[CANCER RESEARCH 57, 4838-4848. November 1. 1997]
`
`Induction of Apoptosis and Cell Cycle Arrest by CP-358,774, an Inhibitor of
`
`Epidermal Growth Factor Receptor Tyrosine Kinasc
`
`James D. Moyer,’ Elsa G. Barbacci, Kenneth K. lwata, Lee Arnold, Bruce Boman, Ann Cunningham,
`Catherine DiOrio, Jonathan Doty, Michael J. Morin, Mikel P. Moyer, Mark Neveu, Vincent A. Pollack,
`Leslie R. Pustilnik, Margaret M. Reynolds, Don Sloan, April Thelenran, and Penny Miller
`Pfizer Central Research, Grown. Connecticut 06340 [J. D. M.. E. G. B.. L A.. A. C.. C. D., J. D.. M. J. M.. M. P. M., M. N., V. A. P.. L R. P.. D. 5.. P. M.I; Oncogene Science
`Inc, Uniorxdale, New York I 1553 (K. K. 1.. M. M. R. A. T. 1; and Storz Cancer Institute, Omaha. Nebraska 68105 (B. 8.]
`
`ABSTRACT
`
`The epidermal growth factor receptor (EGFR) is overexpressed in a
`significant percentage of carcinomas and contributes to the malignant
`phenotype. Cl’-358,774 is it directly acting inhibitor of human EGFR
`tyrosine kinase with an IC,o of 2 nM and reduces EGFR autophosphoryb
`atlon in intact tumor cells with an [C5, of 20 nM. This inhibition is selective
`for EGFR tyrosine kinase relative to other tyrosine kinases we have
`examined, both in ways of isolated kinases and whole cells. At doses of
`100 mg/kg, CP~358.7’74 completely prevents EGF-induced autophosphr»
`rylation of EGFR in human HN5 tumors growing as xenografts in athyrnic
`mice and of the hepatic EGFR of the treated mice. Cl‘-358,774 inhibits the
`proliferation of DiFl human colon tumor cells at subrnicromoiar concen-
`trations iu cell culture and blocks cell cycle progression at the G, phase.
`This inhibitor produces a marked accumulation of retinoblastoma protein
`in its underphosphorylated form and accumulation of p27"“" in DiFi
`cells, which may contribute to the cell cycle block. Inhibition of the EGFR
`also triggers apoptosis in these cells as determined by formation of DNA
`fragments and other criteria. These results indicate that Cl‘-358,774 has
`potential for the treatment of tumors that are dependent on the EGFR
`pathway for proliferation or survival.
`
`INTRODUCTION
`
`EGFR2 is a transmembrane glycoprotein with an external domain
`that binds activating ligands, such as EGF and tumor growth factor oz,
`and an intracellular tyrosine kinase domain that, upon activation,
`phosphorylates both the receptor itself and a variety of “effector"
`proteins such as SHC and phospholipase C7 (1). Activation of this
`signaling cascade triggers DNA synthesis in cells that express the
`EGFR.
`
`Many human tumors, especially squamous carcinomas of the lung
`or head and neck, express high levels of EGFR or tumor growth factor
`or relative to the corresponding normal tissue (2—4). Although over-
`expression of EGFR is the most prevalent alteration of this pathway in
`human tumors, constitutively active mutant EGFR has also been
`reported in gliomas (5), breast tumors (6) and lung tumors (7). This
`suggests that activation of the BGFR may drive the proliferation of
`these tumors and that inhibitors of EGFR may be of use as antitumor
`agents (2, 8——l0).
`Blockade of the EGFR pathway by several methods inhibits the
`proliferation of a variety of tumor cell lines. For example, down-
`regulation of EGFR by antisense expression reduces the proliferation
`
`and invasive properties of a human colon tumor cell Line (ii) and
`blocks proliferation of human rhabdomyosarcoma cells (12). More~
`over, the transformation of cells by overexpression of EGFR in the
`presence of EGF is reversed by expression of a dominant negative
`mutant EGFR (13). lnhibltion of EGFR as an antitumor approach has
`been further substantiated by studies that show that antibodies that
`block EGF binding to the EGFR inhibit tumor cell proliferation in cell
`cultures and tumor xenografts in athymic mice (2, 8, l4~l6). Impor-
`tantly, anti~EGFR antibodies have been mently shown to produce
`complete regressions of established human tumor xenografts in athy-
`rnic mice (17, 18). Clinical trials of a humanized monoclonal antibody
`against the EGFR for the treatment of patients with tumors overex-
`pressing EGFR are in progress (19). Finally, selective low molecular
`weight inhibitors of the EGFR kinase have been shown to inhibit
`EGF-dependent cell proliferation (20-22) and exhibit antitumor ac-
`tivity in a human tumor xenograft model (23). Thus, a subset of
`tumors are dependent, at least in pan, on the EGFR for proliferation.
`Although the evidence above suggests that inhibition of EGFR may
`block proliferation of some tumor cells, EGFR inhibitors must also be
`well tolerated in patients to provide a useful therapeutic index. Al-
`though an inhibitor of EGFR would not be expected to be cytotoxic in
`the manner of the most current chemotherapy, pharmacological inhi~
`bitlon of the EGFR could interfere with the physiological functions of
`EGF and other EGFR ligands. However, targeted “knockout" of the
`mouse EGFR permitted embryonic development and birth of mice
`that survived as long as l8 days (24. 25). This indicates that EGFR is
`dispensable for the proliferation of all critical cell types and essential
`physiological functions. However, various defects such as thin epi-
`dermis, distorted colonic and hepatic epithelium. and low body weight
`were observed in these animals. Mice with the waved-2 phenotype
`express mutant EGFR with markedly impaired klnase activity in vivo
`(26) and, thus, may represent a model of partial inhibition of EGFR.
`These mice are viable and fertile but have hair, skin, and eye abnor-
`malities. Furthermore, early results from clinical trials with the anti-
`EGFR antibody have not revealed any toxicities that would prevent
`further development (19). These results suggest that a partial inhibi-
`tion of EGFR may be reasonably well tolerated in adults or present a
`much different pattern of toxicity than does standard chemotherapy.
`However, many normal cells express EGFR, for example, skin, liver,
`and gastrointestinal epithelium, and may be affected by such an
`inhibitor.
`
`Received 6:26/97; accepted 9/l9I97.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advent:-amen: in accordance with
`18 U.S.C. Section l734 solely to indicate this fact.
`‘To whom requests for reprints should be addressed. at Pfiur Central Research.
`Eastern Point Road. Groton. CT 06340. Email: james_d_moyer@groton.pfizcr.com.
`2 The abbreviations used are: EGFR, epidermal growth factor receptor, EGF, epider-
`mal growth factor; IGF-I. insulin-like growth factor I; IGP-IR. IGF-l receptor: FBS. fetal
`bovine serum: FRE cells, Contact-inhibited Fischer rat embryo cells; POT. poly(glutamic
`scidztyrosine) 4:1: HRP. horseradish peroxidase; BrdUrd. bromodeoxyuridiue; PDGF.
`platcleuderived growth factor; IJFGF. basic fibroblast growth factor; pRB, rerinoblastomn
`gene protein; PARP. poly(ADP-ribosc) polymerase; PI, propidium iodide: TUNEL. ter~
`minal deoxynuclootidyl uansferasc dU'I'i’ nick end labeling;
`lRS—l.
`insulin receptor
`substrate-1; PS. phosphaddylscrine.
`
`Although treatment with anti-EGFR antibodies has produced re-
`gressions of established human tumors in some xenograft tumor
`models (17, 18, 27). inhibition of growth factor pathways may only
`produce a cytostatlc effect if tumor cells survive in a Go state when the
`EGFR pathway is blocked. However. an important recent report by
`Wu el al. (28) showed that an anti-EGFR antibody could trigger
`apoptosis in a human colorectal cell line, DiFi, that overexpresses
`EGFR. Addition of IGF~I was able to protect the cells from the
`apoptotic effect of the antibody. These results, together with recent
`studies of inhibition of lGF—lR function (29. 30), indicate that, in some
`tumors, inhibition of growth factor pathways may kill tumor cells
`4838
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`Research.
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`APOPTOSIS AND CELL CYCLE ARREST BY CP-358,774
`
`rather than only produce cell cycle arrest. Here. we extend these
`findings with a novel EGFR tyrosine kinase inhibitor and demonstrate
`that this inhibitor blocks DiFi cell proliferation by a combination of
`cell cycle arrest and induction of apoptosis.
`
`MATERIALS AND METHODS
`
`Cl’-358,774. [6.7-Bis(2-methoxy—ethoxy)-quinazolin-4-yl]~(3-ethynylphe~
`nyl)amine. molecular formula C22H23N3O4. was prepared as described (CH).
`The structure is shown in Fig. IA. The monohydrocliloride salt form of this
`compound was used in the studies reported here.
`Cell Lines and Culture Conditions. DiFi is a human colorectal carcinoma
`
`cell line derived from a familial adenomatous polyposis patient, as described
`previously, that expresses 5 X 10° EGFRs/cell (I6, 32). DiFi cells were
`maintained in l:1 DMEM:Ham‘s F-12 with 10% FBS. HN5 human head and
`
`neck tumor cells that express L4 X 107 EGFRS/cell (l5, 33) were obtained
`from Dr. M}. O'llare of Haddow Laboratories, The Institute of Cancer
`Research, Sutton (Surry, United Kingdom), and grown in DMEM with 10%
`FBS. MDA—MB~468 human breast cancer cells that express l.5 X 10*’ EGFRs/
`cell (15) were obtained from the American Type Culture Collection (Bethesda,
`MD) and grown in DMEM with 5% FBS. RB cells (34) were maintained in
`DMEM with l0% FBS. All cells were cultured at 37°C in 5% carbon dioxide!
`
`95% air in the presence of 100 units/ml penicillin. l00 pg/ml streptomycin.
`and 2 mM glutamine. DiFi, MDA-MB-468. and HNS cells were tested and
`found to be mycoplasma free using the Gen—Probe kit (Fisher Scientific.
`Pittsburgh. PA).
`Kinase Assays. The EGFR kinase assay is similar to one described previ-
`ously (35). Nunc MaxiSorp 96-well plates were coated by incubation overnight
`at 37°C with l00 p.l per well of 0.25 mg/ml PGT (Sigma Chemical Co.. St.
`Louis, MO) in PBS. Excess PGT was removed by aspiration. and the plate was
`washed 3 times with wash buffer (0. l % Tween 20in PBS). The kinase reaction
`was performed in 50 pit of 50 mM HEPES (pll 7.3). containing l25 mM sodium
`chloride, 24 mM magnesium chloride. O.l mM sodium orthovanadate, 20 pm
`ATP. 1.6 pig/ml EGF. and i5 ng of EGFR. affinity purified from A43l cell
`membranes as described (36). The compound in DMSO was added to give a
`final DMSO concentration of 2.5%. Phosphorylation was initiated by addition
`of ATP and proceeded for 8 min at room temperature. with constant shaking.
`The lcinase reaction was tenninated by aspiration of the reaction mixture and
`was washed 4 times with wash buffer. Phosphorylated PGT was measured by
`25 min of incubation with 50 ptl per well HRP-conjugated PY54 (Oncogene
`Science Inc. Uniondale, NY) antiphosphotyrosine antibody, diluted to 0.2
`trg/ml in blocking buffer (3% BSA and 0.05% Tween 20 in PBS). Antibody
`was removed by aspiration. and the plate was washed 4 times with wash buffer.
`The colorimetric signal was developed by addition of TMB Microwell Perox-
`idase Substrate (Kirkegaard and Perry, Gaithersburg. MD). 50 pl per well. and
`stopped by the addition of 0.09 M sulfuric acid. 50 p.l per well. Phosphoty-
`rosine is estimated by measurement of absorbance at 450 nm. The signal for
`controls was typically 0.64.2 absorbance units. with essentially no back-
`ground in wells without ATP, EGFR, or PGT and was proportional to the time
`of incubation for l0 min.
`
`Conditions for selectivity assays were the same as those for the EGFR
`ltinase assay, except for the addition of l nuvt manganese chloride to the assay
`buffer and a final ATP concentration of l00 ;LM. The reaction was terminated
`by the addition of 50 id of 250 mM EDTA prior to aspiration. For experiments
`comparing inhibition of EGFR to v-abl or c—src kinase. recombinant bacterially
`expressed V-abl (3.4 ng/well) or purified human platelet c—src (l.2 units/well,
`Oncogene Science lnc.) was substituted for the EGFR. For experiments com-
`paring inhibition of EGFR to insulin receptor or lGF~lR. purified recombinant
`proteins were substituted for native EGFR. Baculovirus~expressed cytoplasmic
`domain of the insulin receptor B subunit (l0 units/well) was from Stratagene
`(La Jolla. CA). Recombinant EGFR kinase domain (2 ng).
`lGF~lR ltinase
`domain (3 ng), and web! ltinase were prepared as described below.
`Preparation of Recombinant Kinases. The complete intracellular domain
`of human EGFR (amino acids 6444186) was PCR amplified and subcloned
`into pAcG2T to generate a glutathione S-transferase fusion protein with a
`thrombin cleavage site. Plaque—purified recombinant baculovirus was used to
`infect Sf‘) insect cells for 60 h. The complete intracellular domain of human
`lGF—lR 8 subunit (amino acids 7l l~l377) was PCR amplified and subcloned
`into pAcG2’l“ to generate a glutathione S-transferase fusion protein with a
`thrombin cleavage site. Plaque-purified recombinant baculovirus was used to
`infect High V insect cells for 36 h. For both preparations. active kinase was
`purified using glutathione Sepharose followed by elution with free glutathione.
`The v—ahl was expressed as a Hiso-tagged protein and purified by affinity
`chromatography with the QlAexpness system (Qiagen).
`l.S X l0‘
`FRE Mltogenesis. FRE cells were plated in 96-well plates at
`cells/well in 100 ptl of DMEM with l0% F88. The next day. the medium was
`replaced with l00 (Ll of serum-free medium (RPMI 1640). After l2—20 h.
`growth factors and Brdljrd were added in the presence or absence of CP-
`358.774. The growth factors and their concentrations were as follows: murine
`EGF (Collaborative Biomedical Products). 0.5 ng/ml; PDGF (Genzyme,
`Cambridge, MA).
`l5 ng/ml;
`lGF—l
`(Genzyme). 50 ng/ml; and bFGF
`(Genzyme). 50 ng/ml. After overnight incubation, BrdUrd incorporation was
`measured using the Cell Proliferation Assay Kit (Amersham, Arlington
`Heights. IL) according to the supplier's instructions, except for substitution of
`mphenylenediamine (Pierce Chemical Co., Rockford, ll.) as the peroxidase
`substrate. The color reaction was stopped with 2 N sulfuric acid. and absorb-
`ance was read at 490 nm.
`For calculation of inhibition. all wells were corrected for the background
`signal obtained from cells incubated without Brdllrd. Percent inhibition was
`calculated as follows: I00 - l00l(treated - basal)l(control ~ basalll. where
`basal is signal from cells not stimulated with growth factor. control is signal
`4839
`
`HN
`
`A
`
`1 00
`90
`80
`70
`60
`50
`40
`30
`20
`‘l0
`
`%ofControl(tS.D.)
`
`0.01
`
`
`1 000
`0.1
`1
`10
`100
`[CP-358.774} (MA)
`
`Increasing
`[CP-358,774}
`
`4
`
`PhosphorglationI/V
`
`0
`
`0.0
`
`0.1
`
`0.2
`
`0.3
`
`1l{ATP]
`
`(nun)
`
`l. CP—358.774 inhibits purified EGFR kiuase and is competitive with ATP.
`Fig.
`Phosphorylation of PGT by purified EGFR was musured by immunoassay with antiphos—
`photyrosine antibodies as described in “Materials and Methods.“ A, data points. means of
`six determinations: bars. SD. This result is representative of four independent experi—
`ments. B, ATP competition experiments. CP-358,774 concentrations were: it) nm (0): 3
`nm (A); 1 nm (0); 0.3 nM (A); or 0 nM (Cl). The ATP concentrations tested were 5 mi.
`20 int. 80 p.M. 320 pm. and 3 mM. The K,,, for ATP under these conditions was 7 p.M.
`l‘-‘hosphorylation is measured in arbitrary units, and the data were fit by a linear least
`squares method. This experiment was repeated with identical results.
`
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`Research.
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`APOPTOSIS AND CELL CYCLE ARREST BY CP~358,774
`
`Systems, Minneapolis. MN) and the supplier‘s protocol. Annexin V~FI'I‘C and
`Pl binding were analyzed by flow cytometry using a FACSort. Data were
`collected using logarithmic amplification of both the FL! (FITC) and FL2 (PI)
`channels. Quadrant analysis of coordinated dot plots was done using CellQuest
`software. Unstained cells were used to control for autofluorescence. Singly
`stained cells were used to adjust the photomultiplier voltages and compensa-
`tion settings to eliminate spectral overlap between the FLl and FL2 signals.
`Preparation of Tissue Extracts for Western Blot Analysis. Frozen tumor
`or liver samples were pulverized. extracted with boiling 2>< Laemmli sample
`buffer (37) with 2 mM sodium orthovanadate, placed in a boiling water bath for
`I0 min. and stored at ~80"C until analysis. After the tissue extracts of
`insoluble material were cleared by centrifugation. the protein content of the
`extracts was determined, and the samples were analyzed by Western blotting
`as described above.
`
`RESULTS
`
`from growth factor-stimulated cells. and treated is signal from Cl’~358,774-
`treated, growth factor-stimulated cells.
`Measurement of Phosphotyroslne, pRB Phosphorylation, p27'“"’
`Expression, and PARP Cleavage by Western Blotting. Cells (DiFi or HN5)
`were incubated in the presence or absence of CP~358,774 or cisplatin. After
`24 h. cells were washed with 50 mM Tris-l~lCl. 140 mM sodium chloride, 3.3
`mM potassium chloride. and 500 ,u.M sodium orthovanadate (pH 7.4) and lysed
`by boiling in 2X Laemmli sample buffer (37) with 2 mM sodium orthovanadate
`for 10 min. Cellular protein was determined using the BCA protein assay
`(Pierce Chemicals. Rockford. IL). Equal amounts of total protein (10-20 ug)
`were loaded onto 4—20% Tris-glycine minigels (Integrated Separated Systems,
`Natick. MA) for phosphotyrosine determinations. 7.5% Daiichi Tris-glycine
`minigels for pRB, 12.5% Daiichi Tris~glycine minigels for p27'“"‘. or 4~l2%
`Bis-Tris NuPage minigcls (Novex, San Diego. CA) for PARP. After electro
`phoresis. proteins were transferred to an lrnmobilon-P membrane (Miilipore,
`Bedford, MA) for 2 h at 250 mA. After transfer, membranes were blocked for
`l h or overnight with 5% BSA in TBST [50 mM Tris-I-lCl (pH 7.4). 150 mM
`NaCl. and 0.1% Tween 20} for antiphosphotyrosine blotting or with 4~5%
`nonfat dry milk in TBST. For phosphotyrosine determinations. blots were
`probed with HRP-conjugated antiphosphotyrosine PY20 (ICN, Costa Mesa.
`CA) or liRl’~l’Y54 (Oncogene Science). For pRB detenninations, blots were
`probed with l pig/ml monoclonal antibody G3-245 (Pharlvlingen, San Diego.
`CA). followed by HRP-conjugated goat antimouse (Pharhiingen; H000). The
`identification of the lower band as underphosphorylated pRB was confirmed
`by use of an antibody specific for this form (PharMingen). For p27‘“”’, blots
`were probed with 0.! pg/ml arrti—p27'“”’ Clone 57 (Transduction Labs. Lex-
`ington. KY) followed by HRP—goat antimouse. For PARP, blots were probed
`with anti-PARP. Clone C-2-l0 (Biornol, Plymouth Meeting. PA), followed by
`HR?-conjugated rabbit antimouse lgG (Pierce Chemicals). The M, ll6,000
`PAR? and its M, 85,000 proteolytic fragment were identified by comparison
`with lysates of untreated HL-60 cells or HL-60 cells induced to undergo
`apoptosis with etoposide (Biomol). All HRP-labeled antibodies were detected
`using enhanced chemiluminescence (Amersham) according to the supplier’s
`directions and quantitated by densitometry.
`Cell Cycle Analysis of DiFi Cells. Scmiconflucnt DiFi cells growing in
`six—well plates were incubated for 24 h in DMEM:l-larn's F12 and 0.5% FBS
`containing diluent (0.l25% DMSO). cisplatin. or CP—358.774 in the absence or
`presence of IGF-l. Cells were harvested and incubated in medium containing
`0.2% Triton X-100 and 50 ug/ml Pl for 30 min. according to published
`methods (38). Pl uptake was analyzed by flow cytometry using a FACSort
`(Becton Dickinson, San Jose. CA). Data were acquired using linear arnplili~
`cation of FL2 and analyzed using CellQuest softwa.re (Becton Dickinson). For
`Inhibition of Tyrosine Phosphorylation in Intact Cells. The
`measurement of BrdUrd incorporation, DiFi cells were treated for 24 h with
`addition of EGF to cells that express EGFR leads to a rapid autophos-
`diluent or 1 us: CP-358.774, pulse labeled with I0 ,u.M 8rdUrd, incubated with
`phorylation of the EGFR on tyrosine residues in the COOH terminus,
`FITC-conjugated anti-BrdUrd monoclonal antibody (Becton Dickinson). and
`counterstained with Pl.
`thus providing a facile assay for inhibition of the EGFR tyrosine
`Agarose Gel Analysis of DNA Fragmentation. DiFi cells were treated to
`kinase activity in intact cells. CP-358.774 potently inhibits EGFR
`induce apoptosis as described for cell cycle analysis. After trypsinization of the
`autophosphorylation in I-INS human head and neck tumor cells (Fig.
`cells, DNA was extracted using a TACS Apoptotic DNA Laddering Kit
`3). a cell line that expresses high levels of EGFR (15). Evaluation of
`according to the supplier‘s instructions (Trevigen. Gaithersburg, MD). Isolated
`these Westem blots by densitometry indicates an IC50 for inhibition of
`DNA was quantitated by absorbance at 260 nm. Samples (6 peg/lane) were
`EGFR phosphorylation of 20 nM. At the higher concentrations of the
`electrophorescd on a 1.5% agarose gel containing 0.5 pg/ml P! for 2 h at Ill)
`compound, the extent of autophosphorylation after EGF stimulation is
`V in TAB buffer (40 mM Tris~acetate. l0 mM EDTA, and 20 mM glacial acetic
`lower than that in the controls without EGF, indicating reduction of
`acid. pH 8.4). Gels were visualized on 8 UV transilluminator.
`basal EGFR activity, which may arise from autocrine stimulation.
`TUNEL Analysis. DiFi cells were treated to induce apoptosis as described
`for cell cycle analysis. Cells were harvested. and aliquots of 2 X I0” cells were
`Additional studies indicated that the inhibition of kinase is rapidly
`obtained (<10 min) on addition of CP-358,774 to cell medium and is
`fixed in l% paraformaldehyde. permeabilized in 70% ethanol. and stored at
`-20"C. 3'~OH DNA strand breaks were detected by the TUNEL technique
`rapidly reversed after the inhibitor is washed out and the cells
`using the ApopTag Plus Fluorcscein (FlTC) kit (Oncor, Gaithersburg. MD)
`are incubated in inhibitor-free medium (data not shown). Similarly
`according to the supplier's instructions. FITC-labeled cells were counter-
`potent
`inhibition of EGF-induced EGFR autophosphorylatiorr by
`stained with P1 and analyzed by flow cytometry using a FACSort. Excitation
`Cl’-358,774 was seen with DiFi human colon cancer cells and MDA-
`was at 488 nm with emission read in the FLI (BP 530/30) and FL2 (BP
`MB~468 human breast cancer cells (data not shown).
`SS5/42) channels. Data were acquired using logarithmic amplification of both
`The selectivity of CP-358,774 as an inhibitor of the phosphoryla—
`FL] and FL2 (the FITC and PI signals. respectively). Data were analyzed using
`tion of endogenous intracellular substrates by EGFR tyrosine ltinase
`CellQuest software. Regions of ill versus FL2 dot plots were set using
`can also be demonstrated in intact cells. The phosphorylation of the
`control (cells treated with diluent alone) samples (39).
`adaptor protein SHC (40, 41) upon addition of EGF serves as a
`Annexln V Binding. DiFi cells were treated to induce apoptosis as de-
`scribed for cell cycle. Cells were harvested, and aliquots of 2 X l05 cells were
`convenient physiological marker for EGFR activity. The EGF-
`treated with anrrexin V-FITC and PI using the Apoptosis Detection Kit (R&D
`induced tyrosine phosphorylation of SHC proteins is completely
`4840
`
`Inhibition of EGFR Tyrosine Kinasc by CP-358,774.
`CP-358,774 inhibits purified EGFR ltinase with an ICSO of 2 nM (Fig.
`IA). Kinetic analysis indicates that the inhibition is competitive with
`ATP. A Lineweaver—Burk plot of phosphorylation in the presence of
`varied ATP and inhibitor indicates that the inhibition is reduced at
`
`higher concentrations of ATP and can be restored to the uninhibited
`rate at high ATP concentrations (Fig. 18). The extent of inhibition
`may, therefore. be influenced by the intracellular ATP concentration.
`A replot of the Km“, versus concentration of CF‘-358,774 from the
`data of Fig. 28 indicated a K, for CP~358,774 of 2.7 nrvr. EGFR is more
`sensitive to inhibition by CP-358,774 than are the other tyrosine
`kinases we have examined, and it is >l000—fold more sensitive than
`human c—src or v-abl when compared under identical conditions (Fig.
`2A). Cl’-358.774 is also a potent inhibitor of the recombinant intra—
`cellular (kinase) domain of the EGFR, with an IC50 of I nm (Fig 2B),
`essentially identical to that observed with full-size EGFR (Fig. IA),
`which indicates that the inhibitor binding site is in the kinase domain.
`The kinase domains of the human insulin receptor and IGF—lR are
`much less sensitive to this inhibitor and are essentially unaffected at
`concentrations as high as 10 ptM (Fig. 28). Thus, Cl’-358,774 is a
`potent. selective, and directly acting inhibitor of the EGFR tyrosine
`kinase.
`
`Downloaded from cancerresaacrjournals.org on October 19, 2015. © 1997 American Association for Cancer
`Research.
`
`APOTEX EX. 1016-003
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`
`APOPTOSIS AND CELL CYCLE ARREST BY Cl’-353,774
`
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`Hg. 2. Selectivity of CP-358.774 for EGFR kinase. Inhibition of purified kinases was
`measured as described in “Materials and Methods." Data points. means of triplicate
`determinations; bars. SE. Similar results were observed in two experiments. A. selectivity
`for native human EGFR versus c-src and recombinant v-abl. I. El-‘GR: O. c-src: A, v—abl.
`B. selectivity for recombinant EGFR ltinase domain (I) versus insulin receptor kinase
`domain (A) and IGF-IR kinase domain (I). As a positive control.
`tyrphostin A25
`(Calbiochem. San Diego. CA) produced 50% inhibition of IGF-IR kinase in this assay at
`20 p.M.
`
`blocked by CP-358,774 (Fig. 4, Lane 9), whereas the insulin-induced
`phosphorylation of IRS-l, a prominent insulin receptor tyrosine ki-
`nase substrate, is unaffected (Fig. 4., Lane 4). Staurosporine, a rela-
`tively nonselective kinase inhibitor (42), completely blocked IRS-l
`phosphorylation in response to insulin (Fig. 4. Lane 5) and therefore
`served as a positive control. This experiment demonstrates that CP-
`358,774 markedly inhibits in situ phosphorylation of an endogenous
`substrate of activated EGFR at concentrations that have no effect on
`
`completely inhibited by pretreatment with 100 mg/kg CP~358,774 and
`is reduced by 89 i ll% (mean 1 SE; n = 4) and 61 1 7% at doses
`of 25 and l0 mg/kg, respectively, at l h after an i.p. treatment. The
`doses of CP-358.774 used here (10, 25, and 100 mg/kg) can be
`administered daily for at least 5 consecutive days without lethality in
`mice. These results indicate that CP-358,774 treatment of mice effec-
`tively inhibits both murine EGFR kinase in liver and human EGFR in
`transplanted tumors.
`Inhibition of Tumor Cell Proliferation. The DiFi human colon
`
`tumor cell line expresses high levels of EGFR and is inhibited by the
`anti~EGFR antibody (16, 28, 44). The proliferation of DiFi cells is
`strongly inhibited by CP-358.774 with an ICSO of I00 nM for an 8-day
`proliferation assay (Fig. 6). Cl’-358,774 did not produce a rapid loss
`of viability:
`the percentage of viable cells was 98 t 0.8%
`(mean 1: SD) in untreated cell cultures and 97 : l.7% in cultures
`exposed to l ;.tM CP-358,774 for 24 h. The I-{N5 human head and neck
`tumor cell line (15, 28. 33), which also expresses a very high level of
`the EGFR. is markedly inhibited by Cl’-358.774 at concentrations as
`low as 50 nM and is completely blocked at 250 nM (data not shown).
`In contrast,
`the proliferation of raf-transformed NIH-3T3 cells or
`ras-transformed FRE cells is much less sensitive, with IC,0s for
`proliferation of 7 and 3 p.M, respectively (data not shown).
`CP-358,774 is selective for the EGFR kinase pathway, as evaluated
`in cellular proliferation assays. Unlike tumor cell lines, FRE fibro-
`blasts can be rendered quiescent by incubation in scrum~free medium
`and then triggered to proliferate by addition of defined growth factors
`such as EGF, PDGF. IGF-I, or bFGF. Each of these growth factors
`acts through a cognate transmembrane receptor with tyrosine kinase
`activity (I); thus, FRE cells are a well-defined model, unambiguously
`dependent on the added factors. that can be used to evaluate selectivity
`of tyrosine kinase irmibitors. Cl’-358,774 inhibits EGF-stimulated
`mitogenesis with an IC,0 of 70 nM but only inhibits mitogenesis
`stimulated by the other factors at concentrations of >l p.M (Fig. 7).
`Thus, CP-358,774 is not simply indiscriminately cytotoxic because
`the FRE cells continue to undertake DNA synthesis when stimulated
`with mitogens other than EGF. This indicates that Cl’-358,774 is
`selective for EGFR ltinase relative to other tyrosine kinase-linked
`receptors and that, at concentrations that inhibit EGF-induced prolif-
`eration, it does not effectively inhibit any of the many ltinases or other
`enzymes that are necessary for mitogenesis in response to other
`rnitogens.
`Additional studies were done to further characterize the effects of
`
`Cl’-358.774 on DiFi cell proliferation. Analysis of the cell cycle
`distribution of these cells indicated that
`the cells were partially
`blocked in the G, phase of the cell cycle by this EGFR inhibitor (Fig.
`8 and Table 1). Although the changes were somewhat masked by the
`appearance of an apoptotic cell population discussed below, marked
`
`CP—358,774 [nM]
`EGF
`
`50
`
`phosphorylation of the major physiological substrate of another trans-
`membrane tyrosine kinase, the insulin receptor.
`Inhibition of Murine and Human EGFR in Vivo by CP-358,774.
`Intravenous administration of EGF to mice produces a marked auto-
`phosphorylation of EGFR in liver and other tissues (43), thus provid-
`ing a dynamic assay for EGFR inhibition in vivo. We examined the
`ability of CP-358,774 to block EGFR autophosphorylation in liver and
`HN5 tumors in athyrnic mice, as shown in Fig. 5. As reported, EGFR
`in mouse liver is hypophosphorylated but is rapidly tyrosine phos-
`Fig. 3. Effect of CP—358.774 on tyrosine phosphorylation in HN5 cells. as evaluated
`phorylated when mice receive EGF (Fig. 5B). This phosphorylation is
`with Western blots. HN5 cells U0’ cells/well in 24-well cell culture plates) were exposed
`to the indicated concentrations of CP-358.774-Ol for l h and then stimulated with 50
`inhibited 54% by pretreatment with 10 rnglltg CP-358,774 and is
`ng/ml EGF (Latter 2, 4, 6. 8, and I0) or not (tuner I, 3, 5. 7, and 9). Cell lysates were
`nearly completely inhibited (93%) at 100 mg/kg. Similarly, EGFR in
`prepared alter a 5~min exposure to EGF. and BGFR-associated phosphotyrosine was
`human HN5 tumors is rapidly autophosphorylated in response to EGF
`measured by Western blotting as described in “Materials and Methods." The Cl’-358.774
`(Fig. 5A). Analysis of a larger set of samples prepared as in Fig. 5
`was present at 0 nM (Lanes 1 and 2); 20 nM (Lanes 3 and 4); 50 nM (Lanes 5 and 6); 250
`nM (Lanes 7 and 8); or 1000 rm (Lanes 9 and I0).
`indicates that EGF~induced autophosphorylation of tumor EGFR is
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`APOPTOSIS AND CELL CYCLE ARREST BY CP~358.774
`
`Fig. 4. CP-358,774 sclectivcly inhibits EGFR signaling in HN5
`cells. Following serum starvation for 2 it. HN5 cells were incubated
`for an additional hour in the presence of 012.5% DMSO (Lanes I~3
`and 6-8). l tm CP-358.774—0l (Lanes 4 and 9). or l0 p.M 513030-
`sporine (Lanes 5 and I0). Cells were then stimulated for 5 min with
`l00 nM insulin (Lanes 2-5) or 100 ng/ml EGF (Lanes 7-10) and
`lysed in 1% Triton X-l00 buffer. Ixmes l~5. lysates were subjected
`to immunoprecipitation with antibody to IRS- l. Lanes 6-10, lysates
`were subjected to immunoprecipitation with antibody to SHC. Im-
`munoprecipitates were resolved by SDS~PAGE. transferred to Im-
`rnobilon. and imrnunohlottcd with antiphosphotymsine antibodies as
`described in “Materials and Methods." Lanes 2 and 3. duplicates of
`the insulimtreated controls (no inhibitor); Lanes 7 and 8. duplicates
`of the EGF~treated controls (no inhibitor). Other conditions are
`single lanes.
`
`1234 5678910
`
`IRS-l ---> C O .
`
`EGFR -‘u- .
`
`}SHC
`
`decreases in the percentage of cycling cells in S phase and G2~M and
`an increase in the percentage of cells in G, were observed, indicating
`a G, block. Similar experiments were performed with the HN5 cells:
`48 h of exposure to 1
`;.LM CP-358,774 reduced the S-phase cells from
`55% of the total to 24% and increased the percentage of cells in G,
`from 24 to 56% (data not shown). Thus, for both DiFi and HN5 cells,
`
`inhibition of the EGFR ltinase by CP—358,774 leads to a partial G,
`arrest.
`
`The reduction in S~phase cells by Cl’-358.774 was confirmed
`by flow cytometric measurements of total DNA and incorporated
`BrdUrd, a more specific method for identifying cells in S—phase. The
`percentage of S-phase DiFi cells measured by this procedure de-
`
`A.
`CP358T74
`
`HN5 Tumor
`
`mglkg
`EGF
`
`O
`-
`
`0
`+
`
`1
`+
`
`10
`+
`
`100
`+
`
`EGFFl----
`
`B.
`
`Liver
`
`Fig. 5. CP~3S8.774 pretreatment inhibits EGF-
`i