SCF-induced proliferation Cediranib is a potent inhibitor of VEGFR-1 Cediranib inhibits c-Kit with a similar potency to that To determine the potency of cediranib against VEGFR- with which it inhibits the tyrosine kinase activity of 1 in cells, a cell line stably transfected with full-length VEGFRs (7). The activity of cediranib against c-Kit was VEGFR-1 (AG1-AG-Flt-1) was used. Cediranib inhibited tested in 2 cell lines, M07e and NCI-H526. SCF-stimulated VEGF-A–driven VEGFR-1 phosphorylation with an IC50 c-Kit phosphorylation was inhibited with IC50 values of 3 value of 1.2 nmol/L (Fig. 2A). This is comparable with the and 1 nmol/L, respectively (Fig. 3A and B). MAPK as a cellular potency versus VEGFR-2 (0.5 nmol/L; ref. 7) and downstream signaling marker was also inhibited with an < VEGFR-3 ( 1 nmol/L; ref. 8) and consistent with the IC50 value similar to that for inhibition of receptor phos- primary pharmacology of the compound being that of phorylation (Fig. 3A and B). The relationship between a potent pan-inhibitor of VEGFR-1, VEGFR-2, and inhibition of acute ligand-induced phosphorylation and VEGFR-3 tyrosine kinase activity. SCF-stimulated c-Kit-dependent proliferation was deter- To identify the tyrosine phosphorylation sites on mined using NCI-H526 cells. Cediranib inhibited SCF- VEGFR-1 modulated by ligand-induced autophosphor- stimulated proliferation of NCI-H526 cells after 72 hours ylation and inhibition by cediranib, Phosphoscan was with an IC50 value of 0.013 nmol/L (Fig. 3C), and full done on VEGFR-1 isolated from the AG1-G1 cells treated inhibition being achieved at concentrations between 20 with VEGF-A and with VEGF-A in the presence of 100 and 50 nmol/L. From these experiments, it seems that nmol/L cediranib. Phosphorylated receptor was about 10-fold higher concentrations were required to enriched via a total phospho-tyrosine immunoprecipita- inhibit functional consequences of c-Kit signaling (i.e., tion. The residues phosphorylated on VEGFR-1 in each c-Kit–dependent cellular proliferation) than for the inhi- treated lysate were examined by specifically identifying bition of receptor phosphorylation. phosphorylated peptides corresponding to VEGFR-1 Mutations in c-Kit are associated with certain tumors (Fig. 2A and B; Supplementary Table S1). On stimulation such as gastrointestinal stromal tumors (GIST) and AML with VEGF-A or PlGF, significant induction of phosphor- in which they drive tumor growth. The activity of cedir- ylation of peptides incorporating tyrosine residues anib against a range of common c-Kit mutations was also Y1053, Y1048/Y1053, and Y1048 was observed. Modest determined using a panel of cell lines that either induction of phosphorylation was also detected at resi- expressed mutated c-Kit endogenously or were transi- dues 794 and 1242, but the magnitude of change was ently transfected with mutated receptors. The c-Kit muta- lower (Fig. 2A). The pattern of ligand-induced phosphor- tions assessed were V560G, V559D, W557R, Del 557–558, ylation by both VEGF-A and PlGF was similar, although V654A, T670I, D816V, D816Y, and N822K. To assess the the magnitude of induction was higher with VEGF-A potential of the compound to inhibit phosphorylation of than with PlGF. Serine-phosphorylated peptides were these receptors, cells were incubated in the presence and also detected, although the significance of these modifi- absence of 20 nmol/L of cediranib. Cediranib inhibited cations is unclear (Supplementary Table S1). This shows phosphorylation of c-Kit mutants V560G, V559D, W557R, that under these conditions, the phosphorylation status of and Del 557–558, V654A, and N822K markedly, but it did VEGFR-1 is dynamically regulated on a restricted num- not inhibit constitutive phosphorylation of c-Kit mutants ber of residues on engaging VEGF-A or PlGF, with Y1048 T670I, D816V, and D816Y (Supplementary Table S2). and Y1053 showing the greatest fold changes. To determine which residues were dynamically regu- Inhibition of c-Kit phosphorylation by cediranib lated by cediranib, we compared protein extracts from in vivo cells stimulated with VEGF-A with those from cells Inhibition of c-Kit phosphorylation was examined in stimulated with VEGF in the presence of 100 nmol/L vivo in established NCI-H526 tumor xenografts, following

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AC AG1-G1-Flt-1 0 μ Mw IC50 ( mol/L) 0.0012 ± 0.0008 kDa -250 -10 pVEGFR1

VEGFR1 -250 -20 0 7 22 66 0.1 0.3 0.8 2.5 -30 control VEGF-A + cediranib (nmol/L) control stimulated -40

Fold change vs. VEGF-A vs. change Fold 4 48 53 42 09 9 0 0 2 3 1 1 Y7 Y Y1 Y1 Y 48 Y1053 0 Y1 BD 25 2,000 PlGF 20 VEGFA 1,600 15 1,200 10 800

5 (counts) 400 0 Phospho-VEGFR-1 Fold change vs. control vs. change Fold -5 4 0 9 48 53 42 09 0 0 2 3 Y7 1 ol -A nib Y1 Y1 Y1 Y r GF F a Pl G 48 Y1053 E 0 Cont V cedir Y1 + A F-

VEG

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 minutes in the absence or presence of a range of concentrations of cediranib. Phosphorylation of VEGFR-1 was determined 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 by using a total VEGFR-1 antibody. The IC50 value was determined using MSD ELISA; the mean IC50 SEM observed on a number of experiments is shown. B, to determine the residues on VEGFR-1 that show dynamic changes in phosphorylation on 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 antiphosphotyrosine antibody, and the associated proteins were 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 on activation of VEGFR-1, AG1-G1-Flt1 cells were stimulated with 50 ng/mL VEGF-A in the presence of 100 nmol/L 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.

chronic once-daily dosing of cediranib (17 doses of 6, 3, Cediranib inhibits PDGFR-mediated 1.5, or 0.75 mg/kg) to tumor-bearing mice (Fig. 3D). The autophosphorylation and PDGF-driven dose range examined has been previously determined to proliferation at higher concentrations result in dose-dependent inhibition of a wide range of In recombinant kinase assays, cediranib has been pre- human tumor xenograft models that do not express or viously shown to exhibit lower potency, 10- and 36-fold have a dependency on c-Kit. C-Kit was immunopreci- for inhibition of PDGFR-a and PDGFR-b than for VEGFRs pated, and the samples were analyzed for phosphory- or 2.5- and 19-fold for c-Kit (7). The activity against lated and total receptors. In NCI-H526 tumors, cediranib PDGFR-a and PDGFR-b signaling was further explored reduced phosphorylation of the receptor by greater than using a range of cell types including other tumor cells, 80% at doses as low as 0.75 mg/kg. Although NCI-H526 VSMCs, and fibroblasts (Fig. 4A–F). PDGF-AA and PDGF- tumor growth has been suggested to be dependent on c- BB ligands were used in stimulation assays, the former Kit (11), despite the tumors expressing constitutively pc- inducing homodimerization of PDGFR-a and the latter Kit, no enhanced effects on growth or survival of the homodimerization of PDGFR-b and heterodimerization tumor cells were observed in these experiments (Supple- of PDGFR-a and PDGFR-b. U118MG human glioma mentary Fig. S1) compared with other xenografts not cells express both human PDGFR-a and PDGFR-b. Cedir- expressing c-Kit. anib inhibited PDGF-AA–induced phosphorylation of

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AB NCI-H526 M07e

MW MW IC (μmol/L) 0.001 ± 0.0003 IC (μmol/L) 0.003 ± 0.0020 50 kDa 50 kDa pc-Kit -160 pc-Kit -160

c-Kit -160 c-Kit -160

pMAPK -35 pMAPK -35

-35 -35 MAPK MAPK 0 1 0 1 10 0.1 10 100 0.1 0.01 100 0.01 0.001 0.001 Control Control SCF + [cediranib] (nmol/L) SCF + [cediranib] (nmol/L)

CD NCI-H526 proliferation NCI-H526 tumor PD

300,000 MW Cediranib (mg/kg) kDa Control 6 3 1.5 0.75 μ IC50 ( mol/L) 0.013 ± 0.005 160- pc-Kit 0.25 250,000 (pY719) 160- c-Kit 0.20 200,000

0.15 150,000

0.10 100,000 pc-Kit signal pc-Kit Response (OD) ** 0.05 50,000 *** ** **

0.00 0 0 0 .1 1 5 0 0 0 0 Control 6 3 1.5 0.75 0 1 2 5 1 [Cediranib] (nmol/L) Dose of cediranib (mg/kg)

Figure 3. Cediranib inhibits SCF-driven c-Kit phosphorylation and proliferation in tumor cells. To assess phospho 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 to 120 minutes and then stimulated with 50 ng/mL SCF for 5 to 10 minutes. Representative Western blots showing the inhibition of phosphorylated c-Kit and phosphorylated MAPK in NCI-H526 cells

(A) and M07e cells (B) are shown. All data are representative of at least 3 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, NCI-H526 cells were stimulated with 50 ng/mL cediranib in the presence and absence of cediranib for 72 hours and proliferation was assessed by BrdU incorporation. The mean IC50 SEM observed over a number of experiments is shown. D, to show that 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/d cediranib and the levels of pc-Kit were determined by ex vivo analysis. c-Kit was immunoprecipitated from tumor lysates by using a total c-Kit antibody. c-Kit and phospho c-Kit were detected by Western blotting and chemiluminescence; a representative Western blot is shown. Phospho c-Kit was quantified, and the mean levels in each group SEM are shown. **, P < 0.01; ***, P < 0.001. PD, pharmacodynamics.

PDGFR-a and PDGF-BB–induced phosphorylation of tional consequences of PDGFR activation, its potency was b PDGFR- , with mean IC50 values of 20 and 32 nmol/L, assessed in both PDGF-AA- and PDGF-BB–driven pro- respectively. In C6 rat glioma cells, a similar IC50 value of liferation assays. In human aortic VSMCs, cediranib inhib- 24 nmol/L was observed versus PDGF-AA stimulation of ited PDGF-BB–stimulated proliferation after 48 hours a PDGFR- (Fig. 4C). In NIH 3T3 cells (mouse fibroblast with an IC50 value of 36 nmol/L (Fig. 4G), similar to line), cediranib was slightly more potent, inhibiting the potency versus PDGFR-b phosphorylation in the same PDGF-BB–mediated phosphorylation of PDGFR-b with cells. In MG63 cells, cediranib inhibited PDGF-BB–stimu- an IC50 value of 12 nmol/L (Fig. 4D). Comparable activity lated proliferation with an IC50 value of 63.5 nmol/L was found in smooth muscle cell types. In cultured human (Fig. 4H), similar to the previously reported IC50 value coronary VSMCs, the primary PDGFR is PDGFR-a (data of 40 nmol/L versus PDGF-AA-induced proliferation in not shown). Cediranib inhibited PDGF-AA–stimulated the same cell line (7). receptor phosphorylation with an IC50 value of 15 nmol/L (Fig. 4E). In contrast, in human aortic VSMCs, Cediranib gives differential inhibition of PDGFR the primary PDGFR is PDGFR-b (data not shown). In these signaling in C6 tumors and murine lung tissue in cells, cediranib inhibits PDGF-BB–induced phosphoryla- ligand-induced acute pharmacodynamic assays b tion of PDGFR- with an IC50 value of 23 nmol/L (Fig. 4F). We have previously shown time- and dose-dependent To determine how effectively cediranib inhibits the func- inhibition of VEGFR-2 in murine lung tissue by using a

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AB U118MG U118MG MW μ μ MW IC50 ( mol/L) 0.020 ± 0.008 kDa IC50 ( mol/L) 0.032 ± 0.010 kDa pPDGFR-α -250 pPDGFR-β -250

PDGFR-α -250 PDGFR-β -250 0 0 12 37 12 37 1.4 4.1 4.1 1.4 100 300 100 300 0.15 0.46 0.15 0.46 Control Control PDGF-AA + cediranib (nmol/L) PDGF-BB + cediranib (nmol/L)

CD Figure 4. Cediranib inhibits C6 NIH 3T3 both PDGF ligand–stimulated MW MW phosphorylation of PDGFR-a IC (μmol/L) 0.024 ± 0.004 IC (μmol/L) 0.012 ± 0.0027 50 kDa 50 kDa and PDGFR-b and ligand- α -250 β pPDGFR- pPDGFR- -250 dependent proliferation. -250 To determine the activity of PDGFR-α PDGFR-β -250 cediranib against PDGFRs, a 0 0 12 37 12 37 range of cell lines U118MG (A and 1.4 4.1 1.4 4.1 100 300 100 300 0.15 0.46 0.15 0.46 B), C6 (C), NIH 3T3 (D), human Control Control coronary VSMCs (E), and human PDGF-AA + cediranib (nmol/L) PDGF-BB + cediranib (nmol/L) aortic VSMCs (F) were stimulated with PDGF-AA (50 ng/mL) or PDGF-BB (50 ng/mL) for 5 to 10 EF minutes and then lysed. Inhibition Human coronary VSMC Human aortic VSMC of pPDGFR-b in human aortic VSMCs (D) and inhibition of MW MW μ μ pPDGFR-a in human coronary IC50 ( mol/L) 0.015 ± 0.003 kDa IC50 ( mol/L) 0.023 ± 0.007 kDa VMSCs and C6 cells (E). α -250 -250 pPDGFR- pPDGFR-β Representative Western blots -250 showing pPDGFR-a and PDGFR-α -250 PDGFR-β pPDGFR-b, as well as levels of 7 0 7 0

22 66 total receptors, are shown as 0.3 0.8 2.5 66 22 200 0.3 0.8 2.5 200 indicated. The mean IC50 SEM observed over a number of Control Control experiments is shown. To PDGF-BB + cediranib (nmol/L) PDGF-AA + cediranib (nmol/L) determine the effect of cediranib on PDGF-stimulated proliferation, GH human aortic VSMCs (G) and Human aortic VSMC (PDGF-BB) MG63 (PDGF-BB) MG63 cells (H) were stimulated with 50 ng/mL PDGF-BB for 72 hours in the presence or absence 20 μ μ IC50 ( mol/L) 0.032 ± 0.004 IC50 ( mol/L) 0.0635 ± 0.0009 of cediranib. The mean IC 12 50 SEM observed over a number of experiments is shown. 15 10

8 10 6

5 4 Response (OD x100) Response (BrDu units)Response (BrDu 2 0 0 3 8 5 0 3 3 0 . . . 7 2 6 0 .5 .6 .7 . 2 70 0 0 2 2 6 2 0 1 4 3 1 1 3 1 4 1,1113,333 [Cediranib] (nmol/L) [Cediranib] (nmol/L)

ligand-induced pharmacodynamic assay (12). This addition of exogenous ligand to stimulate receptor phos- approach was taken because the interanimal variability phorylation overcame this issue. Here we used a similar in pVEGFR-2 levels was high, making accurate assess- approach to assess the inhibition of PDGFR activation ment of inhibitor dose responses extremely difficult. The relative to VEGFR-2 to gain greater insight into the effects

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of cediranib on these receptors in vivo. The relative nificantly less active against these receptors than against potency of cediranib versus VEGFR-2 and PDGFR-b the VEGF receptors, or c-Kit. was compared directly in vivo in the same animal. To normalize levels of pVEGFR-2 and stimulate PDGFR-a Discussion and PDGFR-b phosphorylation, animals were injected with both VEGF-A and PDGF-BB immediately before We have previously shown that cediranib is a potent sacrifice. Lungs from animals bearing C6 tumors receiv- inhibitor of VEGFR-2 and VEGFR-3 and that it reduces ing cediranib 6, 3, 1.5, or 0.75 mg/kg for 4 hours were growth of a wide range of tumor models by targeting assessed for levels of pVEGFR-2 (Fig. 5A) and pPDGFR-b tumor vasculature (7, 8, 12). Cediranib has selectivity for (Fig. 5B) 4 hours after dosing. This time point was chosen, VEGFR-2 against a wide range of kinases, including the as we established that the maximal exposure of cediranib PDGFR family members CSF-1R and Flt-3 in cellular occurs between 2 and 3 hours in mice (data not shown). phosphorylation assays (420- and >20,000-fold selectivity, Consistent with previous data (12), VEGFR-2 phosphor- respectively, vs. VEGFR-2; ref. 7). Here we explore the ylation in lung was significantly reduced over a range of pharmacology of cediranib in more depth, examining its doses from 6 mg/kg down to 0.75 mg/kg (Fig. 5A). In activity against VEGFR-1 in cells and the PDGFR family contrast, phosphorylation of PDGFR-b was only signifi- members c-Kit, PDGFR-a, and PDGFR-b in vitro and in cantly inhibited in lung at 6 mg/kg, with no significant vivo within a dose range of 0.75 to 6 mg/kg which has inhibition achieved at lower doses (Fig. 5B). been routinely examined within preclinical tumor xeno- To further explore the relative activity against activity graft experiments. PDGFR-a and PDGFR-b expressed in tumor cells, we Establishing the potency of small molecule inhibitors examined C6 tumor xenografts. C6 cells express both against VEGFR-1 signaling in ligand-induced endothelial PDGFR-a and PDGFR-b.AlthoughPDGFR-a is consti- cell assays has proven challenging due to the low intrinsic tutively phosphorylated in C6 cells in vivo, the injection kinase activity associated with this receptor. Despite this of PDGF-BB results in the additional phosphorylation of low kinase activity, there is some evidence to implicate PDGFR-b, enabling inhibition of both receptors to be VEGFR-1 signaling in pathologic angiogenesis (13–15) as studied in the same tumor. In separate studies (acute and well as in the recruitment of macrophages and myeloid chronic), we have assessed the activity of cediranib prescursor cell recruitment to tumors (16–18), a process against PDGFR-a in C6 tumor xenografts (at 6 hours) which has been linked with resistance to VEGF signaling and established that cediranib gives up to 60% inhibition inhibitors (19–21). The role of the VEGFR-1 kinase of PDGFR-a phosphorylation. Again because of varia- domain in the recruitment of bone marrow–derived cells bility in phosphorylation between tumors, these data into tumors has been confirmed using VEGFR-1 TK / were variable (data not shown). Adopting an acute transgenic mice (22). Consequently, concurrent inhibition ligand stimulation approach allowed us to further exam- of VEGFR-1 and -2 signaling may afford added thera- ine inhibition of both PDGFRs in tumor and lung within peutic benefit. To examine inhibition of VEGFR-1, we the individual animals. In contrast to the modulation of used a cell line derived from a human benign angioma receptor phosphorylation in lung tissue, phosphoryla- into which the full-length receptor was overexpressed by tion of both PDGFR-a (Fig. 5C) and PDGFR-b (Fig. 5D) in stable transfection. This cell line does not express VEGFR- C6 tumors, in the same animals, was partially inhibited 2 and therefore enables a more accurate assessment of by all doses of cediranib examined. activity against VEGFR-1 by avoiding any confounders that could result from VEGFR heterodimerization. The Selectivity against other tyrosine kinase receptors inhibition of VEGF-induced VEGFR-1 phosphorylation Some VEGFR tyrosine kinase inhibitors also have activ- by cediranib, as determined by Western blotting, was ity against other kinases. Selectivity of cediranib against evident at a potency that is comparable with that deter- mutFlt-3 and CSF-1R members of the PDGFR family has mined against VEGFR-2 and VEGFR-3 activation in cel- been shown previously (Wedge and colleagues 2005). In lular assays, thereby confirming that cediranib is a pan- addition to determining the relative activity against VEGFR kinase inhibitor. Although it is clear that VEGFR-1 VEGFR-1, c-Kit, and PDGFRs, we also tested its activity does induce specific signaling (10, 23, 24), there is limited against wild-type Flt-3 and fibroblast growth factor information on the residues involved. We therefore also receptor (FGFR) 1 and FGFR4 (Supplementary used an MS-based method to examine the residues acti- Table S3). The activity against Flt-3 was determined using vated on VEGFR-1 by VEGF or PlGF in AG1-G1-Flt-1 cells OCI-AML-5 cells stimulated with Flt-3 ligand (Flt-3L). and inhibition of the VEGF-induced response by cedir- The activity against FGFR1 and FGFR4 was determined anib. VEGF-A and PlGF were found to induce a broadly by transiently overexpressing the receptor in Cos-1 cells, similar pattern of change in VEGFR-1 increasing the which resulted in constitutive phosphorylation of the phosphorylation of tyrosine residues Y794, Y1048, receptor. Cediranib was inactive against wild-type Flt-3 Y1053, and Y1242. The greatest fold change evident was > m (IC50 value 1 mol/L) and had marginal activity versus at Y1048 and Y1053, which are within the tyrosine kinase m FGFR-1 and -4 (IC50 values of 0.35 and 2.17 mol/L, domain of the receptor. Cediranib treatment abolished all respectively). These data indicate that cediranib is sig- VEGF-stimulated phosphorylation on the 4 residues

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AB Lung pVEGFR-2 Lung pPDGFR-β Cediranib (mg/kg) Cediranib (mg/kg) MW MW kDa kDa Control 6 3 1.5 0.75 Control 6 3 1.5 0.75 250- pPDGFR-β 250- pVEGFR-2 (pY1175) (pY751) 250- 250- PDGFR-β 650,000 VEGFR-2 40,000 600,000 35,000 550,000 500,000 30,000 450,000 25,000 400,000 β signal 350,000 20,000 300,000 250,000

pVEGFR-2 signal *** pPDGFR- 15,000 200,000 *** *** 10,000 ** 150,000 *** 100,000 5,000 50,000 0 0 Control 6 3 1.5 0.75 Control 6 3 1.5 0.75 Dose of cediranib (mg/kg) Dose of cediranib (mg/kg)

CD C6 tumor pPDGFR-α C6 tumor pPDGFR-β

Cediranib (mg/kg) MW Cediranib (mg/kg) MW kDa kDa Control 6 3 1.5 0.75 Control 6 3 1.5 0.75 α 250- 250- pPDGFR- pPDGFR-β (pY720) (pY751) 250- 35,000 250- PDGFR-α 50,000 PDGFR-β

45,000 30,000 40,000

25,000 35,000

β signal 30,000 α signal 20,000 *** 25,000 15,000 20,000 pPDGFR- pPDGFR- *** *** *** 10,000 15,000 *** 10,000 *** 5,000 *** 5,000 ***

0 0 Control 6 3 1.5 0.75 Control 6 3 1.5 0.75 Dose of cediranib (mg/kg) Dose of cediranib (mg/kg)

Figure 5. Cediranib inhibits phosphorylation of PDGFR-b in lung and tumor tissue and phosphorylation of VEGFR-2 in lung and in an established rat C6 tumor xenograft model following a single dose. Acute changes in phosphorylation of VEGFR-2 and PDGFR-a and PDGFR-b were determined following a single dose of cediranib at the doses 6, 3, 1.5, and 0.75 mg/kg. Phosphorylation of PDGFR-a and PDGFR-b and VEGFR-2 was stimulated by injection of PDGF-BB and VEGF-A 5 minutes before sacrifice of the animals. A, levels of pVEGFR-2 in lung lysates were quantitated, and the mean levels SEM are represented. A representative Western blot showing pVEGFR-2 and total VEGFR-2 is inset. B, levels of pPDGFR-b in lung were quantitated, and the mean levels SEM are represented. A representative Western blot showing pPDGFR-b and total pPDGFR-b is inset. B, levels of pPDGFR-a (C) and pPDGFR-b (D) in tumor tissue lysates were quantitated and the mean levels SEM are represented. Representative Western blots showing phosphorylated and total pPDGFR-a and pPDGFR-b are inset. **, P < 0.01; ***, P < 0.001.

described but had greatest effect against Y1048/Y1053, the labile. These data contrast with previous studies that have signal from this peptide sequence being reduced by nearly described a number of VEGFR-1 residues in the C-term- 37-fold when compared with the untreated control, sug- inal tail as being modulated by VEGF-A treatment, in gesting that phosphorylation at this site was the most particular Y1213 (25–27) but also Y1327 and Y1333 (27),

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and Y1309 in response to PlGF stimulation (24). Although cell types is consistent with our previous data, with IC50 peptides indicating phosphorylation at Y1213 were values in the range of 15 to 32 nmol/L being slightly detected in our study, these were not modulated by ligand higher than existing data in MG63 cells (cediranib IC50 activation. These differences may be attributable to the cell values of 5 and 8 nmol/L against PDGFR-a and PDGFR- line examined or technical approach used. We were b, respectively). A previously observed drop-off in unable to develop a pharmacodynamic assay to measure potency of approximately 7-fold between phosphoryla- inhibition of VEGFR-1 activity in vivo, due to both the low tion and proliferation end points for PDGF-AA/PDGFR- level of receptor phosphorylation and the inability to a signaling in MG63 cells was also apparent in the present identify selective phosphorylation-specific antibodies to studies when these cells were stimulated with PDGF-BB. VEGFR-1. However, given the similar potency of cedir- The activity of cediranib against PDGF-BB–induced anib against each VEGF receptor in cells, it would be PDGFR-b phosphorylation and cellular proliferation reasonable to assume that it has the ability to inhibit was more comparable in the primary VSMCs, suggesting VEGFR-1–driven signaling responses in vivo. that PDGFR-mediated signaling responses may be cell- In addition to having activity against the VEGF recep- type dependent. In vivo, cediranib inhibited PDGFR-b tors, cediranib also inhibits the kinase activity of c-Kit. signaling in C6 rat tumor xenografts across the dose range Inhibition of wild-type c-Kit signaling in M07e and NCI- examined, in contrast to an effect being evident only at a H526 cells prevented downstream MAPK phosphoryla- dose of 6 mg/kg in normal lung tissue. This apparent tion. Cediranib inhibited the SCF-induced proliferation of discrepancy is unlikely due to species-specificity differ- NCI-H526 cells and reduced an associated increase in ences, given the high degree of receptor homology AKT phosphorylation (data not shown). However, a between mouse and rat. Although a distribution effect decrease in potency (of 10-fold) was observed for inhi- cannot be ruled out, the tissue concentrations of cediranib bition of SCF-induced proliferation, suggesting that up to in C6 tumors did not exceed those in normal lung tissue 90% of SCF signaling through c-Kit and MAPK needs to (data not shown), nor is the inhibition of PDGFR phos- be suppressed to deliver a cytostatic effect in NCI-H526 phorylation in C6 tumors due to a bystander effect that is cells. In vivo, inhibition (85%) of the constitutive phos- secondary to the antivascular effects of cediranib, because phorylation of c-Kit in established NCI-H526 xenograft compound treatment does not affect the phosphorylation tumors was observed after 17 days of chronically dosing of other receptor tyrosine kinases, such as EGFR in Lovo cediranib at 0.75 to 6 mg/kg. This suggests that cediranib human colorectal tumor xenografts, at doses that signifi- may elicit a pharmacodynamic affect great enough to cantly inhibit tumor growth (data not shown). An alter- influence the phenotypic consequences of c-Kit signaling native explanation for the divergent effect observed in in vivo, although an enhanced antitumor effect was not lung and C6 tumors could relate to the differential regula- observed in xenografts derived from this particular tion or function of PDGFR-b in these tissue compartments, tumor line. the receptor driving significant cellular proliferation in C6 Mutation or aberrant activation of c-Kit and its ligand tumors but not in normal lung tissue. SCF is associated with the progression of numerous Although cediranib inhibited PDGFR-a and PDGFR-b solid and hematologic malignancies, including GIST phosphorylation in C6 tumors, this model did not seem to (28), SCLC (29), and AML (30). Approximately 95% of have increased sensitivity to the antitumor effects of the GIST cases are positive for c-Kit, with 60% to 70% compound. A dose of 3 mg/kg cediranib, which inhibited positive for the c-Kit exon 11 mutations (V560G, PDGFR-a and PDGFR-b phosphorylation by 73% and V559D, W557R,andDel 557–558) against which the c- 76%, respectively, 4 hours after an acute dose, inhibited Kit/PDGFR/Abl kinase inhibitor imatinib shows activ- tumor growth by 52% ( 4% SEM) after 10 to 14 days of ity (31). More recently, secondary mutations of c-Kit continuous once-daily dosing (data not shown), an effect have been identified that confer acquired resistance to not dissimilar to that observed in non–PDGFR-depen- imatininb (32, 33). Cediranib was found to inhibit phos- dent tumor models as a consequence of inhibiting VEGF phorylation of all of the imatinib sensitive c-Kit mutant signaling (5). This finding reinforces the fact that very forms found in GIST, as well as inhibited 2 of the significant inhibition of PDGFR signaling may be secondary point mutations which confer acquired resis- required to prevent phenotypic signaling responses. tance to imatinib (V654A and N822K; refs. 34–37). How- The activity of cediranib against PDGFR-a and ever, cediranib was not active against the T670I PDGFR-b would therefore not be expected to contribute gatekeeper mutation in c-Kit (38) or the D816V/D816Y significantly to an effect on tumor growth or survival, c-Kit mutations (35–37). unless a tumor has a particularly high dependency on Previous data generated in cellular phosphorylation signaling from these receptors. assays showed that cediranib was 10- to 16-fold less This work highlights the significant challenge to accu- active against PDGFR-a and PDGFR-b than against rately describe the relative activity of an ATP-competitive VEGFR, and this margin increased to 100-fold when a inhibitor potent against more than 1 kinase. This requires comparative assessment of ligand-induced proliferation consideration of activity at the recombinant kinase level, was done. The in vitro data generated with cediranib in within multiple cellular phosphorylation and prolifera- this study against receptor phosphorylation in multiple tion assays, and then in vivo potency against the pertinent

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kinase in target tissues. These data are all required to fully AstraZeneca. J.C. Silva and C.L. Farnsworth are employees of Cell Signaling Technology. interpret observations made in preclinical models. The in vivo pharmacodynamic data that show that across the dose range cediranib is primarily a VEGF signaling inhi- Acknowledgments bitor with activity against c-Kit. That a significant drop- The authors thank the staff of CDMG, Inc., for xenograft studies, Susan off in potency is observed between ligand-induced recep- Lovick for statistical analysis, Jonathon Orme for FGFR-1 and FGFR-4 tor phosphorylation and cellular proliferation for c-Kit, assays and analyses, Jonathan Dry for mutation analysis, and James PDGFR-a, and PDGFR-b, but not for VEGFR-2, in Sherwood and John Smith for c-Kit sequencing and mutation analyses. endothelial cell assays (7), combined with the relative order of potency against these targets within a number of Grant Support in vitro assays, suggests that cediranib is primarily a VEGFR inhibitor. The Phosphoscan analysis of VEGFR-1 was done by Cell Signaling Tech- nology and funded by AstraZeneca. The costs of publication of this article were defrayed in part by the Disclosure of Potential Conflict of Interest payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate S.R.Brave,K.Ratcliffe,Z.Wilson,N.H.James,S.Ashton,A.Wain- this fact. wright,J.Kendrew,P.Dudley,N.Broadbent,G.Sproat,S.Taylor,C. Barnes,L.Hennequin,D.J.Ogilvie,J.M.Jurgensmeier,€ S.R. Wedge, and Received October 27, 2010; revised February 15, 2011; accepted S.T. Barry are current or former employees and shareholders of February 23, 2011; published OnlineFirst March 25, 2011.

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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. Brave, Kirsty Ratcliffe, Zena Wilson, et al.

Mol Cancer Ther 2011;10:861-873. Published OnlineFirst March 25, 2011.

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