Introduction

Deregulation of RAS signal transduction, either directly through activating mutations or indirectly through mutations of other oncogenes or tumor suppressor genes, has been implicated in the molecular pathogenesis of myeloid leukemias.^1,2 The importance of RAS is underscored by the positioning of several oncogene and tumor suppressor gene products on this pathway.^1,2,3,4,5 Activated, GTP-bound RAS proteins (H-, K- and N-RAS) transduce mitogenic signals from ligand-stimulated tyrosine kinase, cytokine and heterotrimeric G-protein-coupled receptors by binding and activating effector molecules such as Rafs, MEKK, PI-3K and Ral-GEF, which regulate proliferation, differentiation and malignant transformation.^{3,4,5,6,7,8,9,10,11,12}

RAS proteins require several post-translational modifications (eg prenylation, proteolysis, carboxymethylation and palmitoylation) for membrane binding and full biologic activity.^{3,4,5,6,13,14} Protein prenylation is catalyzed by prenyl transferases that covalently attach either a farnesyl moiety (by farnesyl transferase, FTase) or a geranylgeranyl moiety (by geranylgeranyl transferases I and II, GGTase I and II) to carboxyl-terminal cysteines. While the preferred recognition motif for FTase and GGTase I is a carboxyl-terminal CAAX (where C is cysteine, A is an aliphatic amino acid and X is any amino acid), GGTase II prenylates proteins with carboxyl-terminal CXC, XXCC or CCXX sequences. Although the X position of the CAAX motif determines whether a protein will be a substrate for FTase (X=methionine, serine, cysteine and glutamine) or GGTase I (X=leucine or isoleucine), these two enzymes have some degree of cross-specificity.^13,14

Inhibition of RAS post-translational modification is one strategy to impede oncogenic RAS function.⁵ FTase inhibitors (FTIs) are the most promising new class of these potential cancer therapeutics as they are remarkably specific and cause no gross systemic toxicity in animals.^15,16,17 Some FTIs (eg R115777, BMS-214662, SCH66336 and L-778,123) are presently being evaluated in phase I and II clinical trials in AML and high-risk MDS.^{5,18,19,20,21,22}

RAS mutations in myeloid leukemias predominantly occur in N-RAS and to a lesser extent in K-RAS. Several reports describe decreased efficacy of FTI treatment in cells transformed with K- or N-RAS, in contrast to H-RAS-transformed cells.^23,24,25 Additionally, mutated H-RAS-induced tumors in mouse mammary tumor virus (MMTV)-RAS transgenic mice were found to be more susceptible to FTI treatment vs mutated K- and N-RAS-induced tumors.^26,27,28 This partial resistance may be a consequence of the finding that although all RAS proteins are normally farnesylated in vivo, K- and N-RAS become geranylgeranylated by GGTase I upon FTI treatment.^24,29,30 Geranylgeranylated K- and N-RAS remain associated with the plasma membrane, thus retaining full biologic activity, including transforming ability.^31,32 Limited efficacy of FTI treatment in Nf1-deficient hematopoietic cells suggests that geranylgeranylation of K- and N-RAS in myeloid leukemia cells treated with FTIs may be an important mechanism of resistance.³³ One strategy to overcome FTI resistance is inhibition of both FTase and GGTase I to block RAS processing and thus biologic activity.

Here we report effectiveness of GGTase I inhibitors (GGTIs)^34,35 alone and in combination with FTI on leukemia cell growth, cell cycle progression and induction of apoptosis. GGTI-286, GGTI-298 and GGTI-2147 induced potent, dose-dependent growth inhibition, an accumulation of cells in the G₀/G₁ cell cycle phase and apoptosis in myeloid leukemia cell lines and primary AML cells. In contrast, CAAX-based FTI L-744,832 induced an accumulation of cells in G₀/G₁ or G₂/M. Cotreatment of myeloid leukemia cells with FTI and GGTI resulted in synergistic cytotoxic effects due to increased apoptosis. Cotreatment led to an accumulation of unprocessed N-RAS and inactive N-RAS–RAF complexes. Accumulation of N-RAS protein was not always concomitant with increased N-RAS mRNA levels. Our results suggest that resistance to FTI monotherapy in AML patients may be due to alternative prenylation of N-RAS by GGTase I.

Materials and methods

Cells and antibodies

Cell lines were obtained from the German Collection of Microorganisms and Cell cultures (Braunschweig, Germany).³⁶ Antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) except for diphospho-MEK-1/2 (PP-MEK-1/2) antibodies (New England Biolabs, Frankfurt, Germany). GGTIs were purchased from Calbiochem-Novabiochem (Bad Soden, Germany). FTI L-744,832 was obtained from Biomol Inc. (Plymouth Meeting, PA, USA). Human CD34+ cells were a gift from Cytonet (Hannover, Germany), purified to >98% CD34+ content by magnetic cell sorting (Clini MACS, Miltenyi Biotech, Germany) and cryopreserved in liquid nitrogen.

Primary AML cells

Primary AML cells were obtained from six patients (>18 years) with acute myeloid leukemia (de novo and secondary AML). Diagnosis was based on cell morphology according to FAB criteria complemented by cytochemistry and immunophenotyping. This investigation was approved by the local ethics committee and samples were obtained after informed consent was received. Mononuclear cells were purified by Ficoll-Hypaque gradient centrifugation (Pharmacia LKB, Uppsala, Sweden). Samples contained more than 90% leukemic cells at the time of analysis. Short-term cultures of primary AML cells and purified human CD34+ cells were grown in StemSpan™ serum-free medium (CellSystems Biotech., St Katharinen, Germany) supplemented with cytokines (100 ng/ml rh Flt-3 ligand, 100 ng/ml rh stem cell factor (SCF), 20 ng/ml rh IL-3 and 20 ng/ml rh IL-6).

Cell proliferation assay

Proliferation of primary AML cells was determined using the CellTiter 96^R AQ_ueous one solution reagent (Promega, WI, USA) according to the manufacturer's instructions.

Colony forming assays

Colony forming assays were performed essentially as described.³⁷ Briefly, cells were seeded at 1.0–2.5 10⁵/ml in 96-well plates and treated with inhibitors as indicated. After 4 days, aliquots were plated in 400 l of methylcult H4230 (CellSystems Biotech., St Katharinen, Germany) according to the manufacturer's instructions and incubated for 7–14 days. Primary AML cells and purified CD34+ cells were plated in 400 l MethoCult SF-Bit supplemented with rh SCF, 20 ng/ml rh GM-CSF, 20 ng/ml rh IL-3, 20 ng/ml rh IL-6, 20 ng/ml rh G-CSF and 3 U/ml rh erythropoietin (CellSystems Biotech., St Katharinen, Germany). Aggregates of more than 25 cells were scored as colonies.

Western blot analysis

Cell extracts were prepared and Western blotting was performed as described.^38,39,40 Cellular protein concentrations were determined using the Coomassie dye-binding assay according to Bradford⁴¹ (Bio-Rad Laboratories, Hercules, CA, USA), total cellular protein amounts were adjusted for equal loading in all experiments, separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were probed with specific antibodies and proteins were detected by chemiluminescence (Amersham Biosciences, Little Chalfont, England, UK).

RAS-GTP pulldown assay

RAS-GTP pulldown assays were accomplished as described.^38,42,43,44 The pGEX 2T-RBD construct (encoding a GST fusion protein containing amino acids 51-131 of c-Raf-1) was a gift from J Bos.

Taqman real-time PCR

RNA was isolated from approximately 5 10⁶ cells using the RNeasy Protect Mini-kit (Qiagen, Hilden, Germany). cDNA was prepared from 1 g of RNA with the Omniscript RT Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol, and 1 l of cDNA was used for Taqman real-time PCR. The primers and probe designed for N-RAS with the PRIMER-EXPRESS^® software (Applied Biosystems, Foster City, CA, USA) are: forward primer 5'-GATCCCACCATAGAGGATTCTTACAG-3'; reverse primer 5'-AATACACAGAGGAAGCCTTCGC-3'; and probe 5'-TGCCAT GAGAGACCA ATACATGAGGA-3'. FAM was used as the reporter and TAMRA as the quencher dye. Real-time PCR conditions were as described,⁴⁵ except that the annealing temperature for N-RAS was 59°C. All experiments were carried out in duplicate and several negative controls were included. N-RAS expression relative to GAPDH was calculated by the delta C_T-method.^46,47

Cell cycle analysis

Cell permeabilization (1–2 10⁶ cells) was performed using the GAS-002 kit from Bio Research (Kaumberg, Austria) according to the manufacturer's instructions as described.³⁸ Modfit LT 2.0 software (Verity Software House Inc., Topsham, ME, USA) was used to calculate G₀/G₁, S and G₂M cell cycle phases. Sub-G₀ (debris) fractions were calculated as percentages of total cells.

Detection of apoptosis

The in situ cell death detection kit of Boehringer Mannheim was used to quantify apoptosis.³⁸ The Annexin V-PE/7-Amino-actinomycin (7-AAD) double-staining method was also used to quantitatively determine the percentage of cells actively undergoing apoptosis.³⁸

Analysis of combined drug effects

Synergism, additive effects and antagonism were evaluated by the method of Chou and Talalay⁴⁸ using the CalcuSyn computer program (Biosoft, Cambridge, UK). Fractural survival (f) was calculated by dividing the number of colonies in drug-treated plates by the number of colonies in control plates. Briefly, log ((1/f)-1) was plotted against log(drug dose) to obtain the resulting median effect curves, the X intercept (log IC₅₀) and slope m for each drug alone and for drug combinations. These parameters were then used to calculate doses of individual drugs and the combination required to produce varying levels of cytotoxicity according to the equation Dose_f=Dose_IC50[(1-f)/f]^1/m. For each level of cytotoxicity, the combination index (CI) was calculated according to the equation CI=(D)₁/(D_f)₁+(D)₂/D_f)₂+(D)₁(D)₂/(D_f)₁(D_f)₂, where (D)₁ and (D)₂ are concentrations of the combination required to produce survival f, (D_f)₁ and (D_f)₂ are the concentrations of the individual drugs required to produce f, and =1 or 0 depending on whether the drugs are assumed to be mutually nonexclusive (totally independent modes of action) or mutually exclusive (same or similar modes of action), respectively. Synergy is indicated by CI<1, additivity by CI=1 and antagonism by CI>1.⁴⁸

Results

Effects of GGTase I inhibitors on myeloid leukemia cell growth

Several GGTIs were screened for inhibition of viability and colony formation of myeloid leukemia cell lines. The cell lines tested are shown in Table 1 and included some that express mutationally activated K-RAS, N-RAS or BCR-Abl. Inhibitors employed in this study included (a) the CAAL-based peptidomimetic GGTI-287 and its methyl ester derivative GGTI-286, (b) the CAAL-based peptidomimetic GGTI-297 and its methyl ester derivative GGTI-298, (c) the nonthiol inhibitor GGTI-2133 and its methyl ester derivative GGTI-2147 and (d) CAAX-based FTI L-744,832.

Table 1 - Screening of FTI and GGTI for inhibition of colony formation and cell viability of myeloid leukemia cell lines.

Full table

As it has previously been reported that these compounds cause nonspecific toxicity at concentrations >20 M,⁴⁹ screening for growth inhibition was performed at 20 M. Limited growth inhibitory effect was observed after treatment with 20 M of the nonmethylated GGTIs (GGTI-287, GGTI-297 and GGTI-2133) (Table 1). Growth inhibition greater than 70% was obtained with 20 M of methyl ester derivatives GGTI-286 (9/19), GGTI-298 (14/19) and GGTI-2147 (16/19). Growth inhibition greater than 70% was also observed with the CAAX-based FTI L-744,832 (17/17) (Table 1). At >10–15 M, GGTI-286, GGTI-298 and GGTI-2147 elicited some toxicity toward purified CD34+ human stem cells. DMSO and GGTI-2133 (IC₅₀ 38 M) induced only minor growth inhibitory effects on purified CD34+ cells at concentrations up to 50 M (not shown). FTI L-744,832 caused inhibition of stem cell colony formation with an IC₅₀ value of <1 M. At concentrations below 10 M, GGTI-2147 elicited leukemia-specific cytotoxicity in some cell lines, for example, ML-2 (IC₅₀ 8.2 M), MV4-11 (IC₅₀ 4.2 M), NB-4 (IC₅₀ <1 M) and THP-1 (IC₅₀ <1 M), as colony formation of human CD34+ cells (IC₅₀ 15–20 M) was largely unaffected (Figure 1).

Figure 1.

Inhibition of myeloid leukemia cell and colony growth upon treatment with GGTI-286, GGTI-298, and GGTI-2147. Purified human CD34+ cells and myeloid leukemia cell lines were incubated in liquid suspension cultures with increasing concentrations of GGTI-286, GGTI-298, and GGTI-2147, the CAAX-based farnesyltransferase inhibitor L-744,832 or with DMSO as solvent control. After 4 days, viability of the cells was determined by trypan blue dye assays. Aliquots of the samples were incubated in methylcellulose for an additional 7–14 days in the presence of freshly added inhibitors. The graphs represent the averages of two to eight independent experiments for each cell line. Results are expressed in percentage inhibition and are normalized to solvent (DMSO) controls.

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Effects of GGTIs on cell cycle progression, cell cycle-dependent activation of MEK and induction of apoptosis

NB-4 cells were incubated with 20 M of GGTI-286, GGTI-298, GGTI-2133 and GGTI-2147 (Figure 2). Compared to solvent control (DMSO), 18 h treatment of NB-4 cells with GGTI-286 resulted in a slight increase of the sub-G₀/G₁ fraction (debris), indicating apoptotic DNA fragmentation. Furthermore, GGTI treatment led to an increase in the G₀/G₁ fraction and PP-MEK-1/2⁺ cells in G₀/G₁ and a reduction in the G₂/M fraction and PP-MEK-1/2⁺ cells in G₂/M (Figure 2a). Similar results were obtained for GGTI-298, GGTI-2133 and GGTI-2147 demonstrating that treatment with GGTIs leads to an increase of cells in G₀/G₁ with an initial accumulation of PP-MEK-1/2⁺ cells in G₀/G₁ (Figure 2a).

Figure 2.

Effects of GGTI treatment on cell cycle progression and the cell cycle-dependent activation of MEK-1/2 in myeloid leukemia cells. NB-4 cells were incubated in the absence or the presence of 20 M GGTI-286, GGTI-298, GGTI-2133 or GGTI-2147. After 18 h (a) and 36 h (b), double staining with propidium iodide and an antibody specific for activated PP-MEK-1/2 was performed and analyzed as described in Materials and methods. Representative cell cycle profiles (right) and FACS profiles of the double staining (left) are shown. (a) Untreated NB-4 cells; (b), solvent-treated (DMSO) NB-4 cells; (c) NB-4 cells treated with 20 M GGTI-286; (d) NB-4 cells treated with 20 M GGTI-298; (e) NB-4 cells treated with 20 M GGTI-2133; (f) NB-4 cells treated with 20 M GGTI-2147. Note: According to ModFit, cell cycle fractions (G₀/G₁, S and G₂M phase) are shown in percent of viable cells. Sub-G₀ fractions are expressed as percent debris of total cell counts.

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GGTI-286 treatment for 36 h resulted in an increase of cellular debris, an increase in the G₀/G₁ fraction, and a substantial decrease in PP-MEK-1/2⁺ cells in G₀/G₁ and G₂/M as compared to solvent control (Figure 2b). Similar effects were observed after 36 h incubation with GGTI-298, GGTI-2133 and GGTI-2147 (Figure 2b). However, the extent of GGTI-induced G₀/G₁ accumulation varied between the different GGTIs.

Apoptosis was measured by the TUNEL assay and by an Annexin V-PE/7-amino-actinomycin (7-AAD) assay. In contrast to solvent control (DMSO), GGTI-286 treatment for 48 h resulted in apoptotic DNA fragmentation (67.7%) and exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Similar results were obtained with GGTI-298, GGTI-2133 and GGTI-2147 (Figure 3).

Figure 3.

GGTI-induced apoptosis in myeloid leukemia cells. NB-4 cells were incubated with or without 20 M GGTI-286, GGTI-298, GGTI-2133 or GGTI-2147. After 45 h, cells were harvested, and labeling of DNA strand breaks was performed applying the TUNEL method as described in Materials and methods (left). M1, no DNA fragmentation; M2, DNA fragmentation. Exposure of phosphatidylserine on the outer leaflet of the plasma membrane was detected using an Annexin V-PE/7-AAD double-staining method as described (right). Apoptotic exposure of phosphatidylserine is shown in the upper left (early apoptosis) and upper right squares (late apoptosis). Results are given in percentage of total cell population. (a) Untreated NB-4 cells; (b) solvent control (DMSO); (c) GGTI-286; (d) GGTI-298; (e) GGTI-2133; (f) GGTI-2147.

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Effects of FTI/GGTI cotreatment

In contrast to GGTI treatment, FTI L-744,832 treatment for 36 h resulted in a G₂/M accumulation and an increase of PP-MEK-1/2⁺ cells in G₂/M, but a decrease in G₀/G₁ (Figure 4a). Similar effects were recently observed with CAAX-based FTI-277.³⁸ Cotreatment with 10 M GGTI-286 and 10 M FTI L-744,832 led to alterations in G₀/G₁ and G₂/M and increased cellular debris (sub-G₀/G₁ fraction) (Figure 4a). Co-treatment of NB-4 cells with GGTI-286 also reduced the FTI-induced accumulation of PP-MEK-1/2⁺ cells in G₂/M, suggesting more efficient inhibition of RAS prenylation (Figure 4a).

Figure 4.

Effects of GGTI-286 and FTI L-744,832 cotreatment on cell cycle progression, cell cycle-dependent activation of MEK-1/2 and induction of apoptosis in myeloid leukemia cells. NB-4 cells were incubated with 20 M GGTI-286, 20 M FTI L-744,832 or a combination of 10 M GGTI-286 and 10 M L-744,832. After 36 h, double staining with propidium iodide and an antibody specific for activated PP-MEK-1/2 was performed as described in Materials and methods. Representative cell cycle profiles and FACS profiles of the double staining are shown (a). After 45 h, cells were harvested and labeling of DNA strand breaks was performed applying the TUNEL method as described in Materials and methods (b). M1, no DNA fragmentation; M2, DNA fragmentation. Exposure of phosphatidylserine on the outer leaflet of the plasma membrane was detected using an Annexin V-PE/7-AAD double-staining method as described in Materials and methods. Apoptotic exposure of phosphatidylserine is shown in the upper left (early apoptosis) and upper right squares (late apoptosis). (a) Untreated NB-4 cells; (b) solvent control (DMSO); (c) 20 M GGTI-286; (d) 20 M FTI L-744,832; (e), 10 M GGTI-286 and 10 M L-744,832. According to ModFit, cell cycle fractions (G₀/G₁, S and G₂M phase) are shown as percent of viable cells. Sub-G₀ fractions are expressed as percent debris of total cell counts.

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To overcome resistance caused by geranylgeranylation of K-RAS and N-RAS during FTI treatment, myeloid cell lines were exposed to a fixed ratio (1:1) of FTI L-744,832 and GGTI-286. Cotreatment of NB-4 cells with 10 M GGTI-286 and 10 M FTI L-744,832 induced more apoptosis than treatment with either 20 M GGTI-286 or 20 M FTI L-744,832 alone (Figure 4b).

In agreement with these results, FTI/GGTI cotreatment of HL-60 and other myeloid cell lines resulted in stronger inhibition of colony growth than treatment with FTIs or GGTIs alone (Figure 5). The data were analyzed by the median effect method⁴⁸ to determine whether inhibitory effects were additive, synergistic or antagonistic. For the combination of FTI L-744,832 and GGTI-286, the CI calculated under the assumption that the drugs were mutually exclusive was <1 over much of the range examined, with mean CI values of 0.2720.179 at the IC₅₀, 0.1720.179 at the IC₇₀ and 0.0830.093 at the IC₉₀ (n=4) (Figure 5). Effects of combining FTI L-744,832 with GGTI-286 were also examined in other myeloid cell lines (K562, MUTZ-2, AML-OCI5, M-07e) (Figure 5). The CI values consistently dropped below 1 over much of the range examined (Figure 5), demonstrating synergistic cytotoxic effects.

Figure 5.

Treatment of myeloid leukemia cells with a combination of FTI L-744,832 and GGTI-286 leads to synergistic growth inhibition. Myeloid leukemia cell lines were incubated in liquid suspension cultures with increasing concentrations of FTI L-744,832, GGTI-286 or with a fixed 1:1 ratio of L-744,832 and GGTI-286. After 4 days, viability of the cells was determined by trypan blue dye exclusion assays. Aliquots of the samples were incubated in methylcellulose for an additional 7–14 days in the presence of freshly added inhibitors. Colony formation was plotted relative to solvent-treated (DMSO) control cells. (a) Colony formation (=fraction affected) of HL-60 cells in the presence of increasing concentrations of FTI L-744,832, GGTI-286 or a fixed 1:1 ratio of both inhibitors; (b) median effect plot of data in (a); (c) plot of combination index (CI) vs cytotoxicity calculated from data in (b) under the assumption that agents are mutually exclusive; (d) isobolograms for data in (a); (e) plot of CI calculated from data obtained for HL-60, MUTZ-2, K562, OCI-AML5 and M-07e cells under the assumption that agents are mutually exclusive or nonexclusive (f). Each panel of results was accomplished at least three times.

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To demonstrate synergistic cytotoxicity of FTI/GGTI combination over a wide range of concentrations, THP-1 cells were incubated with increasing concentrations of GGTI-286 and FTI L-744,832. GGTI-286 greatly enhanced the FTI-induced inhibition of THP-1 cell growth in a concentration-dependent manner (Figure 6). Surprisingly, GGTI-286 led to increased proliferation of THP-1 cells when used alone at 0.05–5 M in MTT assays; however, this proliferative advantage was not observed in colony forming assays (Figure 1).

Figure 6.

Synergistic growth inhibition of THP-1 cells upon treatment with combinations of FTI L-744,832 and GGTI-286. THP-1 cells were incubated in liquid suspension cultures with increasing concentrations of FTI L-744,832 alone and in combination with five different concentrations of GGTI-286. After 3 days, viability of the cells was determined using a cell proliferation assay as described in Materials and methods. Cell proliferation is given as percent of cell proliferation normalized to solvent control (DMSO). THP-1 cells harbor an activating N-RAS mutation. GGTI-286 monotherapy led to increased proliferation, but even low concentrations greatly enhanced FTI-induced growth inhibition.

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Effects of FTI L-744,832 and GGTI-286 on RAS prenylation

Expression of all three RAS isoforms (H-, K- and N-RAS) was detected in myeloid cell lysates by Western blotting. In contrast to treatment with GGTI-286 alone (20 M), treatment with FTI L-744,832 alone (20 M) or in combination (10 M each) with GGTI-286 resulted in an electrophoretic mobility shift corresponding to unprocessed H-RAS, indicating FTI-induced inhibition of post-translational prenylation (Figure 7a). Compared to the mobility shifts observed for H-RAS and N-RAS, electrophoretic mobility shifts of K-RAS2B were not consistently observed in all experiments (Figure 7b). K-RAS2A was not detected using an antibody specific for this splice variant (not shown). Interestingly, a strong accumulation of unprocessed N-RAS was observed in cells treated with L-744,832. Furthermore, FTI/GGTI cotreatment led to a substantial increase in unprocessed N-RAS, indicating potent inhibition of N-RAS geranylgeranylation in the presence of FTI by GGTI cotreatment (Figure 7c). Similar results were also observed with Mo7e, MV4-11 and Kasumi-1 cells (not shown).

Figure 7.

SDS-PAGE analyses of H-, K- and N-RAS prenylation in myeloid leukemia cell lines treated with FTI, GGTI or FTI/GGTI combination. (a–c) K-562, THP-1 and NB-4 cells were incubated 24 h alone (lane 1), with DMSO solvent control (lane 2), 20 M, 20  FTI L-744,832 (lane 3), M GGTI-286 (lane 4) or a combination of 10 M FTI L-744,832 and 10 M GGTI-286 (lane 5). Cell lysates were probed for (a) H-RAS, (b) K-RAS2B or (c) N-RAS. (d) Affinity precipitation of unprocessed, activated N-RAS with GST-RBD. NB-4 and THP-1 cells were incubated for 24 h alone (lane 1), with DMSO solvent control (lane 2), 20 M, GGTI-286 (lane 3), FTI L-744,832 (lane 4), or a combination of 10 M FTI L-744,832 and 10 M GGTI-286 (lane 5). Cellular lysates were subjected to affinity precipitation (AP) with GST-RBD as described in "Materials and methods". N-RAS proteins were detected by immunoblotting with N-RAS-specific antibodies. Unprocessed (U) and processed (P) RAS proteins are indicated. Total cellular protein amounts were measured and adjusted for equal loading as described in Materials and methods. All experiments were repeated at least twice and representative immunoblots are shown. H- and K-RAS were not observed to bind GST-RBD under these experimental conditions. No RAS proteins were found to bind to the GST protein when it was not linked to the minimal RAS-binding domain (RBD).

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To identify the presence of GTP-RAS, cell lysates were incubated with the minimal RAS-binding domain (RBD) of C-Raf-1.^42,43,44 Western blotting with antibodies against H-, K- and N-RAS was performed to detect binding of GTP-RAS with GST-RBD. RAS proteins were not observed to bind to GST alone (not shown). While high levels of activated N-RAS were found in NB-4 and THP-1 cell lysates (Figure 7d), activated H- and K-RAS were not detected in these assays (not shown). FTI and FTI/GGTI cotreatment resulted in binding of unprocessed N-RAS to GST-RBD, suggesting that unprocessed N-RAS is capable of forming inactive N-RAS–RAF complexes when GTP-loaded.

To determine if accumulation of N-RAS protein levels observed in myeloid leukemia cell lines upon treatment with FTI alone and in combination with GGTI was linked to FTI-induced alterations in N-RAS gene expression, N-RAS mRNA levels were evaluated in treated and untreated cells by Taqman real-time PCR. While treatment with FTI L-744,832 caused a slight increase in N-RAS mRNA in NB-4 and K562 cells, a decrease was observed in THP-1 cells (1.9- and 2.2-fold increase vs a 2.3-fold reduction, respectively). Treatment with GGTI-286 caused a 9.5-fold increase in NB-4 cells, but only modest changes in N-RAS mRNA in K562 and THP-1 cells (1.8- and 1.3-fold, respectively). FTI/GGTI cotreatment resulted in a 4.2-fold increase of N-RAS mRNA in K562 cells, a 1.4-fold increase in NB-4 cells and a 7.0-fold decrease in THP-1 cells. These changes in N-RAS mRNA levels did not always correlate with N-RAS protein levels.

As FTIs induce multiple effects on cancer cells, it has been suggested that multiple farnesylated proteins may be important in mediating FTI-induced effects.⁵⁰ To evaluate the inhibition of processing of other FTase substrates in myeloid leukemia cells by FTI treatment, we analyzed several farnesylated proteins known to undergo mobility shifts upon FTase inhibition.⁵¹ In most experiments, mobility shifts of the H-RAS-related G-proteins Rap2A and Rheb were observed (Figure 8a and b). While FTI-induced mobility shifts have recently been reported for Lamins A and B as well as RhoB,^50,51,52 no mobility shifts of these proteins were observed in myeloid cells treated with FTI L-744,832 alone or in combination with GGTIs (Figure 8c, not shown). However, FTI treatment resulted in an accumulation of Lamin B, which might be due to cell cycle-dependent or FTI-induced expression. Additionally, CENP-E and CENP-F, which function as centromere-associated kinesin motors and play critical roles in mitosis, were not detected in myeloid cell lysates by Western blotting with commercially available antibodies (not shown).

Figure 8.

SDS-PAGE analyses of Rap2A, Rheb and Lamin B in myeloid leukemia cell lines treated with FTI, GGTI or an FTI/GGTI combination. K-562, THP-1 and NB-4 cells were incubated for 24 h alone (lane 1), with DMSO solvent control (lane 2), 20 M FTI L-744,832 (lane 3), 20 M GGTI-286 (lane 4) or a combination of 10 M FTI L-744,832 and 10 M GGTI-286 (lane 5). Cell lysates were probed for Rap2A (a), Rheb (b), or Lamin-B (c). Unprocessed (U) and processed (P) proteins are indicated. Total cellular protein amounts were measured and adjusted for equal loading as described in Materials and methods.

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Effect of FTI/GGTI cotreatment on primary AML cells

To assess the potential clinical relevance of our findings, the effects of FTI and GGTI treatment on primary AML cells were analyzed. Treatment with FTI L-744,832 or GGTIs alone induced apoptosis in a fraction of cells (Table 2). In agreement with the results using myeloid cell lines, GGTI-2147 was found to be more effective than GGTI-286 or GGTI-298 in inducing apoptosis and inhibiting AML cell growth (Table 2). As shown in Table 2 and Figure 9, FTI/GGTI cotreatment was found to be even more effective in inhibiting cell proliferation and inducing apoptosis than treatment with FTI or GGTI alone. FACS analysis of primary AML blasts cultured in RPMI containing 20% FCS revealed a large portion of cells in G₀/G₁ and very low amounts of PP-MEK⁺ cells in the G₀/G₁ and G₂/M phases. Proliferation of primary AML cells was significantly increased by culturing in StemSpan^TM containing cytokines (SCF, IL-3, IL-6 and Flt-3 ligand) (not shown). GGTI treatment primarily resulted in an increase of cells in G₀/G₁, whereas FTI treatment caused increases of either G₀/G₁ or G₂/M in primary AML cells, suggesting that FTI-induced increase in G₂/M was dependent on AML cell type and/or proliferation. To demonstrate synergism of FTI/GGTI cotreatment in primary AML cells, AML blasts from five AML patients were titrated with increasing concentrations of FTI L-744,832, the most effective GGTase I inhibitor (GGTI-2147) and a fixed ratio (1:1) of both inhibitors. After 72 h inhibitor treatment, cell viability was measured. As shown in Figure 10, the CI values for the FTI/GGTI combination were consistently below 1 over much of the range examined, indicating synergism in all AML cases investigated. Response of primary AML blasts to treatment with FTIs and GGTIs was independent of the presence of N-RAS mutations.

Figure 9.

FTI/GGTI cotreatment leads to induction of apoptosis in primary AML cells. Primary AML cells from patient #5, which have an N-RAS-61 mutation, were incubated with 20 M FTI L-744,832, 20 M GGTIs, or a combination of 10 M L-744,832 and 10 M GGTIs. After 42–45 h, cells were harvested and DNA strand breaks were labeled by the TUNEL method as described in Materials and methods. M1, no DNA fragmentation; M2, DNA fragmentation. Results are given in percentage of total cell population. (a) Solvent-treated (DMSO); (b) 20 M FTI L-744,832; (c) 20 M GGTI-286; (d) 10 M GGTI-286 and 10 M L-744,832; (e) 20 M GGTI-298; (f) 10 M GGTI-298 and 10 M L-744,832; (g) 20 M GGTI-2147; (h) 10 M GGTI-2147 and 10 M FTI L-744,832.

Full figure and legend (87K)

Figure 10.

Treatment of primary AML cells with a combination of GGTI-2147 and FTI L-744,832 leads to synergistic growth inhibition. AML cells were incubated in liquid suspension cultures (StemSpan^TM) with increasing concentrations of GGTI-2147, FTI L-744,832 or with a fixed 1:1 ratio of GGTI-2147 and L-744,832. After 72 h, viability of the cells was determined by trypan blue dye exclusion and MTS proliferation assays. (a) Viability (=fraction affected) of AML cells treated with GGTI-2147, FTI L-744,832 or a fixed 1:1 ratio of both inhibitors; (b) median effect plot of data in (a); (c) plot of combination index (CI) vs cytotoxicity calculated from data in (b) under the assumption that agents are mutually exclusive; (d) isobolograms for data in (a); (e) plot of CI calculated from data obtained for leukemic blasts from different AML patients (1, 2, 3, 5, 6) under the assumption that agents are mutually exclusive or nonexclusive (f). AML blasts from patients 1, 4, 5 and 6 were found to harbor N-RAS mutations (CAA CGA resulting in Q61R, GGT GAT resulting in G12D, CAA CAC resulting in Q61 H and GGT GAT resulting in G12D, respectively). Note: CI values for patient 4 could not be calculated because FTI L-744,832 had no effect on blast viability. However, FTI/GGTI cotreatment was more effective than GGTI alone.

Full figure and legend (180K)

Table 2 - Effect of FTI and GGTI treatment on cell cycle progression and induction of apoptosis of primary AML cells.

Full table

Discussion

RAS deregulation plays an important role in the molecular pathogenesis of myeloid leukemias and has considerable potential implications for therapeutic approaches that target RAS signaling pathways.^{5,15,16,17,18,19,20,21,22} At least four FTIs have recently entered clinical trials (SCH66336, R115777, L-778,123, BMS-214662).^5,18,22 It has been speculated that geranylgeranylation of K-RAS and N-RAS in the presence of FTIs might represent a possible mechanism of FTI resistance.^33,34 Alternative geranylgeranylation of other FTase substrates in the presence of FTIs has not been identified. As the majority of RAS mutations in AML occur in K- and N-RAS, this mechanism of FTI resistance may have therapeutic consequences. The present study addresses this hypothesis by investigating effects of GGTIs alone and in combination with FTI on myeloid leukemia cell growth, cell cycle progression, induction of apoptosis, RAS processing and signaling. Treatment of myeloid leukemia cells with GGTI-286, GGTI-298 and GGTI-2147 caused inhibition of colony formation, a G₀/G₁ enrichment, induction of apoptosis and a decrease in the cell cycle-dependent activation of MEK. While no correlation between susceptibility toward these inhibitors and RAS status (eg mutation or activation) was observed, growth factor-dependent cell lines (Mutz-2, Mutz-3, OCI-AML5, M-07e) were more resistant to GGTI treatment.

Treatment of myeloid cell lines with FTI L-744,832 resulted in strong G₂/M accumulations and a subsequent increase in PP-MEK-1/2⁺ cells in G₂/M. In contrast to NB-4 cells, FTI treatment induced only modest increases of cells in G₀/G₁ or G₂/M in primary AML cells, which is most likely due to the lack of cell cycle progression in the primary cells. In agreement with these results, others have demonstrated a G₀/G₁ block in cells treated with GGTIs and G₀/G₁ or G₂/M blocks after treatment with CAAX-based FTIs, depending on cell type.^{38,53,54,55,56}

While FTI L-744,832 potently inhibited proliferation and induced apoptosis in myeloid leukemia cells, it also displayed cytotoxicity toward purified human CD34+ cells. Recently, we reported similar cytotoxicity of CAAX-based FTI-277 toward CD34+ cells, suggesting that myelotoxicity may be a side effect of treatment with some FTIs.³⁸ While FTI-mediated myelotoxicities have not been reported in recent animal studies with this inhibitor,^{23,26,27,28,34,57,58} mild myelosuppression, neutropenia and anemia have been observed in phase I clinical trials of another FTI, R115777.^19,59

FTI/GGTI cotreatment of myeloid cells caused synergistic cytotoxicity, which correlated with increased accumulation of unprocessed N-RAS but not always with increased N-RAS mRNA levels. FTI-induced inhibition of H-RAS processing was not augmented by coaddition of GGTIs, which underscores the fact that H-RAS, in contrast to K- and N-RAS, is solely farnesylated.^24,29,30,32 Resistance of K-RAS processing to inhibition by FTIs may be due to the higher binding affinity of K-RAS to FTase.^25,32 Others have reported that inhibition of oncogenic K-RAS4B processing required FTI-277 concentrations 100-fold higher than those needed for H-RAS inhibition.⁶⁰ In RAS–RAF binding assays, unprocessed N-RAS bound to the minimal RAS-binding domain of RAF (GST-RBD) (Figure 7). This observation is in agreement with earlier reports describing RAF binding to nonprenylated, GTP-loaded RAS that was produced from bacteria.^40,61,62 Accumulation and RAF binding of unprocessed N-RAS in cells treated with FTIs alone or in combination with GGTIs suggest dominant negative effects of this biologically inactive version of N-RAS. Cytosolic accumulation of inactive RAS–RAF complexes and inhibition of RAS-induced constitutive activation of MAPK have been observed in FTI-277-treated cells overexpressing oncogenic H-RAS.⁶³

The synergistic toxicity of FTI/GGTI cotreatment in myeloid leukemia cells is in agreement with recent reports describing similar synergistic efficiency of FTI/GGTI combination in adrenocortical and human colon cancer cells containing mutant K-RAS.^64,65 In a nude mouse xenograft model, both FTI and GGTI are required to inhibit prenylation of oncogenic K-RAS, but each alone is sufficient to suppress human tumor growth.^34,35 Furthermore, FTI/GGTI cotreatment and treatment with a dual prenylation inhibitor (DPI) with both FTI and GGTI activities, resulted in higher levels of apoptosis in K-RAS-transformed cells relative to FTI and GGTI alone.⁶⁶ While the GGTIs described by Sun et al³⁴ and used in our study were nontoxic in mice, the chemically distinct GGTIs and DPIs used by Lobell et al⁶⁶ revealed strong toxicity in mice, which may be caused by an activity/effect unrelated to GGTase-I inhibitory activity.

In addition to K- and N-RAS, prenylation of other farnesylated and geranylgeranylated proteins may be inhibited by FTI/GGTI cotreatment and contribute to increased apoptosis. As others have suggested that RAS may not be the therapeutically relevant loci of FTI treatment,^50,51,59 effects of FTIs and GGTIs on processing of other FTase substrates were investigated. Our results demonstrate that FTI treatment inhibits processing of the G-proteins Rap2A and Rheb in myeloid leukemia cells. While the biological function of these G-proteins is currently under investigation, it has recently been shown that failure to farnesylate Rheb proteins contributes to enrichment of G₀/G₁ phase cells in S. pombe.⁶⁷

Work of several laboratories previously demonstrated that Lamin B and Prelamin/Lamin A are farnesylated proteins^68,69 and that FTI treatment causes electrophoretic mobility shifts of Lamins A and B.^21,51,70,71 Our immunoblotting studies failed to demonstrate a mobility shift of Lamin B, but elevated protein levels of Lamin B were observed in cells treated with FTI alone and in combination with GGTI.

RhoB, an endosomal Rho protein that functions in receptor trafficking and which has been shown to be both farnesylated (RhoB-F) and geranylgeranylated (RhoB-GG), has been proposed as a key target of FTIs.⁵⁹ FTI treatment of cells overexpressing epitope-tagged RhoB shifts the prenylation status exclusively to RhoB-GG, which induces apoptotic and antineoplastic responses.^59,72,73,74 However, induction of apoptosis by overexpression of either RhoB-F or RhoB-GG in Panc-1 cells suggests that both function similarly and argues against distinct roles for RhoB-F and RhoB-GG.⁷⁵ Electrophoretic mobility of RhoB was not altered by FTI, GGTI or FTI/GGTI cotreatment in our studies, arguing against RhoB involvement.

In conclusion, our results suggest that synergistic cytotoxic effects of FTI/GGTI combination are at least in part, due to increased inhibition of N-RAS prenylation. Regardless of the locus of action, our results demonstrate profound in vitro inhibitory effects of FTIs and GGTIs on myeloid leukemia cell growth, irrespective of RAS mutations, BCR-Abl expression or MAPK activation. Accumulation of unprocessed N-RAS after FTI/GGTI cotreatment of myeloid leukemia cells suggests that alternative geranylgeranylation of N-RAS in the presence of FTI is a mechanism of resistance to FTI monotherapy. However, further studies are needed to evaluate and establish the potential clinical benefits of combining FTIs with GGTIs in myeloid leukemia treatment to overcome the partial resistance of K- and N-RAS to FTI monotherapy.

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Acknowledgements

We thank Natalja Möbius for excellent technical assistance and Dr Kristine A Henningfeld and Dr Thomas Winkler for valuable discussions. We thank Dr Jürgen Krauter and Kerstin Görlich for help with the TaqMan experiments.