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Evaluation of Farnesyl:Protein Transferase and Geranylgeranyl:Protein Transferase Inhibitor Combinations in Preclinical Models

Departments of Cancer Research [R. B. L., C. A. O., M. T. A., C. A. B., H. G. B., J. P. D., M. S. E-H., A. M. K., D. L., E. R., A. I. O., D. C. H., N. E. K.], Drug Metabolism [M. J. B.], Medicinal Chemistry [S. J. d., C. J. D., W. C. L., T. M. W., S. L. G., G. D. H.], and Safety Assessment [S. V. M.], Merck Research Laboratories, West Point, Pennsylvania 19486

	ABSTRACT

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Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor lines having either wild-type or oncogenic Ki-ras, treatment with an FTI/GGTI combination or with a DPI blocks Ki-Ras prenylation and induces markedly higher levels of apoptosis relative to FTI or GGTI alone. We demonstrate that these compounds can inhibit their enzyme targets in mice by monitoring pancreatic and tumor tissues from treated animals for inhibition of prenylation of Ki-Ras, HDJ2, a substrate specific for FPTase, and Rap1A, a substrate specific for GGPTase-I. Continuous infusion (72 h) of varying doses of GGTI in conjunction with a high, fixed dose of FTI causes a dose-dependent inhibition of Ki-Ras prenylation. However, a 72-h infusion of a GGTI, at a dose sufficient to inhibit Ki-Ras prenylation in the presence of an FTI, causes death within 2 weeks of the infusion when administered either as monotherapy or in combination with an FTI. DPIs are also lethal after a 72-h infusion at doses that inhibit Ki-Ras prenylation. Because 24 h infusion of a high dose of DPI is tolerated and inhibits Ki-Ras prenylation, we compared the antitumor efficacy from a 24-h FTI infusion to that of a DPI in a nude mouse/PSN-1 tumor cell xenograft model and in Ki-ras transgenic mice with mammary tumors. The FTI and DPI were dosed at a level that provided comparable inhibition of FPTase. The FTI and the DPI displayed comparable efficacy, causing a decrease in growth rate of the PSN-1 xenograft tumors and tumor regression in the transgenic model, but neither treatment regimen induced a statistically significant increase in tumor cell apoptosis. Although FTI/GGTI combinations elicit a greater apoptotic response than either agent alone in vitro, the toxicity associated with GGTI treatment in vivo limits the duration of treatment and, thus, may limit the therapeutic benefit that might be gained by inhibiting oncogenic Ki-Ras through dual prenyltransferase inhibitor therapy.

	INTRODUCTION

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Ki-ras is the most commonly mutated oncogene in human cancers and, as such, is an attractive pharmaceutical target (1) . All four of the isoforms of Ras, including the two splicing variants of the Ki-ras gene, Ki4A-Ras and Ki4B-Ras, as well as the Ha-Ras and N-Ras isoforms, are M_r 21,000 GTP binding proteins that are modified with an isoprenoid lipid. This lipid modification is normally performed by the enzyme FPTase,⁵ which adds the 15-carbon farnesyl isoprenoid to a cysteine residue 4 amino acids from the COOH-terminus of Ras (2) . Farnesylation occurs on the cysteine that is part of the CA₁A₂X motif, where C is cysteine, A is typically an aliphatic amino acid, and X is typically serine or methionine. Oncogenic forms of Ras, which are transforming to cells because of mutations, which inactivate their GTPase activity, as well as wild-type Ras, require this COOH-terminal prenylation for their biological and/or transforming functions (3, 4, 5, 6) . These findings provided the impetus to develop FTIs as a means of targeting oncogenic Ras for the treatment of cancer. Over the past 8 years many inhibitors of FPTase have been identified, and several of these are currently in clinical trials (7) .

FTIs inhibit the growth of both normal cells and cancer cells containing either wild-type or mutant forms of ras, although cells transformed with oncogenic Ha-ras tend to be much more responsive than cells harboring wild-type ras or oncogenic forms of Ki-ras or N-ras (8, 9, 10) . Furthermore, whereas mammary tumors formed in mice due to expression of a MMTV-Ha-ras transgene undergo dramatic regression when animals are treated with FTIs (11) , tumor growth in transgenic MMTV-Ki-ras and MMTV-N-ras mice is inhibited by FTI treatment, but the tumors do not dramatically regress (12 , 13) . Analysis of oncogenic Ha-Ras from cells treated with an FTI has shown that the prenylation of this protein is indeed blocked by the inhibitor (14) . In order for Ras to activate its downstream signaling effectors such as c-Raf-1, it must be localized and bound to the plasma membrane. Inhibition of Ha-Ras farnesylation prevents the protein from binding to the plasma membrane and leads to the cytosolic sequestration of unactivated c-Raf-1 (15) . Thus, inhibition of Ha-Ras farnesylation may cause accumulation of a dominant-negative form of oncogenic Ha-Ras, a mechanism that could account for the sensitivity of Ha-ras-transformed cells to FTIs.

In contrast to Ha-Ras, Ki- and N-Ras remain prenylated in FTI-treated cells. Whereas all three of the Ras isoforms are substrates for FPTase in vitro, Ki-Ras and N-Ras but not Ha-Ras are also substrates for GGPTase-I, a protein:prenyl transferase related to FPTase (16) . Whereas Ki- and N-Ras are normally farnesylated in cells, they are subject to geranylgeranylation by GGPTase-I in FTI-treated cells (17 , 18) . An oncogenic form of Ki-Ras that contains an altered CAAX motif such that the protein is exclusively geranylgeranylated is capable of transforming rodent cells (6 , 19) , suggesting that FTI treatment does not significantly alter the activity of Ki-Ras. Inhibition of growth of tumor cells containing wild-type Ras, oncogenic N-Ras, or oncogenic Ki-Ras may, therefore, involve inhibition of farnesylated proteins other than Ras, such as RhoB or the mitotic kinesin CENP-E (Ref. 20 ; reviewed in Refs. 21 and 22 ).

GGTIs have been developed by Sebti and Hamilton (reviewed in Ref. 23 ), and have been shown to inhibit the growth of tumor lines in culture and in nude mouse xenograft models (24 , 25) . Human tumor cell lines exposed to a GGTI typically arrest in G₀/G₁ (26 , 27) via a mechanism that involves the p53-independent induction of the cyclin-dependent kinase inhibitor p21 (27, 28, 29) . The mechanism of p21 up-regulation by GGTIs may involve inhibition of geranylgeranylation of the RhoA GTPase, because a constitutively active RhoA mutant represses p21 transcription, whereas a dominant-negative RhoA mutant or treatment with the Rho protein inhibitor ADP-ribosyl transferase C3 protein activates p21 (28) .

Ki-Ras prenylation can be inhibited through treatment with an FTI in combination with a GGTI (24 , 30) , and combination treatment has been shown to inhibit mitogen-activated protein kinase signaling in adrenocortical cells that overexpress Ki-Ras (31) . In this paper, we have explored whether Ki-Ras can be targeted for cancer therapy by combination treatment with an FTI and a GGTI, or through administration of a DPI, which inhibits both FPTase and GGPTase-I. We show that potent inhibitors of FPTase and GGPTase-I, when used in combination, can inhibit the prenylation of Ki-Ras in cells in culture and in tumor and normal tissues in mice, and induce a dramatic apoptotic response in tumor cells in culture. However, administration of GGTIs, either alone or in combination with FTIs, are lethal to mice when continuously infused for >24 h. These studies indicate that it may not be practical to translate FTI-GGTI combination therapy to a clinical setting.

	MATERIALS AND METHODS

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Cell Culture.
PSN-1 human pancreatic tumor cells, containing oncogenic Ki-ras Gly12 to Arg mutation, were obtained from Banyu Pharmaceutical Co. Ltd. (Tsukuba, Japan) and maintained at 37°C in RPMI 1640 (supplemented with 15% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin) with 5% CO₂. The MDA-MB-468 and LS180 tumor lines were obtained from ATCC (Rockville, MD) and cultured according to recommended conditions.

Analysis of Protein Prenylation in Cultured Cells and Animal Tissues.
For cell culture experiments, PSN-1 cells were treated with inhibitors for 24 h and then lysed in RIPA buffer [40 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1 mM EDTA, and protease inhibitors]. Lysates were subjected to SDS-PAGE using either 8% (for HDJ2) or 10–20% (for Rap1A and Ki-Ras) precast Novex Tris-Glycine gels (Invitrogen, Carlsbad, CA) and then immunoblotted using antibodies specific for HDJ2 (Lab Vision Inc., Fremont, CA), Rap1A (Ab sc-65; Santa Cruz Biotechnology, Santa Cruz, CA), or Ki-Ras (c-K-ras Ab-1 clone 234-4.2; Oncogene Research Products, Boston, MA). Blots were developed using alkaline-phosphatase-conjugated anti-IgG (Cappel Laboratories, West Chester, PA) followed by detection with ECF fluorescent alkaline phosphatase substrate (Amersham Pharmacia Biotech, Piscataway, NJ). Blots were scanned on a Storm 840 imager (Amersham Pharmacia Biotech). Unprenylated and prenylated proteins were distinguished by virtue of their different electrophoretic mobilities. The percentage of unprenylated protein was determined by peak integration using ImageQuant software (Amersham Pharmacia Biotech). EC₅₀ values were derived from dose-response curves with a 4-parameter curve fitting equation using SigmaPlot (SPSS Inc., Richmond, CA) software.

For analysis of tissue samples, frozen tissue was minced and then dounce-homogenized in RIPA buffer. (M/H)DJ2 in lysates was analyzed by immunoblotting, as above. For analysis of Ki-Ras, Ras protein was first immunoprecipitated from 1 mg of lysate with the pan-Ras antibody Y13-259 agarose conjugate (Calbiochem, La Jolla, CA), resolved on 16-cm 15% acrylamide, 0.087 bis-acrylamide SDS-PAGE gels, transferred to Immobilon (Millipore Inc., Bedford, MA), and blotted as above. Rap1A in tissue lysates was resolved on 15% acrylamide, 0.087 bis-acrylamide SDS-PAGE gels before blotting.

Cell Cycle and Apoptosis Analysis of Cells in Culture.
All of the flow cytometry experiments involved analysis of 10,000 cells/treatment group, analyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) using the 488-nm Argon laser. For cell cycle analysis, adherent and nonadherent cells were collected by centrifugation, washed in PBS (Ca^2+/Mg²⁺-free), and fixed in 70% ethanol overnight at 4°C. After fixation, cells were collected by centrifugation and stained at 10⁶ cells/ml in PBS (Ca^2+/Mg²⁺-free) containing 50 µg/ml propidium iodide, 100 Kunitz units/ml RNase A, and 0.1% w/v glucose for 30 min at room temperature. Percentages of cells in each phase of the cell cycle were determined using ModFit LT modeling software (Verity Software Inc.).

TUNEL analysis on cells in culture was measured by flow cytometry using the APO-DIRECT kit from BD PharMingen (San Diego, CA). This kit uses TdT to label DNA ends with FITC-dUTP. The manufacturers suggested method was followed, except that the FITC-dUTP labeling reaction with TdT enzyme was extended to 2 h. After FITC-dUTP labeling, the cells were stained with the propidium iodide solution provided with the kit to allow visualization of total DNA content per cell.

FITC-Annexin V binding was measured by flow cytometry as follows. PSN-1 cells were grown in 10-cm culture dishes and treated with compounds for 48 h. Cell monolayers and floating cells were washed in PBS (Ca^2+/Mg²⁺-free) and then incubated for 45 min at 37°C in binding buffer [0.1 M HEPES (pH 7.4), 1.4 M NaCl, and 25 mM CaCl₂] containing a 1:100 dilution of FITC-Annexin V (BD PharMingen). Cell monolayers were then washed in binding buffer, scraped into binding buffer, combined with the FITC-Annexin V stained floating cells from the same drug- treatment group, and analyzed by flow cytometry.

Caspase-3 activity in cell lysates was determined using the ApoAlert Caspase-3 fluorescent assay kit (Clontech, Palo Alto, CA), which measures the release of the 7-amino-4-trifluoromethylcoumarin (AFC) fluorophore from the substrate DEVD-AFC.

Animal Toxicity and Tumor Efficacy Studies.
All of the animal studies were performed according to the NIH Guide for the Care and Use of Laboratory Animals, and experimental protocols were reviewed by the Merck Animal Care and Use Committee. Compounds or vehicle (50% DMSO) were delivered using Alzet 2001D osmotic pumps (Alza Corporation, Palo Alto, CA) for 24-h infusions or with Alzet 1003D osmotic pumps for 72-h infusions. Pumps were implanted in the subcutis of the right flank of 8–12-week-old female nu/nu mice (Charles River Laboratories, Wilmington, MA). At the indicated times, mice were euthanized by CO₂ inhalation and necropsied. Blood was obtained for determination of compound concentration in plasma by heart puncture using heparinized 20-gauge needles. Tissues were immediately excised and quick-frozen in liquid nitrogen for prenylation analysis (pancreas, spleen, and tumor), or were fixed in 10% neutral buffered formalin for histological analysis (femur with bone marrow, spleen, and duodenum) or for TUNEL analysis (tumor). For histology, tissues were embedded in paraffin, sectioned, stained with H&E by routine methods, and examined microscopically.

Drug concentration in plasma obtained from the blood samples was determined by LC-MS/MS. Plasma was prepared from heparinized whole blood by centrifugation at 3000 rpm for 15 min at 4°C. An appropriate internal standard compound was added to the plasma samples, and then 2 volumes of acetonitrile containing 0.1% (w/v) trifluoroacetic acid was added to precipitate plasma proteins. Samples were centrifuged to remove precipitate, diluted with an equal volume of water containing 0.1% trifluoroacetic acid, and analyzed by LC-atmospheric chemical ionization MS/MS. Standard curves were prepared by addition of the appropriate standard solutions and the internal standard to control mouse plasma to yield nominal concentrations ranging from 2–5000 ng/ml. Samples were injected into a Sciex API III Plus Mass Spectrometer interfaced via the Sciex Heated Nebulizer to an LC system consisting of a Leap HTS PAL autosampler and a Hewlett-Packard 1050 pump. LC chromatography was on a Zorbax SB-C8 column (75 x 4.6 mm, 3.5 um) with isocratic elution using 63% acetonitrile/water containing 0.1% trifluoroacetic acid. MS/MS analysis was performed by monitoring the precursor ion of each analyte and a corresponding product fragment ion. After LC-MS/MS analysis, the concentrations of each analyte in the unknown plasma samples were determined by interpolation from the appropriate standard curve using the weighted (1/concentration²) linear regression parameters. The lower limit of quantitation was 2 ng of analyte/ml of plasma with an accuracy and precision of >80%.

For the nude mouse efficacy study, 10⁶ PSN-1 cells in Matrigel were implanted s.c. into nude mice and were established for 2 weeks. Mice were then treated for 24 h with vehicle or inhibitor via implantation of osmotic pumps as described above. Tumors were monitored by daily caliper measurement for 7 days after initiation of treatment, and MGRs of the tumor of each animal were calculated from a plot of tumor volume versus time as described previously (12) . For the MMTV-Ki-ras transgenic mouse model (12) , mice with palpable mammary tumors with a volume of 100–400 mm³ were used for study. Tumors were monitored by caliper measurement performed every other day. For both models, 10–15 animals/treatment group were used to monitor tumor growth, and several (2, 3, 4) animals were euthanized 24-h after pump-implantation for pharmacokinetic studies and for analysis of protein prenylation in tissues. Apoptosis in formalin-fixed, paraffin-embedded tumor sections was assessed by labeling and detection of free 3' DNA ends by the TUNEL method using the TACS 2 TdT kit from Trevigen (Gaithersburg, MD).

	RESULTS

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Inhibition of Prenylation of FPTase and GGPTase-I in Cultured Cells.
Compounds that are potent, selective inhibitors of either FPTase or GGPTase-I, or are inhibitors of both enzymes, were evaluated (Table 1) . The synthesis of these compounds and assessment of their potency against purified enzymes has been, or will be described elsewhere (32, 33, 34) .⁶ To demonstrate that these compounds are selective for their respective enzyme targets in cells, we used an immunoblot method to monitor prenylation inhibition of HDJ2, a protein prenylated exclusively by FPTase (12 , 35) , and Rap1A, a protein prenylated exclusively by GGPTase-I (26) . As is the case for many prenylated proteins, inhibition of prenylation of HDJ2 and Rap1A can be monitored by immunoblotting, because the unprenylated forms of these proteins display reduced mobility in SDS-PAGE relative to their prenylated versions. When PSN-1 human pancreatic tumor cells were exposed to FTI-1, FTI-2, or DPI-1, a mobility shift on SDS-PAGE was observed in HDJ2 (Fig. 1) , reflecting inhibition of FPTase. The incomplete deprenylation of HDJ2 observed after a 24-h treatment with these prenylation inhibitors (Fig. 1) is not attributable to incomplete inhibition of FPTase but rather is attributable to incomplete turnover of the prenylated HDJ2 that existed before treatment, because incubation with the same doses of prenylation inhibitors for 48 h results in complete deprenylation of HDJ2.⁷ Similarly, a mobility shift in Rap1A was observed on exposure to GGTI-1, GGTI-2, or DPI-1, indicative of GGPTase-I inhibition. The specificity of these prenylation inhibitors is illustrated by the lack of inhibition of Rap1A prenylation by FTI-1 or FTI-2, and, likewise, the lack of inhibition of HDJ2 prenylation by GGTI-1 or GGTI-2. Additionally, GGTI-1 is specific for GGPTase-I, because this compound does not inhibit the prenylation of Rab5a or Rab6, proteins which are geranylgeranylated exclusively by GGPTase-II (2) .⁸ As expected, Ki-Ras remains prenylated when cells are treated with FTI-1, FTI-2, GGTI-1, or GGTI-2, but its prenylation is inhibited in cells exposed either to a combination of FTI-1 and GGTI-1, FTI-2 and GGTI-2, or DPI-1 (Fig. 1) . It should be noted that farnesylated Ki-Ras, present in untreated cells, and geranylgeranylated Ki-Ras, present in FTI-treated cells, comigrate in SDS-PAGE (Fig. 1) but can be distinguished by other methods, including LC-MS (36) . Using the method illustrated in Fig. 1 , we determined EC₅₀ values for each compound with respect to inhibition of the three prenylated marker proteins in PSN-1 cells (Table 1) . Similar results were observed in PSN-1 cells, which contain one wild-type and one mutant allele of Ki-ras (G12R), MDA-MB-468 breast carcinoma cells, which are wild type for Ki-ras, and with the LS-180 colon carcinoma line, which harbors the oncogenic Ki-ras G12D mutation.⁸

View larger version (61K): [in this window] [in a new window]   Fig. 1. Inhibition of prenylation in PSN-1 cells. Extracts from cells treated for 24 h with inhibitors were resolved by SDS-PAGE and proteins detected by immunoblotting as described in "Materials and Methods." 1, vehicle; 2, DPI-1 (10,000 nM); 3, FTI-1 (300 nM); 4, GGTI-1 (100 nM); 5, FTI-1 (300 nM) + GGTI-1 (100 nM); 6, FTI-2 (3000 nM); 7, FTI-2 (300 nM); 8, GGTI-2 (100 nM); 9, FTI-2 (3000 nM) + GGTI-2 (100 nM); and 10, FTI-2 (300 nM) + GGTI-2 (100 nM). Positions of the unprenylated (U) and prenylated (P) version of each protein are indicated.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Inhibition of HDJ2 and Ki-Ras prenylation by FTI-1 titrated in the presence of high concentrations of GGTI-1 in PSN-1 cells. Ki-Ras prenylation as a function of FTI-1 dose was determined with either 100 nM GGTI-1 () or 1000 nM GGTI-1 () added along with the FTI. HDJ2 inhibition as a function of FTI-1 dose without added GGTI-1 is shown (); GGTI-1 addition does not affect the HDJ2 dose-response curve.7 Percentage of unprenylated protein was determined as described in Fig. 1 .

View larger version (24K): [in this window] [in a new window]   Fig. 3. Apoptosis induced by the combination of FTI-1 and GGTI-1 or by DPI-1. A, apoptosis measured by TUNEL-flow cytometry. PSN-1 cells were treated for 48 h with vehicle, 300 nM FTI-1, 100 nM GGTI-1, or a combination of 300 nM FTI-1 plus 100 nM GGTI-1, and analyzed by TUNEL as described in "Materials and Methods." There were 10,000 cells/treatment group analyzed. B, apoptosis detected by FITC-annexin staining. PSN-1 cells were treated for 48 h with vehicle, 300 nM FTI-1, 100 nM GGTI-1, or the combination of 300 nM FTI-1 plus 100 nM GGTI-1, stained with FITC-annexin, and analyzed by flow cytometry as described in "Materials and Methods." There were 10,000 cells/treatment group analyzed. C, DPI-1 inhibits prenylation and induces apoptosis in PSN-1 cells. PSN-1 cells were treated with DPI-1 and harvested at 24 h for analysis of prenylation and at 48 h for analysis of apoptosis (subG1 cells) by propidium iodide staining and flow cytometry analysis.

Cell cycle and apoptosis analysis was also performed on the MDA-MB-468 and LS180 cell lines. MDA-MB-468 were arrested in G₁ in response to GGTI-1, but their cell cycle distribution was only marginally affected by FTI-1, whereas LS180 cells showed a modest G₁ arrest in response to either FTI-1 or GGTI-1.⁹ For both MDA-MB-468 and LS180, FTI-1 and GGTI-1 in combination produced a greater apoptotic response than either agent alone, as seen both in the percentage of subG₁ cells and in the induction of Caspase-3 (Table 4) . The kinetics of induction of apoptosis by FTI/GGTI combination treatment differed among PSN-1, MDA-MB-468, and LS-180 cells, with significant levels of apoptosis detectable in the combination by 24, 48, and 72 h, respectively (Tables 3 and 4 ). Although we currently do not have an explanation for the differences in both the magnitude and the time to onset of apoptosis induced by the FTI/GGTI combination treatment in the different cell lines, these results suggest that apoptosis induction by FTI/GGTI combinations is a general phenomenon and is not limited to cell lines with oncogenic Ki-Ras.

When DPI-1 was administered as a 24-h s.c. infusion in nude mice bearing PSN-1 tumors, a dose-dependent inhibition of Ki-Ras prenylation in tumor tissue was observed. Doses of 770 mg/kg or 1920 mg/kg resulted in circulating plasma concentrations of 7.5 and 14.6 µM DPI-1, respectively, and at these doses, the prenylation of HDJ2, Rap1A, and Ki-Ras were all inhibited (Fig. 4) . The 240 mg/kg dose, which achieved a concentration of 2.6 µM DPI-1, maximally inhibited HDJ2 prenylation but did not inhibit Rap1A or Ki-Ras prenylation. Because all three of the doses of DPI-1 resulted in comparable levels of HDJ2 inhibition, we lowered the dose to determine the EC₅₀ for MDJ2 processing inhibition. We were unable to administer higher doses of DPI-1 because of solubility limitations, so we can only estimate EC₅₀ values for inhibition of Rap1A and Ki-Ras. The calculated EC₅₀ for MDJ2 processing inhibition in vivo is 100 nM,¹⁰ and from the data in Fig. 4 , we estimate the Ki-Ras and Rap1A in vivo EC₅₀ values to be 10,000 nM. Thus, the fold differences in EC₅₀ values between the three marker proteins is similar in vivo and in vitro (Table 1) , with inhibition of Ki-Ras and Rap1A prenylation requiring 100-fold higher concentrations of DPI-1 relative to the concentrations required to inhibit HDJ2 prenylation. It should be noted that the absolute EC₅₀ values for prenylation inhibition in vitro are 20–50 fold lower than the in vivo values. The in vivo EC₅₀ could be affected by a variety of parameters such as the binding of the drug to plasma proteins, which might decrease the free concentration of drug and, hence, raise the EC₅₀ values. These studies demonstrate that a high level of inhibition of FPTase and GGPTase-I sufficient to result in Ki-Ras prenylation inhibition can be achieved in vivo with the appropriate dose of DPI-1.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Inhibition of Ki-Ras prenylation in PSN-1 tumor tissue. Mice were treated using 24-h osmotic pumps with the indicated doses of DPI-1 (two mice per group) and euthanized 24 h after pump implantation. Tumor lysates were analyzed by immunoblotting; the Ki-Ras blot is shown in A. The percentage of unprenylated protein for each marker protein was determined from the immunoblots, and the average from the two animals as a function of dose is shown in B.

To investigate the mechanisms involved in GGTI-2-mediated lethality, histopathological analysis of hematopoietic tissues was performed on animals treated for 72 h with FTI-2, GGTI-2, or the drug combination. Tissues were isolated for analysis from animals either at 72 h or 15 days after pump implantation. By 72 h, treatment with FTI-2 at 200 mg/kg/day had caused only a slight cellular depletion in bone marrow and spleen, whereas treatment with GGTI-2 caused a more significant cellular depletion in bone marrow and spleen in a dose-dependent manner.¹¹ The higher doses of GGTI-2 (30–100 mg/kg/day) caused moderate to marked levels of cellular depletion, with only slight effects at the lower (3–10 mg/kg/day) doses.¹¹ The combination of FTI-2 and GGTI-2 caused cellular depletion that was not significantly more severe than with GGTI-2 alone. Additionally, surviving animals in the 3 and 10 mg/kg/day GGTI-2 dose groups showed evidence of hematopoietic recovery 15 days after pump implantation, as evidenced by a moderate to marked bone marrow-myeloid cell hyperplasia and splenic extramedullary myelopoiesis.¹¹ Whereas these studies show a dose-dependent hematopoietic suppression induced by GGTI-2, it is unclear whether this toxicity accounts for the lethality observed with this agent.

To confirm that the lethality of GGTI-2 is attributable to mechanism-based effects of GGTase-I inhibition and not to an unknown ancillary activity of the compound, we assessed the tolerability of DPI-1 and DPI-2. Infusion of DPI-1 at 770 mg/kg/day inhibited Ki-Ras prenylation in both a 24 h (Fig. 4) and 72 h (Table 7) infusion but also caused death in 2 of 3 animals by 7 days after implantation of the 72-h pump and death in a third animal by 13 days after implantation. Similarly, a 72-h infusion of DPI-2 at 1925 mg/kg/day inhibited Ki-Ras prenylation but also caused lethality by 8–9 days after implantation in 3 of 3 animals (Table 7) . A lower dose of DPI-2 (770 mg/kg/day) maximally inhibited HDJ2 prenylation and partially inhibited Rap1a prenylation but did not inhibit Ki-Ras prenylation and did not cause lethality. Because DPI-2 is a well-balanced DPI, we conclude that mice do not tolerate sustained inhibition of FPTase and GGTase-I at levels sufficient to inhibit Ki-Ras prenylation. Furthermore, our results show that high-level FPTase inhibition is generally well tolerated in mice but that high level inhibition of GGPTase-I is not.

	DISCUSSION

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We have found that treatment of tumor cell lines in culture with the combination of an FTI and a GGTI, or with a DPI, can inhibit Ki-Ras prenylation and induce a higher level of apoptosis than is seen when only one prenyltransferase is inhibited. However, inhibition of Ki-Ras prenylation and induction of apoptosis appear temporally separable. Complete deprenylation of Ki-Ras was observed after a 24-h treatment with the FTI-1/GGTI-1 combination in PSN-1, MDA-MB-468, and LS-180 cells (Fig. 1 and data not shown⁸ ), whereas induction of the highest levels of apoptosis required treatment for 48 h or more, particularly in the MDA-MB-468 and LS-180 cell lines (Tables 3 and 4 ). Presumably the prenylation of many different farnesylated and geranylgeranylated proteins is inhibited by combination FTI and GGTI treatment, and inhibition of one or more of these other prenylated proteins could contribute to the apoptosis. Therefore, we view the inhibition of Ki-Ras prenylation as a pharmacodynamic marker for combined inhibition of FPTase and GGTase-I, and not necessarily as the causative mechanism for the observed effects of dual inhibitor treatment.

We attempted to replicate our in vitro results in mouse tumor models. Because our in vitro studies indicated that continuous treatment for 24 h or more with an FTI and a GGTI will result in high level apoptosis (Table 5) , we delivered the compounds using osmotic pumps to achieve continuous prenyltransferase inhibition in vivo. We were able to demonstrate inhibition of Ki-Ras in tumor and normal mouse tissues with either a combination of an FTI and a GGTI, or with a DPI. However, we found that FTI/GGTI combination treatment is poorly tolerated. The lethality we observed with both DPIs and with GGTI monotherapy indicates that GGTIs are toxic, particularly at the higher doses, which are required to achieve inhibition of Ki-Ras prenylation in the presence of an FTI. By using a pharmacodynamic assay to measure prenyltransferase inhibition in vivo, we have observed that the lethality induced by structurally distinct GGTIs correlates with the amount of GGPTase-I inhibition in vivo, and, therefore, we can conclude with some certainty that the lethality is attributable to GGPTase-I inhibition. Additional experimentation will be required to identify the GGTase-I substrates which contribute to the observed toxicities of GGTIs when inhibited. The lethality induced by GGTIs might be attributable in part to myelosuppressive effects.¹¹ However, in our development of FTIs, we have observed that infusion of mice with a variety of different FTIs can induce myelosuppression to a comparable or greater extent than was observed with GGTI-2 or the DPIs described here, without causing lethality.¹⁵ Therefore, we suggest that the GGTI-induced lethality could be because of other mechanisms in addition to the myelosuppressive effect of GGTIs. In fact, in many of our experiments involving nude mice treated with the GGTIs and DPIs described here, as well as with structurally related compounds not described in this report, we often observed visual signs of edema in the treated animals.¹² Additionally, we have treated beagle dogs by continuous infusion with GGTI-1, GGTI-2, and DPI-1, as well as with GGTIs and DPIs not described here, and have observed significant, acute toxicities within the first 48 h of treatment, including moderate to severe edema and decreased blood pressure.¹⁶ These toxicities were related to the dose of compound and the extent of Rap1A prenylation inhibition observed in mononuclear WBCs from the treated animals.⁸ We considered the possibility that the edema observed on treatment of mice and dogs with GGTIs could be attributable to effects on nitric oxide production, because nitric oxide is a key regulator of blood pressure homeostasis (39) , and it has been reported that a GGTI causes a super-induction of nitric oxide synthase in cultured vascular smooth muscle cells (40) . However, we were unable to detect an induction of either nitric oxide or nitric oxide synthase in tissues from mice treated with GGTIs.¹⁶

The poor tolerability of GGTI treatment limited the treatment time to relatively short (24 h) infusions. Whereas we were able to inhibit Ki-Ras prenylation by treatment with DPI-1 for 24 h, this did not induce a statistically significant increase in tumor cell apoptosis. Because longer treatment times were not tolerated, we could not explore whether more extended treatments might have resulted in greater levels of apoptosis and potentially greater antitumor efficacy. Using two different tumor models, we found that a selective FTI was as efficacious as a DPI. Although the interpretation of these results was complicated by the fact that both the FTI and the dual inhibitor remained at concentrations high enough to significantly inhibit FPTase for at least 6 days after the infusion, we conclude that inhibition of FPTase and GGPTase-I for a 24-h period does not measurably enhance the antitumor efficacy relative to what is obtained when only FPTase is inhibited.

We found that a 72-h continuous infusion of 3 mg/kg/day of GGTI-2 results in approximately half-maximal inhibition of the GGTase-I substrate Rap1A in pancreatic tissue (Table 6) . This dose of GGTI-2 was tolerated and produced only slight bone marrow and spleen cell depletion.¹¹ A higher dose of GGTI-2 (10 mg/kg/day) produced near maximal inhibition of Rap1A prenylation but resulted in the death of 1 of 3 mice (Table 6) . These data show that it is possible to measurably inhibit GGTase-I in vivo but that there is a fairly narrow range of tolerability for this GGTI. We have not addressed whether partial inhibition of GGTase-I by the tolerated dose of GGTI-2 is sufficient to inhibit tumor growth as monotherapy in our tumor models. We also found that DPI-2, which is nearly optimal in its balance of FTI and GGTI activities for inhibition of Ki-Ras prenylation, also induces lethality in mice at concentrations that inhibit the prenylation of both Rap1A and Ki-Ras. A 72-h infusion of DPI-2 at 770 mg/kg/day resulted in circulating plasma concentrations of 2.4 µM, partially inhibited Rap1A prenylation, and was tolerated by the mice but did not significantly inhibit Ki-Ras prenylation. In contrast, the 1925 mg/kg/day dose of DPI-2, which resulted in only slightly higher plasma concentrations (3.4 µM), significantly inhibited both Rap1A and Ki-Ras prenylation but was lethal after a 72-h infusion. These results illustrate a very narrow range of tolerability for this compound and suggest that whereas DPI treatment might potentially be more efficacious than FTI monotherapy, the dual inhibitor approach might have a very narrow therapeutic index when applied in a clinical setting. Ki-ras remains an attractive therapeutic target for cancer, but our results suggest that dual FTI/GGTI therapy is not the best approach for targeting this oncogene.

Our observations of lethality associated with GGTI treatment appear to be at odds with those of Sun et al. (24 , 25) , who reported that peptidomimetic inhibitors of GGTase-I, GGTI-297, and GGTI-2154 partially inhibited the growth of two human tumor cell lines in mouse xenograft models. In these studies, Sun et al. (24 , 25) did not report any toxicities associated with GGTI treatment. Conceivably, the GGTIs and DPIs reported here, which are chemically distinct from the inhibitors reported by Sun et al. (24 , 25) , could contain an activity unrelated to GGTase-I inhibitory activity, which caused the toxicities observed in our studies. However, we disfavor this possibility because our studies with GGTI-2 and DPI-2 (Tables 6 and 7 ) established a clear relationship between toxicity and GGTase-I inhibition. In contrast, the papers by Sun et al. (24 , 25) did not report any measurement of GGPTase-I inhibition from treated animals. Because our studies with GGTI-2 showed that partial inhibition of GGTase-I is tolerated (Table 6) , the doses of GGTI-297 and GGTI-2154 used by Sun et al. (24 , 25) may have been tolerated because of only partial inhibition of GGPTase-I. We have not performed head-to-head comparisons of our GGTIs and the GGTIs described by Sun et al. (24 , 25) , but additional dose-response studies measuring toxicity and Rap1A prenylation inhibition as a function of the dose of GGTI-297 or GGTI-2154 would provide more information as to whether toxicity is inherent to the GGTI class of compounds. Whereas the results of Sun et al. (24 , 25) suggest that GGTIs might be useful as antiproliferative agents in a clinical setting either alone or in combination with other chemotherapeutic agents (25) , our work suggests that GGTIs might have a very narrow therapeutic index.

	ACKNOWLEDGMENTS

 
We thank Dr. Sherri Motzel for expert advice and assistance with the animal studies.

	FOOTNOTES

 
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.

¹ To whom requests for reprints should be addressed, at Department of Cancer Research, Merck Research Laboratories, WP16-3, West Point, PA 19486. Phone: (215) 652-8813; Fax: (215) 993-3398; E-mail: rob_lobell{at}merck.com

² Present address: Pfizer Inc., Ann Arbor, MI 48105.

³ Present address: Exelixis Pharmaceuticals Inc., South San Francisco, CA 94080.

⁴ Present address: GlaxoSmithkline Inc., King of Prussia, PA 19406.

⁵ The abbreviations used are: FPTase, farnesyl:protein transferase; GGPTase-I, geranylgeranyl:protein transferase type I; FTI, FPTase inhibitor; GGTI, GGPTase-I inhibitor; DPI, dual prenyltransferase (FPTase and GGPTase-1) inhibitor; MMTV, mouse mammary tumor virus; MS/MS, tandem mass spectrometry; LC, liquid chromatography; MGR, mean growth rate; TdT, terminal deoxynucleotidyltransferase; TUNEL, terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling.

⁶ S. J. deSolms, "Dual FPTase and GGTPTase-1 inhibitors as potential cancer chemotherapeutic agents," manuscript in preparation.

⁷ M. Abrams and R. Lobell, unpublished observations.

⁸ D. Liu and R. Lobell, unpublished observations.

⁹ J. Davide, and R. Lobell, unpublished observations.

¹⁰ E. Rands, H. Bhimnathwala, and C. Omer, unpublished observations.

¹¹ S. V. Machotka, unpublished observations.

¹² E. Rands, and C. Omer, unpublished observations.

¹³ A. Kral, unpublished observations.

¹⁴ A. Kral and H. Bhimnathwala, unpublished observations.

¹⁵ C. Omer, R. Lobell, and N. Kohl, unpublished observations.

¹⁶ C. Buser, unpublished observations.

Received 6/ 1/01. Accepted 10/18/01.

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