6
ICANCERRESEARCH 55, 5310—5314, November15,1@@5l ABSTRACT i'as oncogenes are present in several types of cancers but are most frequently described in colon and pancreatic carcinomas. Consequently, i-asis being targeted for drug development as a means to develop therapies for these types of cancer. The ras protein is posttranslationally modified by the addition of a farnesyl group, followed by cleavage of the COOH terminal 3 amino acids and methylation of the prenylated cystelne. Be cause the posttranslational addition of farnesyl is obligatory not only for the remaining modifications to take place but also for ms control of cell growth, inhibitors of farnesylatlon are being developed as potential anti tumor agents. In this report, a new peptidomimetic inhibitor of farnesyl tranferase is described. This compound, B956, and its methyl ester B1086, inhibit the formation of colonies in soft agar of 14 human tumor cell lines expressing different ras oncogenes at concentrations between 0.2 and 60 MM. Higher concentrations of B956 (10-80 pM) were required to Inhibit colony formation by 5 tumor cell lines without nsa mutations. B9561B1086 at 100 mg/kg also Inhibited tumor growth by EJ-l human bladder card noma, HT1OSO human tibrosarcoma, and to a lesser extent by HCT116 human colon carcinoma xenografts in nude mice. Furthermore, Inhibition of tumor growth by B956 is shown to be correlated with inhibition of ma posttranslational processing in the tumor. Thus, peptidomimetic inhibi tors of ms famnesylation have the potential to be developed as therapy for ms-dependent tumors. INTRODUCTION The ras proto-oncogene is involved in the control of cell prolifer ation and differentiation. In normal cells, ras switches between mac tive GDP-bound and active GTP-bound states (1). Binding of growth factors like epidermal growth factor or platelet-derived growth factor to their receptors results in the activation of the ras proteins by promoting the exchange of bound GDP for GTP (2). The activated ras, by a still unknown mechanism, triggers a phosphorylation cascade involving raf, mitogen-activated protein kinase kinase, and mitogen activated protein kinase, and ending in the activation of nuclear transcription factors. The final result is stimulation of cell growth or differentiation. Inactivation of ras occurs when the bound GTP is hydrolyzed to GDP, a reaction stimulated by GTPase-activating pro tein. The cycle is thus closed, and ras remains in inactive form until a new growth signal arrives (for reviews, see Refs. 3 and 4). Oncogenic ras protein has lost the ability to switch between the inactive and active states and remains permanently activated. Al though there are several ways in which ras can remain activated, the most common seems to be mutations at codons 12, 13, or 61, which result in loss of the ras-GTPase activity. Whereas it is not clear what triggers the specific mutations that activate ras, the result is a consti tutively activated protein that constantly sends growth signals to the cell nucleus. The presence of oncogenic forms of ras has been detected in 20—30%of all types of human cancers, in up to 50% of colon cancer, and in more than 80% of pancreatic carcinomas (5). The Received 7/13/95; accepted 9/19/95. 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. I To whom requests for reprints should be addressed, at Eisai Research Institute, 4 Corporate Drive, Andover, MA 01810. high incidence of ras mutations in colon and pancreatic carcinomas has motivated a great deal of research on ways to inhibit the activity of the ras oncogene. Signal transduction by ras is dependent on its plasma membrane localization (reviewed in Ref. 6). ras is localized at the plasma membrane after posttranslational farnesylation of the cysteine in the CAAX consensus sequence at the COOH terminus of the protein (7, 8). Farnesylation is followed by proteolytic cleavage of the last three amino acids, methylation of the now famnesylated cysteine, and, in the case of H- and N-ras, palmitylation of upstream cysteines. Several reports have indicated that famesylation is critical for the subsequent modifications, as well as membrane localization and function, of the ras protein (9—12). Therefore, inhibition of famesylation is being sought by several laboratories as a possible mechanism for inhibition of tumor growth (13). In this report, we describe in vivo data obtained with a potent new peptidomimetic inhibitor of farnesylation, B956. This compound inhibits the growth oftumors induced in nude mice by ras-transformed cells, including 3 human xenografts expressing dii ferent types of oncogenic ras. B956 also inhibits the ability of several human tumor cell lines to form colonies in soft agar, a common assay for tumorigenicity. Furthermore, it is shown that inhibition of tumor growth is correlated with inhibition of ras posttranslational processing in the tumor. Although differences in sensitivity to the inhibitor were observed in different cell lines, inhibition of ras famnesylation as antitumor drug therapy appears promising. MATERIALS AND METHODS Cells. NIH 3T3 cells transformed with H-ras (61L) (zHl, or pZIP-ras-F) have been described previously (11). NIH 3T3 cells transformed with K-ras (12V) (DK1) were prepared by infection with retrovirus carrying K-ras 4B (12V) clone. Infected cells were selected with G418, and single clones were isolated, grown, and tested for K-ras expression, growth in soft agar, and tumor formation in nude mice. A single clone (DK1) was chosen for these studies. El-i cells were kindly provided by Dr. C. Der (University of North Carolina, Chapel Hill, NC). The remaining cell lines were obtained from American Type Culture Collection (Rockville, MD) or Dainippon Seiyaku (Osaka, Japan) as indicated in Table 1. All cells were maintained as recom mended. Soft Agar Colony Growth. Cells at the densityindicatedin Table 1 were seeded on 24-well plates in 0.2 ml of 0.33% Noble agar (DLFCO Laboratories) in tissue culturemediumsupplementedwith serum over 0.4 ml of0.66% Noble agar in the same culture medium.The top agar layer was overlaidwith 0.2 ml of 0.66% Noble agar in culture medium. T24 and MlApaca-2 cells were seeded in 0.2 ml Matrigel (Collaborative, MA.) and covered with an upper layer of Matrigel in culturemedium.In all cases, the upperagar or Matrigellayer was coveredwith 0.2 ml of culture medium supplemented with serum and 2.5% DMSO or 2-fold serial dilutions of B956. After 10—21 days at 37°C, 0.2 ml of 3.3 mg/mi 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide in PBS was added and incubatedfor 1 h. The number and size of the stainedcolonieswere analyzedwith an image analyzer program (Spectrum; Mitani Corp., Fukui, Japan). Tumor Implantation Female BALB/c-nu/nu mice (5—6weeks of age; Japan SLC, Inc., Shizuoka, Japan) were housed in barrier facilities on a 12-h light/dark cycle, with food and water ad libitum. Cells (1 X 10@zHl, 5 X 106 El-i, 1 X 106irrl08o, and 8 x 106 HCF116) in 0.1 ml HBSS were injected s.c. into the flank of the animal at day 0. At day 1, mice were separated into control (n = 10) and treatment (n = S each) groups. B956 or its methyl ester 5310 Inhibition of Human Tumor Xenograft Growth by Treatment with the Farnesyl Transferase Inhibitor B956 Takeshi Nagasu, Kentaro Yoshimatsu, Cheryl Rowell, Michael D. Lewis, and Ana Maria Garcia' Department of Cancer Research. Eisai Company, Limited, Tsukuba. 300-26 Japan IT. N., K. Y.J, and Eisai Research institute, Andover, Massachusetts 01810 (C'. R., M. D. L, A. M. G.J on April 20, 2021. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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ICANCERRESEARCH55,5310—5314,November15,1@@5l

ABSTRACT

i'as oncogenes are present in several types of cancers but are mostfrequently described in colon and pancreatic carcinomas. Consequently,i-asis being targeted for drug development as a means to develop therapiesfor these types of cancer. The ras protein is posttranslationally modifiedby the addition of a farnesyl group, followed by cleavage of the COOHterminal 3 amino acids and methylation of the prenylated cystelne. Because the posttranslational addition of farnesyl is obligatory not only forthe remaining modifications to take place but also for ms control of cellgrowth, inhibitors of farnesylatlon are being developed as potential anti

tumor agents. In this report, a new peptidomimetic inhibitor of farnesyltranferase is described. This compound, B956, and its methyl ester B1086,inhibit the formation of colonies in soft agar of 14 human tumor cell linesexpressing different ras oncogenes at concentrations between 0.2 and 60MM.Higher concentrations of B956 (10-80 pM) were required to Inhibitcolony formation by 5 tumor cell lines without nsa mutations. B9561B1086at 100 mg/kg also Inhibited tumor growth by EJ-l human bladder cardnoma, HT1OSO human tibrosarcoma, and to a lesser extent by HCT116human colon carcinoma xenografts in nude mice. Furthermore, Inhibitionof tumor growth by B956 is shown to be correlated with inhibition of maposttranslational processing in the tumor. Thus, peptidomimetic inhibitors of ms famnesylation have the potential to be developed as therapy forms-dependent tumors.

INTRODUCTION

The ras proto-oncogene is involved in the control of cell proliferation and differentiation. In normal cells, ras switches between mactive GDP-bound and active GTP-bound states (1). Binding of growthfactors like epidermal growth factor or platelet-derived growth factorto their receptors results in the activation of the ras proteins bypromoting the exchange of bound GDP for GTP (2). The activated ras,by a still unknown mechanism, triggers a phosphorylation cascadeinvolving raf, mitogen-activated protein kinase kinase, and mitogenactivated protein kinase, and ending in the activation of nucleartranscription factors. The final result is stimulation of cell growth ordifferentiation. Inactivation of ras occurs when the bound GTP ishydrolyzed to GDP, a reaction stimulated by GTPase-activating protein. The cycle is thus closed, and ras remains in inactive form untila new growth signal arrives (for reviews, see Refs. 3 and 4).

Oncogenic ras protein has lost the ability to switch between theinactive and active states and remains permanently activated. Although there are several ways in which ras can remain activated, themost common seems to be mutations at codons 12, 13, or 61, whichresult in loss of the ras-GTPase activity. Whereas it is not clear whattriggers the specific mutations that activate ras, the result is a constitutively activated protein that constantly sends growth signals to thecell nucleus. The presence of oncogenic forms of ras has beendetected in 20—30%of all types of human cancers, in up to 50% ofcolon cancer, and in more than 80% of pancreatic carcinomas (5). The

Received 7/13/95; accepted 9/19/95.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I To whom requests for reprints should be addressed, at Eisai Research Institute, 4

Corporate Drive, Andover, MA 01810.

high incidence of ras mutations in colon and pancreatic carcinomashas motivated a great deal of research on ways to inhibit the activityof the ras oncogene.

Signal transduction by ras is dependent on its plasma membranelocalization (reviewed in Ref. 6). ras is localized at the plasmamembrane after posttranslational farnesylation of the cysteine in theCAAX consensus sequence at the COOH terminus of the protein (7,8). Farnesylation is followed by proteolytic cleavage of the last threeamino acids, methylation of the now famnesylated cysteine, and, in thecase of H- and N-ras, palmitylation of upstream cysteines. Severalreports have indicated that famesylation is critical for the subsequentmodifications, as well as membrane localization and function, of theras protein (9—12).Therefore, inhibition of famesylation is beingsought by several laboratories as a possible mechanism for inhibitionof tumor growth (13). In this report, we describe in vivo data obtainedwith a potent new peptidomimetic inhibitor of farnesylation, B956.This compound inhibits the growth oftumors induced in nude mice byras-transformed cells, including 3 human xenografts expressing diiferent types of oncogenic ras. B956 also inhibits the ability of severalhuman tumor cell lines to form colonies in soft agar, a common assayfor tumorigenicity. Furthermore, it is shown that inhibition of tumorgrowth is correlated with inhibition of ras posttranslational processingin the tumor. Although differences in sensitivity to the inhibitor wereobserved in different cell lines, inhibition of ras famnesylation asantitumor drug therapy appears promising.

MATERIALS AND METHODS

Cells. NIH 3T3 cells transformed with H-ras (61L) (zHl, or pZIP-ras-F)have been described previously (11). NIH 3T3 cells transformed with K-ras(12V) (DK1) were prepared by infection with retrovirus carrying K-ras 4B

(12V) clone. Infected cells were selected with G418, and single clones were

isolated, grown, and tested for K-ras expression, growth in soft agar, and

tumor formation in nude mice. A single clone (DK1) was chosen for thesestudies. El-i cells were kindly provided by Dr. C. Der (University of North

Carolina, Chapel Hill, NC). The remaining cell lines were obtained fromAmerican Type Culture Collection (Rockville, MD) or Dainippon Seiyaku

(Osaka, Japan) as indicated in Table 1. All cells were maintained as recommended.

Soft Agar Colony Growth. Cells at the densityindicatedin Table 1 wereseeded on 24-well plates in 0.2 ml of 0.33% Noble agar (DLFCO Laboratories) in

tissueculturemediumsupplementedwith serumover 0.4 ml of0.66% Noble agarin the sameculturemedium.The top agar layerwas overlaidwith 0.2 ml of 0.66%Noble agar in culture medium.T24 and MlApaca-2cells were seeded in 0.2 mlMatrigel (Collaborative, MA.) and covered with an upper layer of Matrigel in

culturemedium.In all cases, the upperagaror Matrigellayerwas coveredwith0.2ml of culture medium supplemented with serum and 2.5% DMSO or 2-fold serial

dilutions of B956. After 10—21 days at 37°C, 0.2 ml of 3.3 mg/mi 3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide in PBS was added andincubatedfor 1 h. The numberand size of the stainedcolonieswere analyzedwithan image analyzerprogram (Spectrum;Mitani Corp., Fukui, Japan).

Tumor Implantation Female BALB/c-nu/nu mice (5—6weeks of age;Japan SLC, Inc., Shizuoka, Japan) were housed in barrier facilities on a 12-hlight/dark cycle, with food and water ad libitum. Cells (1 X 10@zHl, 5 X 106El-i, 1 X 106irrl08o, and 8 x 106HCF116) in 0.1 ml HBSS were injecteds.c. into the flank of the animal at day 0. At day 1, mice were separated intocontrol (n = 10) and treatment (n = S each) groups. B956 or its methyl ester

5310

Inhibition of Human Tumor Xenograft Growth by Treatment with the Farnesyl

Transferase Inhibitor B956

Takeshi Nagasu, Kentaro Yoshimatsu, Cheryl Rowell, Michael D. Lewis, and Ana Maria Garcia'

Department of Cancer Research. Eisai Company, Limited, Tsukuba. 300-26 Japan IT. N., K. Y.J, and Eisai Research institute, Andover, Massachusetts 01810 (C'. R., M. D. L,A. M. G.J

on April 20, 2021. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Table 1 Human tumar celllinesras

mutationCellline―Tissue of origin(Ref.)No. ofcells/wellH520

(A)COLO32ODM(D)C0L0205 (A)HT29 (A)WiDr (D)El-iT24 (D)A-427 (A)A549(D)SW480 (A)SW620 (A)DLD-l (A)HCTI16 (A)H460 (A)HCT-15 (A)MIA PaCa-2 (A)HT-1080 (D)THP-1 (A)HL-60 (A)Lung

ColonColonColonColonBladderBladderLungLungColonColonColonColonLungColonPancreasFibrosarcomaLeukemiaLeukemia(27)

(28)(28)(29)(30)

H-12V (31)H-12V (32)K-12D (33)K-12S (33)K-12V (34)K-12V (35)K-13D (23)K-13D (23)K-61H (36)K-13D (37)K-12C (38)N-61K (39)N-12D (40)N-61L (41)2

X 10@1 x i0@4 x i0@2 x i0@1 X i0@1 X 10@1 X i0@5 X i0@5 X 1024 X 10@1 X i0@1 X 10@1 x io@1 X 1(92 X1 X5 X i(9

2.5 X iO11 X i0@

B956 IN}IIB@ON OF TUMOR GROWTH

B956 inhibits H- and K-ras posttranslational processing by 50% at 05and 25 ELM,respectively. In this report, these studies have been extendedto the effect of B956 on anchorage-independent growth of 19 humantumor cell lines, as well as the 2 model cell lines zHl and DK1. The panelof human tumor cell lines tested is summarized in Table 1, where thepresence and identity of ras mutation is indicated. This group of 19 celllines represents a variety of tissues, as well as ras mutations. Treatmentwith B956 resulted in inhibition of the number and size of coloniesformed. The concentration of B956 that reduced the number of coloniesby 50% is shown in Fig. 2. There is a similar inhibition pattern for theformation of colonies in soft agar as that seen for inhibition of ranprocessing (namely, cell lines expressing different types of ras showdifferences in sensitivity to the inhibitor). Tumor cell lines expressingH-ras oncogene seem to be the most sensitive, followed by cell linesexpressing mutated N-ras. The more resistant tumor cell lines are theones expressing mutant K-ras as well as cells without ras mutations. TheICso for inhibition of colony formation among the human cell linesexpressing K-ras fluctuates between 1.7 @Mfor the most sensitive(HCI'116) and 50 @LMfor the least sensitive (HCT15). In contrast, H-mstransformed cell lines are inhibited by submicromolar concentration ofB956, whereas for N-ras-transformed cells, 50% inhibition occurs atconcentrations around 5 p.MB956.

In contrast to other reports (14, 15), transformed cell lines without rasmutations were inhibited in their capacity to form colonies in soft agar atconcentrations between 16 and 80 @MB956. Approximately one-half ofthe K-ms-transformed cell lines tested were inhibited at the same concentmations of B956. We have previously shown a similar result withanother inhibitor, B581 (11). This compound at 100 @.LMmhibited colonyformation of NIH 3T3 fibroblasts transformed either with the rafoncogene or mutant H-ras modified by geranylgeranylation or myristylationby about 50% (1 1). The fact that non-ras-mutated cell lines are inhibitedin their colony-forming capacity is not unexpected if other farnesylatedproteins, like lamins or other yet unidentified proteins, are considered.However, the involvement of these proteins in the control of cell growthis not clear. In summary, transformants expressing either H- or N-rasoncogenes are very sensitive to inhibition by the ETase inhibitor B956. Incontrast, only 50% of K-ras mutant cell lines are inhibited at relativelylow (i.e., below 10 ,tM) concentrations of inhibitor. The remaining 50%of the K-ras-transformed cells tested were inhibited by concentrations ofB956 that also inhibit non-ras-mutated cell lines (i.e., between 10 and100 SM).

a Origin of cells: A, American Type Culture Collection (Rockville, MD); D, Dainippon

Seiyaku (Osaka, Japan).

H2N@

B956 R=H

HCT-15

A549H460

SW620

SW480

I 00.

_.@:@

C0

C8C 1

8CoIL,0)Co

ft@

I.

H-ras N-ras K-ras normalmutations

Fig. 2. Soft agar colony formation by human tumor cell lines. The indicated cell lineswere grown in soft agar as described in @‘Materialsand Methods.―At 14—21days, colonieswere stained and counted. Cell lines are grouped according to the mutant form of ras theyharbor: H-, N-, or K-ras, or normal (no ras mutation), as indicated on the abscissa. Theconcentration of B956 that inhibited the number of colonies to one-half is plotted as theordinate for each cell line. Arrows, IC50 for inhibition of colony formation by H-ras (61L)or K-ras (12V) NIH 3T3-transformed fibroblasts (zHl and DK1 cells, respectively).

5311

B1086 R=MeFig. I . Structure of farnesyl transferase inhibitors.

B1086 were dissolved in saline containing 2% Tween 80 just before use andwere injected i.p. Treatment was started on day 1 at the concentrations andtreatment schedules indicated in the figure legends. Control animals receivedvehicle. Tumor volume was measured on the days indicated, and body weightwas determined twice weekly. Tumor volume was calculated according to thefollowing equation: tumor volume (mm3) = (Length X Width2)/2.

After 2—3weeks, mice were sacrificed, and tumor weights were determined.Significance was determined by Student's t test.

InhibitIon of i-asPosttranslatlonal Processing. Tumors were induced in 6nude mice by zHl cells as above. At day 6, when the tumor was visible, B956was administered i.p. to 3 mice, at a concentration of 200 mg/kg every 12 h fora total of 7 injections. After determining tumor size, the mice were sacrificed,and the tumor was minced and treated with collagenase (1.2 mg/ml) and DNase(0.3 mg/ml). A single cell suspension was obtained by filtration through anylon mesh. The cells were washed with cold PBS and lysed by sonication in5 mM Tris-HC1(jH 7.5)-S mM EDTA-1 mM phenylmethylsulfonyl fluoride.Cell debris was removed by centrifugation at 12,000 X g for 5 mm, followedby cytosol and membrane fractionation by centrifugation at 100,000 X g for 30mm. Protein (50 pg/lane) was separated by SDS-PAGE and ras was identifiedby Western blotting with F235—1.7.1antibody (Oncogene Science).

RESULTS

Inhibition of Anchorage-independent Growth of Human Tumor Cell Lines. B956 (Fig. 1) is a new and potent inhibitor of fl'@2activity in vitro.The concentration to inhibit H-ms farnesylation by 50%is in the nanomolar range (IC50 = 11 nM@).In whole cells (zHl or DK1),

2 The abbreviation used is: FTase, farnesyl protein transferase.

3 Unpublished observations.

C0L0205

HT-29••H520I. COIO32ODM

DLD-1 WuDrMlApaca-2

A427

HCT116

HT1O8O r;-iHL6O@ •J

ThP-1

EJ@1F;@H-T24

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grew slowly and were very sensitive to treatment with B1086. Theeffect of the inhibitor was dose dependent. At the maximum dose of100 mgfkg, no apparent tumor growth was observed, and 1 mouse of5 had no visible tumor at the end of the experiment. Tumors inducedby EJ-1 cells were found to be quite resistant to conventional antitumor drugs (such as cisplatin and doxorubicin; data not shown). However, the FFase inhibitor B1086 at the dose of 100 mg/kg inhibitedtumor growth completely.

The effect of B1086 on HT-1080-induced tumors is not as strikingas that observed against FJ-1 xenografts. However, there is a statistically significant difference between the tumor growth of treated andcontrol animals. The tumors in B1086-treated animals grew moreslowly, and the effect was dose dependent (Fig. SB). At the end of theexperiment, tumors of animals treated with 100 mg/kg were 40%smaller than those of control animals (Fig. SB). HT-1080 xenograftsinduce cachexia to the animals; therefore, long experiments are notpossible. Animals bearing tumors in the control group, as well as inthe 25 and 50 mg/kg B1086 treatment groups, showed signs ofnecrosis and bleeding at the tumor site. However, animals treated with100 mg/kg B1086 looked healthier than untreated animals.

The human tumor carcinoma cell line HCfl 16 was the leant sensitive of the tumor xenografts tested (Fig. SC), despite the fact thatwhen grown in soft agar it was more sensitive to the inhibitor thanwere HT1O8O cells (Fig. 2). Changes in growth properties in vitro andin vivo could account for this difference. Treatment with 100 mg/kgB1086 significantly reduced tumor size by only 20% (P < 0.05).Reduction of tumor growth rate was observed between days 11 and17. By the end of the experiment (i.e., 21 days), no significantdifference between the tumor size of treated and control animals was

@1'4:16

!@E@days after implantation

Fig. 3. Effect of B956 on tumor growth induced by H-ms (61L)-transformed NIH 3T3fibroblasts. At day 0, zHl cells were injected s.c. into the flank of nude mice. Thefollowing day, mice were randomized, and at the times indicated, (arrows) they receivedi.p. injections of vehicle (V) or 125 mg/kg (A) or 250 mg/kg (•) B956 in saline containing

2% Tween 80. Tumor size was determined every 2 or 3 days as indicated. Points, tumorvolume; bars, SD.

Inhibition of Tumor Growth Induced by ms-transformed 3T3Fibroblasts. Nude mice were implanted s.c. with H-ras-transformedNIH 3T3 fibroblasts and treated systemically by two daily i.p. injections of 125 or 250 mg/kg B956 for 5 days. Tumors were measuredevery 3—4days until mice were sacrificed at day 14.The results (Fig.3) clearly show that B956 treatment inhibits tumor growth in adose-dependent manner. Tumor growth resumes after a delay ofseveral days, depending on the dose of B956 used. The rate of tumorgrowth after the delay is similar to that of untreated animals. No signsof toxicity were observed, and all the animals gained weight normallyduring the experiment.

Inhibition of ran Processing Associated with Inhibition of Tumor Growth. To determine whether the inhibition of tumor growthwas in fact due to inhibition of ran processing, ran localization wasdetermined after B956 treatment. For this purpose, mice given implantations of ZH1 cells were treated with B956 6 days after implantation, when the tumor was visible. After a 3-day treatment with thefarnesyl transferase inhibitor, a significant reduction of tumor growthwas observed (Fig. 4A). At this time, tumors from control and B956-treated animals were excised, homogenized, and separated into membrane and cytosol fractions. Fully processed ras localizes to theplasma membrane, whereas unprocessed ran, because of the lack ofprenyl anchor, is not membrane associated but accumulates in thecytosol (16). As expected, all the ras protein was detected in themembrane fraction of tumor cells from control animals (Fig. 4B, Lane1). In contrast, in the tumors from B956-treated animals, ran with aslower mobility accumulated in the cytosol (Fig. 4B, Lane 4), andalmost no ras was detected in the membrane fraction (Fig. 4B, Lane3). To our knowledge, this is the first demonstration that the decreasein tumor growth induced by a farnesyl transferase inhibitor is correlated with inhibition of ran farnesylation.

Inhibition of Growth of Human Tumor Xenografts. Preliminaryexperiments with the methyl ester prodrug B956, compound B1086,indicated that the latter was at least twice an potent at inhibiting tumorgrowth induced by 3T3 fibroblasts expressing the H-ras oncogene.Therefore, B1086 was used to test whether tumors induced by humantumor cell lines expressing oncogenic ran protein were also sensitiveto FTase inhibitors. Nude mice were given implantations s.c. ofhuman tumor cells expressing H-ras (El-i, bladder carcinoma; Fig.5A), N-ras (HT-1080 fibrosarcoma; Fig. SB) or K-ras (HCT 116colon carcinoma; Fig. SC) oncogenes. Tumors induced by El-i cells

B956 1NHIB@ON OF TUMOR GROWTH

EE

E

600

500

@300

0 200

E

@100

BI 2 3 4

Fig. 4. ras posttranslational processing and inhibition of tumor growth. A, zHl cellswere implanted s.c. into nude mice. At day 6, B956 was administered i.p. at 200 mg/kgfor 7 injections, every 12 h. The tumor volume of control (0) or treated (@)animals wasdetermined at days 6 and 9. Columns, mean; bars, SD. B, at day 9, tumors were removedand fractionated into membrane (Lanes 1 and 3) and cytosol (Lanes 2 and 4). Protein fromeach fraction (50 pg) was separated by gel electrophoresis and identified by westernimmunoblot with F235-1.7.1 antibody. Lanes I and 2, tumors from control, untreatedanimals; Lanes 3 and 4, tumors from B956-treated animals.

5312

DaysafterImplantation

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z,u'J

400A300....:::

•...@ .12001000B800...600

400.,

,,,3@@::P•@'200

0@ . .0 2 4 Ô@ 10 12 14 16

1200

1000

800

600

400

2flfl

C

,..@.

B956 INHIBITION OF TUMOR GROWTH

peptidomimetic B956 and its methyl ester prodrug B1086 are potentnot only against model cell lines of ras-transformed cells but also

____________________________________ against tumor cell lines of human origin that express different ras

oncogenes. In our in vivo model system, the potency of B1086 wascomparable to that of L739,749 (Ref. 21; data not shown). It is also

shown that inhibition of tumor growth by this FTase inhibitor isassociated with inhibition of ras posttranslational processing in the

__________________________________ tumor (Fig. 4). As it has been indicated by others (21), the use of

Vfase inhibitors appears to be safe and has potential for use as____________________________________ long-term therapy: after 18 daily administrations of B1086, no signs

of toxicity were evident.Whereas the ester prodrug was about 10 times more potent than the

acid in intact cells, presumably because of a higher membrane permeability, there was only a 2-fold increase in potency in vivo. Preliminary

_________________________________ pharmacokinetic data4 indicates that the ester prodrug is not very stable

in circulation because it is hydrolyzed to the acid within 5 mm of________________________________ administration.Thus,theesterprobablyincreasestheprodrugpermeabil

ity across the epithelial barrier, but its rapid hydrolysis results in a higher_______________________________ circulatingconcentrationoftheactivebutlesspermeableform,B956.It

was also found that the inhibitor accumulates in the tumor at similar__________________________________ levels as in plasma, but its clearance from the tumor is slower.

The effect of B1086 or B956 on tumor growth was transient. Once thedrug wan removed, the tumors resumed growth at approximately thesame rate as untreated controls, indicating a cytostatic and not a cytotoxic

______________________________ effect.However,theresultswithEl-ixenografts(Fig.5A)suggestthatwith a more optimized treatment strategy, the inhibition of tumor growthcould be irreversible and complete. In this case, a longer treatmentschedule was devised to accomodate the slow growth rate of EJ-1 tumors.At the end of the experiment, one mouse in the higher dose group wastumor free, whereas the remaining 4 animals did not show apparent tumorgrowth. Although it is possible that tumors would grow during a longerobservation period, this result suggests that an adequate treatment sched

________________________________ ulemighteliminatetumorgrowthcompletely.Furthersupportforthisidea comes from the experiment where treatment of animals with agrowing tumor resulted in a significant reduction of tumor growth (Fig.4A). In most of our experiments, drug treatment is started soon aftertumor implantation because under this condition tumors are more sensitive. However, this experiment shows that the growth of an alreadypresent tumor can be slowed down and possibly stopped by FTase

IJ@@ ;@@ : : inhibitors using an appropriate dosage regimen.

0 5 10 15 20 25 One piece of puzzling information is derived from data with a panelof human tumor cell lines expressing different ras oncogenes. Whenthese cell lines are treated with B956 in a colony-forming assay,differences in sensitivities are observed. We have shown previouslythat increasing concentrations of inhibitor are required to inhibitposttranslational processing of different prenylated proteins (18).However, in this cane, differences in sensitivities are observed amongcell lines expressing the same ras oncogene. This was especially clearfor cells expressing the K-ras oncogene, where a large number of celllines was tested. It is possible that a wide range in sensitivity to FTaseinhibitors will be observed in cells with H- or N-ras mutations if morecell lines are studied. On the other hand, whereas other reports haveshown lack of effect of FTase inhibitors on non-ras-transformed celllines (i.e., raf-, mos-, or sir-transformed cell lines; Refs. 14 and 17),the data presented here indicate that about 50% of the K-ras-transformed cell lines tested are an sensitive an non-ras-transformed celllines. This discrepancy could perhaps be eliminated if more cell linesare studied in different laboratories.

Available data indicate that if a cell line expresses a ras oncogene,its growth is governed by the presence of activated ras (23). However,

E0

a..0E

I-

Daysafter ImplantationFig. 5. Effect of Bl086 on human tumor xenografts. El-i (A), HT1O8O(B), or

HCTI 16 (C) cells were implanted s.c. into nude mice at day 0 as described in “Materialsand Methods.―At day I, mice were separated into control (n = 10) and treatment (n = 5)groups, and i.p. administrations at the times indicated by arrows were started. Controlanimals (U) received vehicle. Treated animals received 25 (•),50 (A), or 100 (0) mg/kgB1086 in saline containing 2% Tween 80. Tumor volume was measured every 3—4daysas shown. Points, tumor volume (mm3); bars, SD.

observed (Fig. SC). The size of tumors in untreated animals reached

saturation at 17 days, probably because of the cachexia induced in theanimals by these tumor cells. However, as observed above forHT1O8O xenografts, the cachexic effect of HCT1 16 cells was significantly decreased in animals treated with 100 mg/kg B1086.

DISCUSSION

The number of reports on new synthetic inhibitors of FTase activityin vitro is growing (Refs. 14, 15, and 17—20contain just a fewexamples of inhibitors). However, there are few reports demonstratingin vivo efficacy (21, 22). In the present report, it is shown that the 4 Unpublished observations.

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B956 INHIBITION OF TUMOR GROWTH

CAAL motif and a second signal are sufficient for plasma membrane targeting of rasproteins. EMBO J., 10: 4033—4039,1991.

17. James, G. L, Goldstein, J. L., Brown, M. S., Rawson, T. E., Somers, T. C.,McDowell, R. S., Crowley, C. W., Lucas, B. K., Levinson, A. D., and Marsters, J. C.,Jr. Benzodiazepine peptidomimetics: potent inhibitors of ras farnesylation in animalcells. Science (Washington DC), 260: 1937—1942,1993.

18. Garcia, A. M., Rowell, C., Ackermann, K., Kowalczyk, J. J., and Lewis, M. D.Peptidomimetic inhibitors of Ras farnesylation and function in whole cells. J. Biol.Chem., 268: 18415—18418,1993.

19. Nigam, M., Seong, C. M., Qian, Y. M., Hamilton, A. D., and Sebti, S. M. Potentinhibition of human tumor p21(ras) farnesyltransferase by A1A2-lackingp21(ras)CA1A2X peptidomimetics. J. Biol. Chem., 268: 20695—20698,1993.

20. Hara, M., Akasaka, K., Akinaga, S., Okabe, M., Nakano, H., Gomez, R., Wood, D.,Uh, M., and Tamanoi, F. Identification of Ras farnesyltransferase inhibitors bymicrobial screening. Proc. NatI. Acad. Sci. USA, 90: 2281—2285,1993.

21. Kohl, N. E., Wilson, F. R., Mosser, S. D., Giuliani, E., deSolms, S. J., Conner, M. W.,Anthony, N. J., Holtz, W. J., Gomez, R. P., Lee, T. J., Smith, R. L, Graham, S. L,Hartman, G. D., Gibbs, J. B., and Oliff, A. Protein farnesyltransferase inhibitors blockthe growth of ras-dependent tumors in nude mice. Proc. Natl. Acad. Sci. USA, 91:9141—9145,1994.

22. Kohl, N. E., Omer, C. A., Conner, M. W., Anthony, N. J., Davide, J. P., Desolms,S. J., Giuliani, E. A., Gomez, R. P., Graham, S. L, Hamilton, K., Handt, L K.,Hartman, G. D., Koblan, K. S., Kral, A. M., Miller, P. J., Mosser, S. D., Oneill, T. J.,Rands, E., Schaber, M. D., Gibbs, J. B., and Oliff, A. Inhibition of farnesyltransferaseinduces regression of mammary and salivary carcinomas in ms transgenic mice. Nat.Med., I: 792—797,1995.

23. Shirasawa, S., Furuse, M., Yokoyama, N., and Sasazuki, T. Altered growth of humancolon cancer cell lines disrupted at activated Ki-ras. Science (Washington DC), 260:85—88,1993.

24. Pendergast, A. M., Quilliam, L A., Cripe, L D., Bassing, C. H., Dai, Z., Li, N.,Batzer, A., Rabun, K. M., Der, C. J., Schlessinger, J., and Gishizky, M. BCR-ABLinduced oncogenesis is mediated by direct interaction with the SH2 domain of theGRB-2 adaptor protein. Cell, 75: 175—185,1993.

25. Puil, L, Liu, J., Gish, G., Mbamalu, G., Bowtell, D., Pelicci, P. G., Arlinghaus, R.,and Pawson, T. Bcr-Abl oncoproteins bind directly to activators of the Ras signallingpathway. EMBO J., 13: 764—773, 1994.

26. Sawyers, C. L., McLaughlin, J., and Witte, 0. N. Genetic requirement for Ras in thetransformation of fibroblasts and hematopoietic cells by the Bcr-Abl oncogene.J. Exp. Med., 181: 307—313,1995.

27. Mitsudomi, T., Steinberg, S. M., Nau, M. M., Carbone, D., D'Amico, D., Bodner, S.,Ole, H. K., Linnoila, R. I., Mulshine, J. L., Minna, J. D., and Gazadar, A. F. p53 genemutations in non-small-cell lung cancer cell lines and their correlation with thepresence of ras mutation and clinical features. Oncogene, 7: 171—180,1992.

28. Sirakawa, S., and Sasazuki, 1. Analysis of molecular mechanism in colorectaltumorigenesis. Fukuoka Igaku Zasshi, 84: 25—35,1993.

29. Kahn, S. M., Jiang, W., Culbertson, A, Weinstein, I. B., Williams, G. M., Tomita, N.,and Ronai, Z. Rapid and sensitive nonradioactive detection of mutant K-ras genes via“enriched―PCR amplification. Oncogene, 6: 1079—1083, 1991.

30. Jenkins, D. C., Rowley, J., Wilkinson, J., Skinner, R., Holmes, L. S., Linstead, D. J.,Rapson, E. B., Stables, J. N., and Topley, P. A panel of tumors with constitutive K-rasmutations for chemotherapeutic studies. Proc. Am. Assoc. Cancer. Res., 31: 2562,1990.

31. Tabin, C. J., Bradley, S. M., Bargmann, C. I., Weinberg, R. I., Papageorge, A. G.,Scolnick, E. M., Dhar, R., Lowy, D. R., and Chang, E. H. Mechanism of activationof a human oncogene. Nature (Lund.), 3(X):143—149,1982.

32. Reddy, E. P., Reynolds, R. K., Santos, E., and Barbacid, M. A point mutation isresponsible for the acquisition of transforming properties by the T24 human bladdercarcinoma oncogene. Nature (Lund.), 300: 149—152,1982.

33. Valenzuela, D. M., and Groffen, J. Four human carcinoma cell lines with novel mutationsin position 12 of c-K-ras oncogene. Nucleic Acids Res., 14: 843—852,1986.

34. Capon, D. J., Seeburg, P. H., McGrath, J. P., Hayflick, J. S., Edman, U., Levinson,A. D., and Goeddel, D. V. Activation of Ki-ras2 gene in human colon and lungcarcinomas by two different point mutation. Nature (Lund.), 304: 507—513,1983.

35. Bos, J. L, Verlaan-de Vries, M., Marshall, C. J., Veeneman, G. H., van Boom, J. H.,and van der Eb, A. J. A human gastric carcinoma contains a single mutated and anamplified normal allele of the Ki-ras oncogene. Nucleic Acids Res., 14: 1209—1217,1986.

36. Mitsudomi, T., Viallet, J., Mulshine, J. L., Linnoila, R. I., Minna, J. D., and Gazadar,A. F. Mutations of ras genes distinguish a subset of non-small-cell lung cancer celllines from small-cell lung cancer cell lines. Oncogene, 6: 1353—1362,1991.

37. Peinado, M. A., Fernandez-Renart, M., Capella, G., Wilson, L., and Perucho, M.Mutations in the p53 suppressor gene do not correlate with c-K-ms oncogenemutations in colorectal cancer. Int. J. Oncol., 2: 123—134,1993.

38. Berrozpe, G., Schaeffer, J., Peinado, M. A., Real, F. X., and Perucho, M. Comparativeanalysis of mutations in the p53 and K-ras genes in pancreatic cancer. Int. J. Cancer,58: 185—191,1994.

39. Brown, R., Marshall, C. J., Pennie, S. G., and Hall, A. Mechanism of activation of anN-ras gene in the human fibrosarcoma cell line HT1O8O. EMBO J., 3: 1321—1326,1984.

40. Janssen, J. W. G., Steenvoorden, A. C. M., Lyons, J., Anger, B., Bolike, i. U., Boa,J. L., Seliger, H., and Bartram, C. R. RAS gene mutation in acute and chronicmyelocytic leukemias, chronic myeloproliferative disorders, and myelodysplasticsyndrome. Proc. NatI. Acad. Sci. USA, 84: 9228—9232, 1987.

41. Murray, M. J., Cunningham, J. M., Parada, L F., Lebowitz, P., and Weinberg, R. TheHL-60 transforming sequence: a ras oncogene coexisting with altered myc genes inhematopoietic tumors. Cell, 33: 749—757, 1983.

5314

tumor cell lines are characterized by the presence of multiple mutations in different genes. It is possible that unidentified mutationsdownstream of ran could maintain cell growth, even if ran signaling isdisrupted by ETase inhibitors. Alternatively, mutations in pathwaysparallel to ras could be sufficient to maintain the transformed phenotype. The above alternatives might explain why a ras-transformed cellline could be resistant to growth inhibition by farnesylation inhibitors.The opposite (namely, activation of factors upstream of ras) couldexplain why cells without mutant forms of ras are sensitive to theseinhibitors. For instance, an activated growth factor receptor or unidentified oncogenes upstream of ras could activate normal ran continuosly. One example of the latter is found in chronic myelogenousleukemias, where bcr/abl is presumed to activate ras by phosphorylation of the grb2 adaptor protein (24—26).In such cases, cell growthcould be reduced by inhibition of ran farnesylation. These hypothesesdeserve further testing. They suggest that, at the clinical level, it mightbe necessary to test for ras dependency of tumor growth beforedeciding on a treatment strategy. Whereas this might reduce thenumber of treatable tumors where the ras oncogene is present, it willadd other tumors that are not obvious targets at the moment.

In summary, the data presented here confirm the hypothesis that,for ras-dependent tumors, inhibition of ras farnesylation results ininhibition of ras function and, therefore, inhibition of tumor growth. Itseems plausible that a tumor might be ras dependent, even in theabsence of the ras oncogene, thus broadening the spectrum of tumortargets for FTase inhibitors.

ACKNOWLEDGMENTS

We thank Drs. K. Tsukidate, H. Suzuki, and Y. Oda for providing thepreliminary pharmacokinetic data.

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1995;55:5310-5314. Cancer Res   Takeshi Nagasu, Kentaro Yoshimatsu, Cheryl Rowell, et al.   the Farnesyl Transferase Inhibitor B956Inhibition of Human Tumor Xenograft Growth by Treatment with

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