[CANCER RESEARCH 64, 7099 –7109, October 1, 2004]
`
`BAY 43-9006 Exhibits Broad Spectrum Oral Antitumor Activity and Targets the
`
`RAF/MEK/ERK Pathway and Receptor Tyrosine Kinases Involved in Tumor
`
`Progression and Angiogenesis
`
`Scott M. Wilhelm,1 Christopher Carter,1 LiYa Tang,1 Dean Wilkie,1 Angela McNabola,1 Hong Rong,1 Charles Chen,1
`Xiaomei Zhang,1 Patrick Vincent,1 Mark McHugh,1 Yichen Cao,1 Jaleel Shujath,1 Susan Gawlak,1 Deepa Eveleigh,1
`Bruce Rowley,1 Li Liu,1 Lila Adnane,1 Mark Lynch,1 Daniel Auclair,1 Ian Taylor,1 Rich Gedrich,1
`Andrei Voznesensky,1 Bernd Riedl,1 Leonard E. Post,2 Gideon Bollag,2 and Pamela A. Trail1
`
`1Bayer Pharmaceuticals Corporation, West Haven, Connecticut; and 2Onyx Pharmaceuticals, Richmond, California
`
`ABSTRACT
`
`The RAS/RAF signaling pathway is an important mediator of tumor
`cell proliferation and angiogenesis. The novel bi-aryl urea BAY 43-9006 is
`a potent inhibitor of Raf-1, a member of the RAF/MEK/ERK signaling
`pathway. Additional characterization showed that BAY 43-9006 sup-
`presses both wild-type and V599E mutant BRAF activity in vitro.
`In addition, BAY 43-9006 demonstrated significant activity against several
`receptor tyrosine kinases involved in neovascularization and tumor
`progression,
`including vascular endothelial growth factor receptor
`(VEGFR)-2, VEGFR-3, platelet-derived growth factor receptor ␤, Flt-3,
`and c-KIT. In cellular mechanistic assays, BAY 43-9006 demonstrated
`inhibition of the mitogen-activated protein kinase pathway in colon, pan-
`creatic, and breast tumor cell lines expressing mutant KRAS or wild-type
`or mutant BRAF, whereas non–small-cell lung cancer cell lines expressing
`mutant KRAS were insensitive to inhibition of the mitogen-activated
`protein kinase pathway by BAY 43-9006. Potent inhibition of VEGFR-2,
`platelet-derived growth factor receptor ␤, and VEGFR-3 cellular receptor
`autophosphorylation was also observed for BAY 43-9006. Once daily oral
`dosing of BAY 43-9006 demonstrated broad-spectrum antitumor activity
`in colon, breast, and non–small-cell lung cancer xenograft models. Immu-
`nohistochemistry demonstrated a close association between inhibition of
`tumor growth and inhibition of the extracellular signal-regulated kinases
`(ERKs) 1/2 phosphorylation in two of three xenograft models examined,
`consistent with inhibition of the RAF/MEK/ERK pathway in some but not
`all models. Additional analyses of microvessel density and microvessel
`area in the same tumor sections using antimurine CD31 antibodies dem-
`onstrated significant inhibition of neovascularization in all three of the
`xenograft models. These data demonstrate that BAY 43-9006 is a novel
`dual action RAF kinase and VEGFR inhibitor that targets tumor cell
`proliferation and tumor angiogenesis.
`
`INTRODUCTION
`
`Many of the processes involved in tumor growth, progression, and
`metastasis are mediated by signaling pathways initiated by activated
`receptor tyrosine kinases (RTKs; ref. 1). RAS functions downstream
`of several RTKs, and activation of RAS signaling pathways is an
`important mechanism by which human cancer develops (2). Consti-
`tutive activation of the RAS pathways occurs through mutational
`activation of the RAS oncogene or of downstream effectors of RAS
`(3). RAS activation can also be exploited by overexpression of a
`variety of RTKs, including those for the epidermal (EGFR), platelet-
`derived (PDGFR), or vascular-endothelial (VEGFR) growth factors
`(4 –9). In this way, the majority of human tumors, not just those with
`
`Received 4/26/04; revised 7/14/04; accepted 7/29/04.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`Requests for reprints: Scott M. Wilhelm, Dept. Cancer Research, Bayer Pharmaceu-
`ticals Corporation, 400 Morgan Lane, West Haven, CT 06516. Phone: (203) 812-2961;
`Fax: (203) 812-6923; E-mail: scott.wilhelm.b@bayer.com.
`©2004 American Association for Cancer Research.
`
`RAS mutations, depend on activation of the RAS signal transduction
`pathways to achieve cellular proliferation and survival (4).
`RAS regulates several pathways that synergistically induce cellular
`transformation, including the well-characterized RAF/MEK/ERK cas-
`cade. RAF kinases are serine/threonine protein kinases that function in
`this pathway as downstream effector molecules of RAS. RAS local-
`izes RAF to the plasma membrane, where RAF initiates a mitogenic
`kinase cascade that ultimately modulates gene expression via the
`phosphorylation of transcription factors (3), which can have profound
`effects on cellular proliferation and tumorigenesis.
`The RAF kinase family is composed of three members: ARAF,
`BRAF, and Raf-1 (also termed c-Raf). BRAF is reportedly mutated in
`70% of malignant melanomas (10), in 33% of papillary thyroid
`carcinomas (11), and in lower frequencies in other cancers (12). The
`V599E mutant form of BRAF activates the RAF/MEK/ERK pathway
`in human melanoma cells in vitro, and small interfering RNA silenc-
`ing of V599E BRAF, but not Raf-1, inhibits soft agar growth of these
`cells (13). In addition, transformation of a melanocyte cell line with
`V599E BRAF activates the mitogen-activated protein kinase (MAPK)
`pathway. BAY 43-9006, a RAF kinase and VEGFR-2 inhibitor, and
`U0126, a MAP kinase kinase (MEK) inhibitor, block MAPK activa-
`tion and inhibit cell proliferation in mutant BRAF- and KRAS-
`transformed melanocytes (14).
`Recent evidence suggests that Raf-1 and BRAF participate in the
`regulation of endothelial apoptosis and, therefore, angiogenesis, a
`process essential for tumor development and metastasis (15, 16).
`Selective delivery of mutant Raf-1 to tumor blood vessels induces
`endothelial cell apoptosis, which inhibits angiogenesis and results in
`regression of established tumors (16). Mice deficient in BRAF or
`Raf-1 die during embryogenesis because of severe vascular defects
`and increased apoptosis that could be due, in part, to effects on
`endothelial cell survival (17, 18).
`Angiogenesis is a tightly regulated multistep process that involves
`the interaction of multiple growth factors expressed as multiple iso-
`forms, including VEGFs, basic fibroblast growth factor, and PDGFs.
`VEGF also regulates vascular permeability. Vessel stabilization
`through pericyte recruitment and maturation is primarily driven by
`PDGF (19). Several antiangiogenic agents are currently being inves-
`tigated in clinical trials (20 –24); however, because of the complex
`interactions between tumor cells, the invading stroma, and new blood
`vessels, a therapeutic agent targeting a single molecular entity might
`have limited efficacy across a spectrum of tumor types (25, 26).
`BAY 43-9006 is a novel bi-aryl urea that has been previously
`shown to inhibit Raf-1 and tumor cell line proliferation and tumor
`growth in several human tumor xenograft models (27, 28). Here, we
`demonstrate that BAY 43-9006 inhibits another member of the RAF
`family, wild-type (wt) BRAF and V599E BRAF. In addition, BAY
`43-9006 demonstrates potent
`inhibition of certain proangiogenic
`RTKs, including VEGFR-2, PDGFR-␤, and VEGFR-3. BAY 43-9006
`also substantially inhibits tumor growth of several human tumor
`
`7099
`Amerigen Exhibit 1106
`Amerigen v. Janssen IPR2016-00286
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`

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`BAY 43-9006, A DUAL ACTING RAF KINASE AND VEGFR INHIBITOR
`
`xenograft models, even in the absence of MAPK pathway inhibition.
`Taken together, these data suggest that BAY 43-9006 functions as a
`novel dual action RAF kinase and VEGFR inhibitor targeting both
`the RAF/MEK/ERK pathway and RTKs that promote tumor angio-
`genesis.
`
`MATERIALS AND METHODS
`
`Preparation of BAY 43-9006
`
`The chemical name of BAY 43-9006 is N-(3-trifluoromethyl-4-chlorophenyl)-
`N‘-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea, and the structural for-
`mula is shown in Table 1. For in vitro experiments, BAY 43-9006 was dissolved
`in DMSO. For in vivo experiments, BAY 43-9006 was dissolved in Cremophor
`EL/ethanol (50:50; Sigma Cremophor EL, 95% ethyl alcohol) at 4-fold (4⫻) of the
`highest dose, foil wrapped, and stored at room temperature. This 4⫻ stock solution
`was prepared fresh every 3 days. Final dosing solutions were prepared on the day
`of use by dilution of the stock solution to 1⫻ with water. Lower doses were
`prepared by dilution of the 1⫻ solution with Cremophor EL/ethanol/water (12.5:
`12.5:75).
`
`Biochemical Assays
`
`In vitro Assays with Recombinant Raf-1 (Residues 305– 648), BRAF
`(Residues 409 –765), V599E BRAF (Residues 409 –765), MEK-1, and Ex-
`tracellular Signal-Regulated Kinase (ERK)-1. COOH-terminal kinase do-
`mains of Raf-1 (residues 305– 648) and BRAF (residues 409 –765) were
`generated by PCR. The BRAF (residues 409 –765) V599E mutation was
`introduced using the QuikChange Site-directed Mutagenesis kit (Stratagene,
`
`Table 1 BAY 43-9006 inhibits the RAF/MEK/ERK pathway and receptor tyrosine
`kinases involved in tumor angiogenesis
`
`Biochemical assay†
`Raf-1‡
`BRAF wild-type§
`V599E BRAF mutant¶
`VEGFR-2
`mVEGFR-2 (flk-1)
`mVEGR-3
`mPDGFR-␤
`Flt-3
`c-KIT
`FGFR-1
`ERK-1, MEK-1, EGFR, HER-2, IGFR-1, c-met, PKB,
`PKA, cdk1/cyclinB, PKC␣, PKC␥, pim-1
`Cellular mechanism储
`MDA MB 231 MEK phosphorylation (human breast)
`MDA MB 231 ERK 1/2 phosphorylation (human breast)
`BxPC-3 ERK 1/2 phosphorylation (human pancreatic)
`LOX ERK 1/2 phosphorylation (human melanoma)
`VEGFR-2 phosphorylation (human, NIH 3T3 cells)
`VEGF–ERK 1/2 phosphorylation (human, HUVEC)
`PDGFR-␤ phosphorylation (human HAoSMC)
`mVEGFR-3 phosphorylation (mouse, HEK-293 cells)
`Flt-3 phosphorylation (human ITD, HEK-293 cells)
`Cellular proliferation
`MDA MB 231 (10% FCS)
`PDGF-BB HAoSMC (0.1% BSA)
`
`IC50
`(nmol/L) ⫾ SD (n)*
`
`6 ⫾ 3 (7)
`22 ⫾ 6 (7)
`38 ⫾ 9 (4)
`90 ⫾ 15 (4)
`15 ⫾ 6 (4)
`20 ⫾ 6 (3)
`57 ⫾ 20 (5)
`58 ⫾ 20 (3)
`68 ⫾ 21 (3)
`580 ⫾ 100 (3)
`⬎10,000
`
`40 ⫾ 20 (2)
`90 ⫾ 26 (7)
`1,200** ⫾ 165 (2)
`880** ⫾ 90 (2)
`30 ⫾ 21 (3)
`60** ⫾ 26 (2)
`80 ⫾ 40 (3)
`100 ⫾ 80 (2)
`20 ⫾ 10 (2)
`
`2,600 ⫾ 810 (3)
`280 ⫾ 140 (5)
`
`* IC50 mean ⫾ SD; (n ⫽ number of trials).
`† Kinase assay were carried out as described in Materials and Methods at ATP
`concentrations at or below Km (1 to 10 ␮mol/L).
`‡ Lck activated NH2-terminal–truncated Raf-1.
`§ NH2-terminal–truncated BRAF (wild-type).
`¶ NH2-terminal V599E-truncated BRAF (mutant).
`储 Cellular mechanism assays (RTK autophosphorylation and RAF/MEK/ERK path-
`way) were performed in 0.1% BSA using phospho-specific antibodies or 4G10 for
`VEGFR-3 as described in Materials and Methods.
`** Activated phospho-ERK 1/2 was quantitated with phospho-ERK 1/2 immunoassay
`(Bio-Plex; Bio-Rad, Inc.).
`
`La Jolla, CA) according to the manufacturer’s protocol. Recombinant baculo-
`viruses expressing Raf-1 (residues 305– 648), BRAF (residues 409 –765), and
`V599E BRAF (residues 409 –765) were purified as fusion proteins as de-
`scribed previously (29). Full-length human MEK-1 was generated by PCR and
`purified as a fusion protein from Escherichia coli lysates (30).
`To test compound inhibition against various RAF kinase isoforms, BAY
`43-9006 was added to a mixture of Raf-1 (80 ng), wt BRAF, or V599E BRAF
`(80 ng) with MEK-1 (1 ␮g) in assay buffer [20 mmol/L Tris (pH 8.2), 100
`mmol/L NaCl, 5 mmol/L MgCl2, and 0.15% ␤-mercaptoethanol] at a final
`concentration of 1% DMSO. The RAF kinase assay (final volume of 50 ␮L)
`was initiated by adding 25 ␮L of 10 ␮mol/L ␥-[33P]ATP (400 Ci/mol) and
`incubated at 32°C for 25 minutes. Phosphorylated MEK-1 was harvested by
`filtration onto a phosphocellulose mat, and 1% phosphoric acid was used to
`wash away unbound radioactivity. After drying by microwave heating, a
`␤-plate counter was used to quantify filter-bound radioactivity. Activated
`MEK-1 and ERK-1 were purchased from Upstate Biotechnology (UBI, Wal-
`tham, MA) and assayed according to manufacturer’s instructions.
`In vitro Assays
`for Murine (m)VEGFR-2 (flk-1), mVEGFR-3,
`mPDGFR-␤, Flt-3, c-KIT, EGFR, HER2, c-MET, c-yes, FGFR-1, and
`IGFR-1. mVEGFR-2 (flk-1; residues 785-1367), human VEGFR-2 (KDR)
`kinase domain, mPDGFR-␤ (residues 560-1098), mVEGFR-3 (residues 818-
`1363), EGFR (residues 669-1210), HER2/neu (residues 691-1255), and
`FGFR-1 (residues 398 – 882) were expressed and purified from Sf9 lysates as
`described previously (29, 31). Flt-3, c-KIT, insulin growth factor receptor
`(IGFR)-1, VEGFR-2, c-MET, and cdk-1/cyclin B were purchased from Pro-
`qinase (Freiburg, Germany). Activated protein kinase (PK)B, PKA, LCK, and
`c-yes were purchased from Calbiochem, Inc. (San Diego, CA) and assayed
`according to the manufacturer’s instructions. BAY 43-9006 was assayed
`against recombinant pim-1 at Proqinase and PKC␣ and PKC␥ at Pan Labs
`(Bothell, WA).
`Time-resolved fluorescence energy transfer assays for mVEGFR-2 (flk-1),
`mVEGFR-3, mPDGFR-␤, Flt-3, c-KIT, EGFR, HER2, c-MET, c-yes, LCK,
`and IGFR-1 were performed in 96-well opaque plates in the time-resolved
`fluorescence energy transfer format. Final reaction conditions were as follows:
`1 to 10 ␮mol/L ATP, 25 nmol/L poly GT-biotin, 2 nmol/L Europium-labeled
`phospho (p)-Tyr antibody (PY20; Perkin-Elmer, Wellesley, MA), 10 nmol/L
`APC (Perkin-Elmer), 1 to 7 nmol/L cytoplasmic kinase domain in final
`concentrations of 1% DMSO, 50 mmol/L HEPES (pH 7.5), 10 mmol/L MgCl2,
`0.1 mmol/L EDTA, 0.015% Brij-35, 0.1 mg/mL BSA, and 0.1% ␤-mercapto-
`ethanol. Reactions volumes were 100 ␮L and were initiated by addition of
`enzyme. Plates were read at both 615 and 665 nmol/L on a Perkin-Elmer
`VictorV Multilabel counter at ⬃1.5 to 2.0 hours after reaction initiation. Signal
`was calculated as a ratio: (665 nm/615 nmol/L) ⫻ 10,000 for each well. Signal
`to noise was generally 4 to 8-fold in each assay.
`For IC50 generation, compounds were added before the enzyme initiation. A
`50-fold stock plate was made with compounds serially diluted 1:3 in a 50%
`DMSO/50% distilled water solution. Final compound concentrations ranged
`from 10 ␮mol/L to 4.56 nmol/L in 1% DMSO. The data were expressed as
`percent inhibition ⫽ 100 ⫺ [(signal with inhibitor ⫺ background)/(signal
`without inhibitor ⫺ background)] ⫻ 100.
`
`Cellular Mechanistic Assays
`
`Tumor Cell Lines and Reagents. The MDA-MB-231 human mammary
`adenocarcinoma cell line was obtained from the National Cancer Institute.
`These cells were maintained in DMEM (Invitrogen, Inc., Carlsbad, CA),
`supplemented with 1% L-glutamine (Invitrogen, Inc.), 1% HEPES buffer
`(Invitrogen, Inc.), and 10% heat-inactivated fetal bovine serum. The Colo-205,
`HT-29, and DLD-1 human colon carcinomas and the NCI-H460 and A549
`human non–small-cell lung cancer (NSCLC) carcinoma lines were obtained
`from and propogated as recommended by the American Type Tissue Culture
`Collection Repository (Manassas, VA).
`Cellular MEK 1/2, ERK 1/2, and PKB Activation and Bio-Plex pERK
`Immunoassay. Tumor cell lines were plated at 2 ⫻ 105 cells per well in
`12-well tissue culture plates in DMEM growth media (10% heat-inactivated
`FCS) overnight. Cells were washed once with serum-free media and incubated
`in DMEM supplemented with 0.1% fatty acid-free BSA (Sigma, St. Louis,
`MO) containing various concentrations of BAY 43-9006 in 0.1% DMSO for
`120 minutes to measure changes in basal pMEK 1/2, pERK 1/2, or pPKB.
`
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`BAY 43-9006, A DUAL ACTING RAF KINASE AND VEGFR INHIBITOR
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`Cells were washed with cold PBS (PBS containing 0.1 mmol/L vanadate) and
`lysed in a 1% (v/v) Triton X-100 solution containing protease inhibitors.
`Lysates were clarified by centrifugation, subjected to SDS-PAGE, transferred
`to nitrocellulose membranes, blocked in TBS-BSA, and probed with anti-
`pMEK 1/2 (Ser217/Ser221; 1:1000), anti-MEK 1/2, anti-pERK 1/2 (Thr202/
`Tyr204; 1:1000), anti-ERK 1/2, anti-pPKB (Ser473; 1:1000), or anti-PKB pri-
`mary antibodies (Cell Signaling Technology, Beverly, MA). Blots were
`developed with horseradish peroxidase (HRP)-conjugated secondary antibod-
`ies and developed with Amersham ECL reagent on Amersham Hyperfilm.
`A 96-well pERK immunoassay, using the laser flow cytometry (Bio-Rad,
`Hercules, CA) platform, was developed to measure BAY 43-9006 –mediated
`inhibition of basal pERK 1/2 in tumor cell lines. MDA-MB-231, LOX, and
`BxPC-3 cells were plated at 50,000 cells per well. One day after plating, tumor
`cells in DMEM with 0.1% fatty acid-free BSA were incubated for 2 hours with
`BAY compounds diluted to a final concentration of 3 ␮mol/L to 12 nmol/L in
`0.1% DMSO. Cells were incubated washed, lysed, and directly transferred to
`assay plate or frozen at ⫺80°C until processed. Tumor cell lysates were
`incubated with ⬃2000 of 5-␮m Bio-Plex beads conjugated with an anti-ERK
`1/2 antibody. The next day, biotinylated pERK 1/2 sandwich immunoassay
`was performed, beads were washed three times during each incubation, and
`phycoerythrin-streptavidin was used as a develop reagent. The relative fluo-
`rescence units of pERK 1/2 were detected by counting 25 beads with Bio-Plex
`flow cell (probe) at high sensitivity. The IC50 was calculated by taking
`untreated cells as maximum and no cells (beads only) as background using an
`Excel spreadsheet-based program.
`VEGFR-2 Autophosphorylation and MAPK Phosphorylation in Hu-
`man Umbilical Vascular Endothelial Cells (HUVECs) and NIH 3T3
`VEGFR-2–Transfected Cells. Subconfluent HUVECs (ATCC or Cambrex)
`were cultured in growth factor deprived culture medium for 24 hours. Cells
`were serum starved by replacing media with basal media (EBM-2) containing
`0.2% BSA for 1 hour. BAY 43-9006 was added to the cells with serum-free
`media for 1 hour followed by VEGF165 treatment (final concentration of 30
`ng/mL) for 10 minutes.
`NIH 3T3 cells transfected with VEGFR-2 were obtained from Dr. Mas-
`abumi Shibuya (Institute of Medical Science, University of Tokyo, Tokyo,
`Japan) and plated at 1 ⫻ 106 cells/well in 6-well tissue culture plates in
`DMEM, 10% fetal bovine serum, and 1.5 mg/mL G418. After 6 hours, culture
`medium was changed to 2 mL per well of 0.2% BSA/DMEM and incubated for
`14 hours. Cells were preincubated with compound added in 0.1% BSA/PBS for
`30 minutes followed by stimulation with 30 ng/mL VEGF165 for 10 minutes.
`Cells were washed and lysed with buffer containing 0.3% Triton X-100 and
`protease inhibitors (Complete Protease Inhibitor Tablets). Twelve ␮g of pro-
`tein from control and treated cell lysates were electrophoresed under reducing
`conditions and transferred onto nitrocellulose membranes. Blots were probed
`with anti-pVEGFR-2 (pTyr-1054 and pY1059) antibodies (PC460; Biosource,
`Inc., Camarillo, CA) or anti-VEGFR-2 antibody (sc-315; Santa Cruz Biotech-
`nology, Santa Cruz, CA). Blots were developed with HRP-conjugated second-
`ary antibodies and developed with Amersham ECL reagent on Amersham
`Hyperfilm.
`To monitor the effects of BAY 43-9006 on VEGF and basic fibroblast
`growth factor-dependent MAPK activation, exponentially growing human
`endothelial cells (HUVECs; Cambrex, East Rutherford, NJ) were seeded at
`25,000 cells per well in 96-well plates in growth medium with (EBM-2 MV;
`Cambrex) and incubated at 37°C in 5% CO2. Sixteen hours after plating, the
`cells were changed to serum-free RPMI 1640 containing 0.1% fatty acid-free
`BSA, and cells were preincubated at different concentrations of BAY43-9006.
`Cells were stimulated for 10 minutes with 50 ng/mL of either VEGF or basic
`fibroblast growth factor and processed for as described above for Bio-Plex
`pERK 1/2 immunoassay from tumor cell lysates.
`PDGFR-␤ Autophosphorylation and Cell Proliferation in Human Aor-
`tic Smooth Muscle Cells (HAoSMCs). A total of 1 ⫻ 105 HAoSMCs (P3-P6;
`Clonetics) were plated in 12-well cluster plates in 1 mL volume per well of
`SGM-2 (Clonetics). Cells were rinsed the next day with D-PBS (Life Tech-
`nologies, Inc.), then serum starved in 500 ␮L of smooth muscle cell basal
`media (Clonetics) with 0.1% BSA (Sigma) overnight. Diluted compounds
`ranged from 10 ␮mol/L to 1 nmol/L in 0.1% DMSO. Media was removed, and
`100 ␮L of each dilution were added to cells for 1 hour at 37°C. Cells were then
`stimulated with 10 ng/mL PDGF BB ligand (Leinco) for 7 minutes at 37°C.
`The media was decanted and 150 ␮L of isotonic 25 mmol/L bicine pH 7.6 lysis
`
`buffer (M-PER from Pierce) with protease inhibitor tablet (Complete; EDTA-
`free), and 0.2 mmol/L Na vanadate were added. Cells were lysed, and 15 ␮L
`of agarose-conjugated anti-PDGFR-␤ antibody (sc-339; Santa Cruz Biotech-
`nology) were added. Next day, beads were rinsed, boiled in 1⫻ LDS sample
`buffer, run on 3 to 8% gradient Tris-Acetate gels (Invitrogen), and transferred
`onto nitrocellulose. Membranes were probed with anti-pPDGFR-␤ (Tyr857)
`antibody (sc-12907; Santa Cruz Biotechnology) and then the secondary goat
`antirabbit HRP IgG (Amersham). Positive bands were visualized using en-
`hanced chemiluminescence. Subsequently, membranes were stripped and rep-
`robed with SC-339 (Santa Cruz Biotechnology) for total PDGFR-␤.
`The PDGF-dependent bromodeoxyuridine incorporation assay measures the
`ability of compounds to inhibit the induction of DNA synthesis by PDGF in
`serum-starved HAoSMCs. For the assay, HAoSMCs (4 ⫻ 103/well) were
`plated in complete SMBM media in 96-well tissue culture plates, incubated
`overnight, and then serum starved for 16 hours in SMBM containing 0.1%
`BSA (SF-SMBM). Fresh SF-SMBM was then added to the cells. Dilutions of
`BAY 43-9006 in SF-SMBM were added in a dose range from 10 ␮mol/L to
`4.57 nmol/L 1 hour before the addition of 10 ng/mL PDGF-BB. Cells were
`incubated for 24 hours and processed using the BrdUrd ELISA kit from
`Amersham.
`mVEGFR-3 and Human Flt-3 (ITD) Receptor Autophosphorylation
`Assays. The human embryonic kidney (HEK-293)-Flt-3 (ITD) cells (CRL-
`1573; American Type Cell Culture) were plated at 2.5 to 5 ⫻ 105 cells/well in
`6-well plates (RPMI ⫹10% FBS). The following day, cells were treated with
`inhibitors (3 ␮mol/L to 10 nmol/L) for 2 hours in serum-free RPMI media.
`Cells were lysed, and 10 ␮g of proteins per lane were loaded on 3 to 8%
`Tris-Acetate NuPAGE gels (Invitrogen). Gels were transferred to nitrocellu-
`lose membranes, blocked, probed with anti-pFlt-3 monoclonal antibody (3466;
`Cell Signaling), washed, and probed with secondary HRP-conjugated anti-
`mouse IgG (Amersham). Membranes were washed, developed with ECL
`Western blot detection reagent (Amersham), and exposed on Hyperfilm ECL
`film (Amersham). For total Flt-3 quantification, membranes were stripped with
`RESTORE buffer
`(Pierce) and blotted as described above using anti-
`Flt-3 polyclonal antibodies (sc-479; Santa Cruz Biotechnology) and HRP-
`conjugated antirabbit IgG (Amersham).
`For mVEGFR-3 studies, HEK-293 cells were transiently transfected with
`pcDNA3.1 vector (Invitrogen) containing a full-length cDNA of murine
`VEGFR3. Two days after transfection, cells were exposed to test compounds
`(3 ␮mol/L to 10 nmol/L in 0.1% DMSO) for 30 minutes at 37°C. Cells were
`washed, lysed in Triton-lysis buffer, pelleted, and 10 ␮g of total protein were
`loaded on 3 to 8% Tris-Acetate NuPAGE gels (Invitrogen). Gels were trans-
`ferred to nitrocellulose membranes, which were probed with anti-mVEGFR-3
`(ADI) or anti-p-mVEGFR-3 (4G10; Upstate Biotechnology) antibodies and
`secondary HRP-conjugated antimouse or antirabbit IgG, respectively (Amer-
`sham).
`
`Tumor Cell Proliferation
`
`Tumor cells were trypsinized and plated in 96-well plates at 3000 cells per
`well in complete media with 10% FCS. Cells were incubated overnight at
`37°C, and the next day, compounds were added in complete growth media over
`a final concentration range of 10 ␮mol/L to 10 nmol/L in 0.1% DMSO. Cells
`were incubated with test compounds for 72 hours at 37°C in complete growth
`media, and cell number was quantitated using the Cell TiterGlo ATP Lumi-
`nescent assay kit (Promega). This assay measures the number of viable cells
`per well by measurement of luminescent signal based on amount of cellular
`ATP.
`
`Tumor Xenograft Experiments
`
`Female NCr-nu/nu mice (Taconic Farms, Germantown, NY) were used for
`all studies. The mice were housed and maintained in accordance with Bayer
`Institutional Animal Care and Use Committee and state and federal guidelines
`for the humane treatment and care of laboratory animals and received food and
`water ad libitum.
`Tumors for all but the DLD-1 model were generated by harvesting cells
`from mid-log phase cultures using Trypsin-EDTA (Invitrogen, Inc.). Three to
`five million cells were injected s.c. into the right flank of each mouse. DLD-1
`tumors were established and maintained as a serial in vivo passage of s.c.
`fragments (3 ⫻ 3 mm) implanted in the flank using a 12-gauge trocar. A new
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`BAY 43-9006, A DUAL ACTING RAF KINASE AND VEGFR INHIBITOR
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`generation of the passage was initiated every three weeks, and studies were
`conducted between generations 3 and 12 of this line.
`Treatment was initiated when tumors in all mice in each experiment ranged
`in size from 75 to 144 mg for antitumor efficacy studies and from 100 to 250
`mg for studies of microvessel density and ERK phosphorylation. All treatment
`was administered orally once daily for the duration indicated in each experi-
`ment. Tumor weight was calculated using the equation length ⫻ (width)2)/2.
`Treatments producing ⬎20% lethality and/or 20% net body weight loss were
`considered toxic.
`
`Detection of Tumor Microvessels and Activated ERK 1/2
`
`Immunostaining of paraffin sections of tumors with murine anti-CD31
`antibodies was performed on the Dako Autostainer, model LV (DakoCytoma-
`tion). Sections were deparaffinized and hydrated, and endogenous peroxidase
`activity was blocked with 3% H2O2. Antigen retrieval was performed using
`Dako Target Retrieval Solution (S1699, DakoCytomation). Sections were
`blocked with an avidin/biotin block (Vector Laboratories) and rabbit serum.
`Immunostaining was performed using a goat Vectastain ABC elite kit (Vector
`Laboratories) and 3,3⬘-diaminobenzidine as the chromagen (DakoCytoma-
`tion), according to the manufacturer’s protocol with the following modifica-
`tions: (a) 2 ⫻ 30-minute incubations were performed with primary antibody;
`and (b) after incubation with the secondary antibody, the slides were rinsed in
`buffer, distilled water, and then buffer again. Sections were incubated with
`anti-CD31 antibodies [PECAM-1 (M-20) SC-1506 goat polyclonal; Santa
`Cruz Biotechnology] diluted 1:750 in Dako Antibody Diluent (DakoCytoma-
`tion, Carpinteria, CA) or goat IgG (1:750; Jackson ImmunoResearch Labora-
`
`tories, Inc., West Grove, PA) as a negative control. Sections were counter-
`stained with Mayer’s hematoxylin for 1 minute and washed with water.
`Immunohisotchemical localization of activated ERK 1/2 was determined
`from paraffin sections. Sections were then placed in heated (95°C) 0.1M
`citrate buffer (pH 6.0) for 35 min, brought to RT for 30 min, and then
`blocked with 1.5% H2O2. Sections were stained using primary pERK 1/2
`antibody (phospho-p44/42 Cell Signaling) diluted 1:100 with Dako Anti-
`body Diluent. Staining was performed using the Envision Plus HRP (3,3⬘-
`diaminobenzidine) System from Dako (4011) according to the manufac-
`turer’s protocol. The slides were counterstained with Mayer’s hematoxylin
`for 1 min and washed with water.
`
`Quantification of Microvessels
`
`Histologic slides were blind-coded during the assessment. The tissue
`sections were viewed at ⫻100 magnification (⫻10 objective lens and ⫻10
`ocular lens; 0.644 mm2 per field). Tissue image was captured with a digital
`camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Four fields per
`section were randomly analyzed, excluding peripheral surrounding connec-
`tive tissues and central necrotic tissues. Total tissue area analyzed in each
`section was 2.576 mm2.
`Area and number of CD31 positive objects were quantified using the software
`ImagePro Plus version 3.0 (Media Cybernetics, Silver Spring, MD). Percentage of
`microvessel area (MVA, %) in each field was calculated as [(area of CD31-
`positive objects/measured tissue area) ⫻ 100%]. Microvessel density (MVD,
`number/mm2) in each field was calculated as (number of CD31-positive objects/
`0.644 mm2). Mean values of MVA and MVD in each group were calculated from
`
`Fig. 1. BAY 43-9006 inhibits activation of the
`RAF/MEK/ERK pathway in most but not all human
`tumor cell lines. A. MDA-MB-231 cells were incu-
`bated with various concentrations of BAY 43-9006 or
`DMSO. Cell lysates were subjected to Western blot
`analysis for phosphorylated (p) and total (T) MEK 1/2
`(top box), ERK 1/2 (middle box), and PKB (bottom
`box). Changes in total MEK 1/2, ERK 1/2 levels, or
`PKB were not observed. The MEK inhibitor U0126
`(10 ␮mol/L) was used in all Western blot experiments
`as a control for detecting RAF/MEK/ERK pathway
`inhibition (Lane 1: MEK-1). B. Subconfluent human
`tumor cells were incubated with BAY 43-9006 for 2
`hours, lysed, and processed for Western blotting. Ac-
`tivated ERK 1/2 was detected with anti-pERK anti-
`bodies. Changes in total ERK 1/2 levels were not
`observed. U0126 was used at 10 ␮mol/L (Lane 1:
`MEK-1).
`
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`BAY 43-9006, A DUAL ACTING RAF KINASE AND VEGFR INHIBITOR
`
`four tumor samples. Data were analyzed statistically with one-way ANOVA
`followed by Fisher’s PLSD (StatView, version 4.5; Abacus Concepts, Inc., Berke-
`ley, CA). P ⬍ 0.05 was considered significant.
`
`RESULTS
`
`BAY 43-9006 Inhibition of the MAPK Pathway. BAY 43-9006
`is a synthetic molecule that can be broadly defined as a bi-aryl urea
`(Table 1), which was originally identified through inhibition of Raf-1
`kinase biochemical and cellular mechanistic assays (27, 32). BAY
`43-9006 was additionally profiled against wt BRAF and V599E
`mutant BRAF (Table 1). Biochemical assays were performed in which
`varying concentrations of BAY 43-9006 were tested for the capacity
`to inhibit MEK-1 phosphorylation by the catalytic domains of Raf-1,
`BRAF, and V599E BRAF. As shown in Table 1, BAY 43-9006
`potently inhibited Raf-1 (IC50, 6 nmol/L), wt BRAF (IC50, 22 nmol/
`L), and V599E mutant BRAF (IC50, 38 nmol/L) but did not signifi-
`cantly inhibit MEK-1 or ERK-1 activity (IC50, ⬎10,000 nmol/L).
`
`The ability of BAY 43-9006 to block activation of the MAPK pathway
`was examined by measuring ERK 1/2 phosphorylation in several tumor
`cell lines by Western blot analysis or Bio-Plex pERK immunoassay.
`Genotyping of each cell line revealed mutations in KRAS (Mia PaCa 2,
`HCT 116, A549, and NCI-H460), V599E BRAF (LOX melanoma,
`HT-29), or both KRAS and G463V BRAF (MDA-MB-231), suggesting
`that transformation of these cells is driven, in part, by disruption of the
`MAPK pathway. Results show that BAY 43-9006 inhibits ERK phos-
`phorylation in most of these cell lines, independent of which mutation
`caused aberrant activation of the RAS/RAF pathway (Fig. 1).
`In MDA-MB-231 breast cancer cells, BAY 43-9006 completely
`blocked activation of the MAPK pathway (Fig. 1A). Cells were
`preincubated with BAY 43-9006 at concentrations ranging from 0.01
`to 3 ␮mol/L, and dose-dependent inhibition of basal MEK 1/2 and
`ERK 1/2 phosphorylation (IC50, 40 and 100 nmol/L, respectively) was
`observed (Fig. 1A). BAY 43-9006 had no effect on the PKB pathway
`in MDA-MB-231 cells, demonstrating selectivity for inhibition of the
`MAPK, but not the PKB, pathway in these cells (Fig. 1A).
`
`tyrosine kinases
`receptor
`Fig. 2. BAY 43-9006 targets
`(VEGFR-2 and PDGFR-␤) involved in tumor angiogenesis. A,
`VEGF-stimulated VEGFR-2 autophosphorylation in HUVECs. B,
`VEGF-stimulated VEGFR-2 autophosphorylation in NIH 3T3
`VEGFR-2 cells. C, PDGF-BB–stimulated PDGFR-␤ phosphoryla-
`tion in HAoSMCs. D, PDGF-BB–stimulated bromodeoxyuridine
`(BrdUrd) proliferation assay in HAoSMCs.
`
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`BAY 43-9006, A DUAL ACTING RAF KINASE AND VEGFR INHIBITOR
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`BAY 43-9006 inhibition of pERK was also observed for the human
`pancreatic (Mia PaCa 2) and colon (HCT 116 and HT-29) tumor cell
`lines by Western blot analysis (Fig. 1B) and in the human LOX
`melanoma and pancreatic BxPC-3 cell lines using the Bio-Plex pERK
`immunoassay (Table 1). However, BAY 43-9006, at concentrations as
`high as 10 ␮mol/L, had no effect on inhibition of ERK 1/2 phospho-
`rylation in the two NSCLC cell lines (Fig. 1B).
`BAY 43-9006 Targets Receptor Tyrosine Kinases Involved in
`Tumor Progression and Angiogenesis. Additional characterization
`of BAY 43-9006 in biochemical assays demonstrated potent inhibi-
`tion of several RTKs, including human and murine VEGFR-2 (IC50,
`90 and 15 nmol/L, respectively), mVEGFR-3 (IC50, 20 nmol/L),
`mPDGFR-␤ (IC50, 57 nmol/L), Flt-3 (IC50, 58 nmol/L), c-KIT (IC50,
`68 nmol/L), and FGFR-1 (IC50, 580 nmol/L; Table 1). By contrast,
`EGFR, IGFR-1, c-met, and HER-2 RTKs were not inhibited by BAY
`43-9006 (IC50, ⬎10,000 nmol/L). Other kinases tested included PKB,
`PKA, cdk1/cyclinB, PKC␣, PKC␥, and pim-1, which were all insen-
`sitive to inhibition by BAY 43-9006 (Table 1).
`Inhibition of VEGFR-2 autophosphorylation by BAY 43-9006 was
`examined in two cell culture systems, HUVECs, and NIH 3