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Molecular and Cellular Biology, October 2008, p. 6496-6509, Vol. 28, No. 20
0270-7306/08/$08.00+0     doi:10.1128/MCB.00477-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Inhibition of Polysome Assembly Enhances Imatinib Activity against Chronic Myelogenous Leukemia and Overcomes Imatinib Resistance{triangledown}

Min Zhang,1 Wuxia Fu,1 Sharmila Prabhu,1 James C. Moore,1 Je Ko,1 Jung Woo Kim,1,{dagger} Brian J. Druker,2 Valerie Trapp,1 John Fruehauf,1 Hermann Gram,3 Hung Y. Fan,1,4,5 and S. Tiong Ong1,5*

Division of Hematology/Oncology, Department of Medicine, University of California at Irvine, Irvine, California,1 Howard Hughes Medical Institute, Oregon Health and Science University Cancer Institute, Portland, Oregon 97239,2 Novartis Institutes for Biomedical Research, Basel, Switzerland,3 Department of Molecular Biology and Biochemistry, School of Biological Sciences,4 Cancer Research Institute, University of California at Irvine, Irvine, California 926975

Received 22 March 2008/ Returned for modification 2 May 2008/ Accepted 28 July 2008


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ABSTRACT
 
Dysregulated mRNA translation is implicated in the pathogenesis of many human cancers including chronic myelogenous leukemia (CML). Because our prior work has specifically implicated translation initiation in CML, we tested compounds that could modulate translation initiation and polysomal mRNA assembly. Here, we evaluated the activity of one such compound, CGP57380, against CML cells and explored its mechanisms of action. First, using polysomal mRNA profiles, we found that imatinib and CGP57380 could independently, and cooperatively, impair polysomal mRNA loading. Imatinib and CGP57380 also synergistically inhibited the growth of Ba/F3-Bcr-Abl and K562 cells via impaired cell cycle entry and increased apoptosis. Mechanistically, CGP57380 inhibited efficient polysomal assembly via two processes. First, it enhanced imatinib-mediated inhibition of eukaryotic initiation factor 4F induction, and second, it independently impaired phosphorylation of ribosomal protein S6 on the preinitiation complex. We also identified multiple substrates of the mTOR, Rsk, and Mnk kinases as targets of CGP57380. Finally, we found a novel negative-feedback loop to the mitogen-activated protein kinase/Mnk pathway that is triggered by CGP57380 and demonstrated that an interruption of the loop further increased the activity of the combination against imatinib-sensitive and -resistant CML cells. Together, this work supports the inhibition of translation initiation as a therapeutic strategy for treating cancers fueled by dysregulated translation.


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INTRODUCTION
 
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the presence of the Philadelphia chromosome (26), which gives rise to the t(9;22) translocation, and the resulting fusion oncogene Bcr-Abl. The constitutive tyrosine kinase activity of Bcr-Abl is required for the transforming properties of Bcr-Abl, and the inhibition of Bcr-Abl kinase by imatinib mesylate (imatinib) has resulted in durable responses in early-stage CML (32). However, patients with late-stage disease respond much less well to imatinib therapy and remain at high risk for developing drug resistance (8, 31) and for death from progressive disease (19, 30). These observations make a strong case for the use of non-cross-resistant drug combinations to combat late-stage disease. There is, thus, a pressing need to understand other pathways and cellular processes that are dysregulated by Bcr-Abl, particularly those that represent "drugable" targets.

Because prior work from our group has shown that Bcr-Abl modulates the activity of several molecules which regulate the initiation of cap-dependent mRNA translation (14, 23), we turned our attention to the therapeutic potential of targeting this process in CML. Specifically, we were interested in small molecules that might interfere with translation initiation and with the efficient assembly of polysomal mRNA, since both processes have been implicated in several cancer types (15), in addition to CML (22).

In mammalian cells, initiation is both the rate-limiting step for the process of mRNA translation and the convergence point for signaling pathways that convey the message from extracellular stimuli to a distal set of proteins that regulate this process (24). In healthy cells, a critical control point is represented by the amount of eukaryotic initiation factor 4E (eIF4E) available to form the cap-binding complex eIF4F. eIF4F consists of (i) eIF4E, which binds the cap structure present at the 5' end of mRNAs; (ii) eIF4G, a scaffolding protein; and (iii) eIF4A, an RNA-dependent ATPase and RNA helicase. Formation of eIF4F then allows recruitment of the translational machinery to mRNA and includes assembly of the preinitiation complex (PIC) that comprises eIF3, 40S ribosomal subunits, and the ternary complex (eIF2/Met-tRNA/GTP) (16). The availability of eIF4E is determined primarily by the phosphorylation status of 4E-BP1, a negative regulator of eIF4E. 4E-BP1 is phosphorylated by the serine/threonine kinase mTOR when the latter forms a complex with raptor that is called mTORC1, which is in turn activated by phosphatidylinositol 3-kinase (PI3K)/Akt signaling. In addition to regulation by the PI3K/Akt/mTOR pathway, mitogen-activated protein kinase (MAPK) signaling also has recently been described as a regulator of the efficiency of polysomal mRNA assembly by activation of extracellular-regulated kinase (ERK)-mediated activation of Rsk. This activation is thought to occur via Rsk-dependent phosphorylation of ribosomal protein S6 (rpS6) at Ser235/236 (25), which in turns facilitates recruitment of rpS6 to the PIC and the efficient assembly of polysomal mRNA. Thus, at least two signaling pathways that are essential for Bcr-Abl-mediated transformation, MAPK and PI3K/Akt (6), share a final common path to proteins involved in the regulation of translation initiation.

It was therefore of interest to determine if pharmacologic interruption of translation initiation and polysomal mRNA assembly might provide evidence to support the hypothesis that these processes contribute to the transformation by Bcr-Abl. Accordingly, we set out to test a novel small molecule, CGP57380, which had been previously found to impair efficient polysome assembly (18) in CML cells and to determine its mechanism of action. First, we found that imatinib and CGP57380 impaired translation initiation via distinct mechanisms and that this impairment resulted in synergistic activity against CML cells. Next, we uncovered putative targets of CGP57380 and describe a novel negative-feedback loop to MEK/ERK that is triggered by CGP57380 exposure. Finally, we show that pharmacologic interruption of the MEK/ERK feedback loop further increases the activity of imatinib and CGP57380 against imatinib-sensitive and -resistant CML cells. Because dysregulated mRNA translation has been implicated in the pathogenesis of several other cancer types (15), our results also have wider application to the therapy of cancer in general.


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MATERIALS AND METHODS
 
Cell culture, cell lines, and patient samples. Parental Ba/F3 cells and Ba/F3 cells stably transfected with wild-type p210 (Ba/F3-Bcr-Abl) and imatinib-resistant mutants have been previously described (12). K562 cells were obtained from ATCC. Peripheral blood (PB) and bone marrow (BM) samples were obtained with appropriate consent and institutional review board approval from patients at the University of California at Irvine. Primary cells were grown at 37°C in serum-free medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with growth factors at concentrations found in stroma-conditioned medium (5). CGP57380 was obtained from Novartis (Basel, Switzerland) (11). LY294002 and rapamycin were commercially obtained (Sigma, St. Louis, MO). BI-D1870 was purchased from the Division of Signal Transduction Therapy at the University of Dundee.

Polysome analysis. Ba/F3-Bcr-Abl or K562 cells (2 x 107) were treated with dimethyl sulfoxide (DMSO) or drug for 4 h. Cycloheximide was added to a final concentration of 0.1 mg/ml for 5 min before collection. Cells were then lysed in extraction buffer (15 mM Tris-HCl [pH 7.4], 15 mM MgCl2, 200 mM NaCl, 0.5 mg/ml heparin, 0.1 mg/ml cycloheximide, 1% Triton X-100, 40 U/µl RNasin [Invitrogen], and 5 mM dithiothreitol). Extracts were fractionated on a 10% to 50% sucrose gradient composed of extraction buffer lacking Triton X-100, RNasin, and dithiothreitol and centrifuged at 35,000 rpm for 150 min in a SW41 model rotor at 4°C. The polysome profile was visualized with an ISCO density gradient fraction system (Lincoln, NE). Areas under the curves corresponding to polysomes and monosomes were calculated using ImageJ software (http://rsb.info.nih.gov/ij/index.html).

Tetrazolium-based proliferation assays. Cells (5 x 103) were plated in triplicate with DMSO or inhibitors in a 100-ml volume in a 96-well plate at 37°C. After 48 or 72 h, CellTiter 96 AQueous One solution (Promega, Madison, WI) was added to each well and processed per the manufacturer's instructions. The optical density for each condition was calculated as a percentage of the untreated control after subtracting for background absorbance. To assess drug synergy, a dose-response analysis was performed according to the method of Chou, using CalcuSyn software (version 1.1; Biosoft, Ferguson, MO) (4).

Flow cytometric analysis of cell cycle and apoptosis. Cells (1 x 106) were incubated with DMSO or inhibitors for 48 h and then washed and fixed with ice-cold 80% ethanol. After 1 h, the cells were washed twice in phosphate-buffered saline and stained with 20 mg/ml propidium iodide (PI) in phosphate-buffered saline with 50 mg/ml RNase A, 10% Na citrate, 10% NP-40 at room temperature for 30 min. Cells were analyzed by flow cytometry, and cell cycle analysis was performed using FlowJo software. For apoptosis assays, 1 x 106 cells were incubated with DMSO or inhibitors for 48 h, stained with annexin V-fluorescein isothiocyanate (BD PharMingen, Carlsbad, CA) and PI per the manufacturer's instructions and then analyzed by flow cytometry.

Immunoblot and antibodies. Cells (4 x 106) were incubated with an equivalent volume of DMSO or inhibitor and lysed in Laemmli buffer with protease and phosphatase inhibitors. Lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The following antibodies were used: 4E-BP1, β-actin (Sigma), cyclin D1, cyclin D2 (catalog no. M-20; Santa Cruz Biotechnology, Santa Cruz, CA), cyclin D3 (catalog no. D-7; Santa Cruz Biotechnology), eIF4G (catalog no. N-20; Santa Cruz Biotechnology), activated caspase-3, Bcl-XL, c-Abl, and CDK4. In addition, antibodies to the phosphorylated and total forms of the following proteins were also employed: Mnk1 (Santa Cruz Biotechnology), ERK, eIF4E, S6K1, and rpS6. Antibodies were from Cell Signaling Technology (Beverly, MA) unless otherwise stated. Immunoblotting was performed according to the manufacturers’ instructions. Immunoreactive bands were visualized by chemiluminescence (Pierce, Rockford, IL) using a FluorChem SP imaging system (Alpha Innotech, San Leandro, CA).

CD34+ cell selection. PB or BM mononuclear cells were isolated by Ficoll-Hypaque (Sigma, St. Louis, MO) density gradient centrifugation. CD34+ cells were then selected by immunomagnetic bead-based column separation according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA), and a purity value of >95% was confirmed by flow cytometry.

Cap-binding assay. The cap-binding assay was performed as previously described (1, 23). Briefly, after a 4-h incubation with DMSO or inhibitors, cells were lysed by three freeze-thaw cycles, and lysates were incubated with a suspension of 7-methyl-GTP-Sepharose beads (Amersham, Piscataway, NJ). After 1 h, the beads were pelleted and washed three times with GTP-containing buffer, and cap-bound proteins were eluted with buffer containing 7-methyl-GTP. Cap-bound material was then subjected to SDS-PAGE and visualized using immunoblots.


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RESULTS
 
Imatinib mesylate and CGP57380 cooperatively inhibit mRNA recruitment to polysomes in Bcr-Abl-containing cells. CGP57380 was originally identified as an inhibitor of Mnk1 kinase from an in vitro screen and has a 50% inhibitory concentration of 2.2 µM against Mnk1 kinase (11). Importantly, CGP57380 also exerts minimal cellular toxicity against untransformed cells, even at concentrations as high as 30 µM (C. Tschopp and H. Gram, unpublished data). Since then, others have also demonstrated the ability of CGP57380 to negatively affect polysomal mRNA assembly (18). Because of these interesting properties and because our prior work had implicated a role for Bcr-Abl-mediated translation initiation in transformation (14, 23), we tested the ability of CGP57380, together with imatinib, to impair polysomal mRNA assembly in CML cell lines. In the first series of experiments, we analyzed the polysome profile of drug-exposed Ba/F3-Bcr-Abl cells. Consistent with imatinib's ability to inhibit eIF4F induction (23), we found that brief (4-h) exposures to imatinib decreased the total amount of polysome-bound mRNA. This effect is appreciable as a decrease in the amplitude of the peaks representing the polysome fraction within the sucrose gradient and is associated with a reciprocal increase in the amplitude of the 80S monosome peaks (Fig. 1A). CGP57380 exerted a similar effect, and the combination of the two drugs was additive. Importantly, these results were replicated in the human CML cell line, K562, demonstrating that the phenomenon is not cell line specific (Fig. 1B). To quantify these findings, we also calculated the ratio of the area representing the polysome fraction to that of the polysome fraction plus the 80S monosome (Fig. 1C, P/P + 80S). For both cell lines, the data confirmed the additive effect of both drugs in decreasing the polysome/monosome-plus-polysome ratio and are consistent with the impairment of polysomal mRNA loading in Bcr-Abl-expressing cells. Closer inspection of the profiles also revealed that drug treatment resulted in a shift toward smaller polysomes, i.e., a decrease in the mRNA ribosome density. We quantified this effect by plotting the amplitude changes of the peaks corresponding to the lowest and highest density polysomes (Fig. 1D). In Ba/F3-Bcr-Abl cells, imatinib and CGP57380 treatment resulted in a 60% and 70% increase for low-density ribosomes, respectively, compared to a 41% and 41% decrease for high-density ribosomes, respectively. In K562 cells, imatinib and CGP57380 treatment resulted in a 20% and 40% increase for low-density ribosomes, respectively, compared to a 50% and 69% decrease, respectively, for high-density ribosomes. Together, these data demonstrate that both drugs impair polysome loading on mRNA, and data are consistent with a negative effect on translation initiation.


Figure 1
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  		FIG. 1. Imatinib and CGP57380 cooperatively impair polysome assembly in CML cells. (A) Ba/F3-Bcr-Abl cells were incubated with DMSO (D), 1 µM imatinib (IM), 10 µM CGP57380 (C), or both inhibitors for 4 h, and cellular extracts were obtained. Extracts were then fractionated over a 10 to 50% sucrose gradient, and the absorbance at 254 nm (A254) of polysomes (P) and monosomes (M) was continuously monitored. OD, optical density. The peak corresponding to the 80S monosome is also highlighted (80S-M), as are the peaks representing two 80S ribosomes per mRNA strand (2-80S) and nine 80S ribosomes per mRNA strand (9-80S). Results are representative of five independent experiments. (B) K562 cells were treated identically, except they were exposed to inhibitors for 16 h prior to cell lysis. Results are representative of five independent experiments. (C) To quantify the decrease in the polysome/monosome ratio, as well as to take into account minor differences in loading, the areas under the 80S peak (80S-M) and polysome peaks (P) were obtained using ImageJ software. The ratio of P/P + 80S-M was then normalized to that of DMSO-treated cells, and the data were plotted as percentages of DMSO-treated controls. (D) The percentage changes in the polysome peak amplitude for low (2-80S, two 80S ribosomes per mRNA strand) and high density (9-80S, nine 80S ribosomes per mRNA strand) are plotted as a bar graph for both Ba/F3-Bcr-Abl and K562 cells.

CGP57380 acts synergistically with imatinib to impair growth of Bcr-Abl-transformed cells via effects on cell cycle entry and apoptosis. To determine if the combination of CGP57380 and imatinib had activity against Bcr-Abl-transformed cells, we evaluated the combined activity of both drugs in the Ba/F3 system and in K562 cells at 72 h by using a tetrazolium-based (MTS) assay, which measures cell viability. For both of these cell lines, combination index plots of the MTS assays demonstrated values of <1, indicating synergistic activity between the two agents (Fig. 2A). The assay was also repeated at 48 h and demonstrated similar results (data not shown).


Figure 2
Figure 2
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  		FIG. 2. Synergistic activity of CGP57380 and imatinib against Bcr-Abl-expressing cells is associated with decreased proliferation and increased apoptosis. (A) Ba/F3-Bcr-Abl cells (left panel) or K562 cells (right panel) were incubated with increasing concentrations of imatinib and CGP57380 for 72 h. Effects on cell viability were determined by the MTS assay, and the combination index (CI) was determined. Values of <1.0 correspond to synergy. The numbers adjacent to each point indicate the concentrations of imatinib and CGP57380 that were used (nM imatinib/µM CGP57380). Results are representative of three independent experiments. (B, top panel) Parental Ba/F3 and Ba/F3-Bcr-Abl cells were treated with DMSO (D), 1 µM imatinib (IM), 10 µM CGP57380 (C), or both (IM+C) for 48 h and harvested. Cells were fixed with paraformaldehyde and then stained with PI and analyzed on a FACSCalibur flow cytometer. The relative percentages of cells in G0/G1, S, and G2/M were calculated using FlowJo software. Cells treated with 5 µM imatinib served as a positive control. Error bars indicate the standard deviation (s.d.) calculated from three independent experiments. (B, bottom panel) Cell lysates were prepared from parental Ba/F3 and Ba/F3-Bcr-Abl cells treated as described in the legend to panel B and used for Western analysis. Antibodies to cyclin D1, cyclin D2, cyclin D3, and CDK4 were used to assess the level of each protein, and antibody to β-actin was used as a loading control. Results are representative of three independent experiments. The dividing lines between the CGP57380 and IM lanes indicate that an unrelated intervening lane was removed. The four remaining lanes are from a single original immunoblot. (C) Parental Ba/F3 and Ba/F3-Bcr-Abl cells were treated with DMSO (D), 1 µM imatinib (IM), 10 µM CGP57380 (C), or both (IM+C) for 48 h and harvested. Cells were analyzed for apoptosis by using flow cytometry-based analysis of live cells by PI staining (error bars indicate the standard deviation calculated from three independent experiments) (top panel) or by detection of activated caspase-3 (A-Cas-3) by Western analysis (bottom panel). Results are representative of two independent experiments. (D) Cell lysates were prepared from K562 and primary blast phase CML cells (BP-1) after 24 h of incubation with DMSO (D), 2 µM imatinib (IM), 10 µM CGP57380 (C), or both (IM+C). Western analysis was performed with lysates, using antibodies to activated caspase-3 and β-actin as loading controls. Results are representative of two independent experiments.

Because the MTS assay reflects changes in both cell proliferation and survival, direct measurements of CGP57380's effects on both of these parameters were carried out. Using flow cytometry and PI staining, we treated the Ba/F3-Bcr-Abl and control parental Ba/F3 cells with inhibitors and performed cell cycle analysis. As expected, imatinib treatment of Ba/F3-Bcr-Abl cells (but not parental Ba/F3 cells) increased the proportion of cells at G0/G1, while single-agent treatment with CGP57380 had a small effect on inhibition of cell cycle entry in both cell lines. However, in combination with imatinib, CGP57380 exposure resulted in an additive effect only in the Ba/F3-Bcr-Abl cells (Fig. 2B, top panel). Since the D-type cyclins, together with CDK4, regulate G1/S transition, levels of each of these proteins were measured by immunoblotting of inhibitor-treated cells. In Ba/F3-Bcr-Abl cells, treatment with imatinib alone decreased levels of all three cyclins, while CGP57380 decreased cyclin D1 and D3 but not D2 (Fig. 2B, lower panel). However, when the drugs were used in combination, the effect was to substantially decrease cyclins D1 and D3 but not D2. A similar but smaller effect of the two drugs on CDK4 expression was also seen.

To determine if the potentiation of imatinib's effects on CML cells was also associated with enhanced apoptosis, Western- and flow cytometry-based analyses of apoptosis were performed. In Ba/F3-Bcr-Abl cells, the addition of CGP57380 enhanced the apoptotic effect of imatinib across a wide dose range but, in contrast, had little effect on imatinib-treated parental cells (Fig. 2C, upper panel). In addition, immunoblotting using antibody to activated caspase-3 demonstrated that CGP57380 administered alone had minimal effects on the Ba/F3-Bcr-Abl cells (Fig. 2C, lower panel). However, in cells treated with the combination, levels of activated caspase-3 were increased over that of cells exposed to imatinib alone. The effect of CGP57380 on K562 and primary blast phase (BP) CML cells exhibited similar characteristics, in that CGP57380 produced a minimal proapoptotic effect by itself but greatly enhanced the ability of imatinib to activate caspase-3 (Fig. 2D, upper panel). Together, these data suggest that CGP57380 induces G0/G1 arrest by decreasing D-type cyclin expression and, in cooperation with imatinib, promotes cell death via activating caspase-3.

CGP57380 independently inhibits phosphorylation of rpS6 on the PIC and cooperates with imatinib to inhibit eIF4F induction. The ability of both imatinib and CGP57380 to impair polysome assembly (Fig. 1) suggested that these agents were interfering with cap-dependent mRNA translation at the point of translation initiation. Because induction of eIF4F is essential for translation initiation and the subsequent recruitment of the 43S subunit to the mRNA strand, we assessed the integrity of the eIF4F 7-methylguanosine-cap complex in Ba/F3-Bcr-Abl cells treated with imatinib and/or CGP57380. Here, we used the cap analog, m7-GTP-Sepharose, to capture eIF4E and its binding partners from whole-cell lysates (1, 23). Following a GTP wash and elution with m7-GTP, cap-bound fractions were assessed for the presence of 4E-BP1 and eIF4G by using immunoblotting, and the integrity of eIF4F was noted as an increase in the ratio of eIF4G to 4E-BP1 in the cap-bound fractions. In Ba/F3-Bcr-Abl cells, imatinib treatment resulted in an increase in cap-bound 4E-BP1 and a corresponding decrease in cap-bound eIF4G, as previously described (Fig. 3A, lane 2 versus 1) (1, 23). Interestingly, despite the equivalent effects shown by CGP57380 on the inhibition of polysome assembly (Fig. 1A), CGP57380 treatment had little effect on eIF4F integrity compared to that in imatinib-treated cells (Fig. 3A, lane 3 versus 1). Consistent with these observations, we found that imatinib exerted a greater ability than CGP57380 to inhibit 4E-BP1 phosphorylation in whole-cell lysates (Fig. 3A, lane 6 versus 7). However, when both agents were combined, we found that eIF4F induction was almost completely inhibited, which was appreciable as a disappearance of cap-bound eIF4G, as well as a corresponding increase in cap-bound 4E-BP1 (Fig. 3A, lane 4). Together, these results suggested two features of CGP57380's effect on the cap-binding complex. First, CGP57380 was modulating polysome assembly via a mechanism that was independent of an effect on eIF4F integrity. Second, CGP57380 was also able to cooperate with imatinib to inhibit eIF4F induction more completely.


Figure 3
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  		FIG. 3. CGP57380 enhances the effect of imatinib on the inhibition of eIF4F induction and independently inhibits phosphorylation of rpS6 on the PIC. (A) Ba/F3-Bcr-Abl cells were incubated with DMSO (D), 1 µM imatinib (IM), 10 µM CGP57380 (C), or both inhibitors (IM+C) for 4 h, and cell lysates were made. Lanes 1 to 4, cap-bound proteins were brought down by incubation of cell lysates with m7-GTP-Sepharose, washed with GTP-containing buffer, and eluted with m7-GTP. The amounts of cap-bound eIF4G and 4E-BP1 were then assessed by immunoblotting with the appropriate antibodies. The antibody to 4E-BP1 detects all forms of 4E-BP1, including the {alpha}, β, and {gamma} forms, which correspond to increasingly phosphorylated 4E-BP1. As expected, the most phosphorylated, {gamma}, form is unable to bind the cap analog but is present in the whole-cell lysate (lanes 5 to 8). Lanes 5 to 8, the effects of inhibitors on 4E-BP1 and rpS6 phosphorylation were also assessed using immunoblots to whole-cell lysates. Anti-β-actin antibody was used as a loading control. Results are representative of four independent experiments. (B) Cell lysates were prepared from Ba/F3-Bcr-Abl, K562, and primary magnetic bead-selected CD34+ blast phase CML (BP-6C) cells after a 24-h incubation with DMSO (D), 2 µM imatinib (IM), 10 µM CGP57380 (C), or both (IM+C). Western analysis was performed with lysates, using antibodies to phospho-rpS6, total rpS6, and β-actin as loading controls. Results are representative of three independent experiments. (C) The cap-bound and whole-cell extracts shown in panel A were also probed with antibody to phospho-rpS6 to evaluate the amount of phosphorylated rpS6 on the cap-bound complex and in whole cells.

To further explore the mechanism by which CGP57380 might inhibit efficient polysome assembly, we took note of a recent finding by Roux and colleagues, who demonstrated that Rsk-mediated phosphorylation of rpS6 at Ser235/236 promotes cap-dependent mRNA translation (25). Specifically, they found that phosphorylation of rpS6 at this residue increased the affinity of rpS6 for the 7-methylguanosine-cap complex and promoted polysome assembly and cap-dependent translation. Accordingly, we assessed the phosphorylation status of rpS6 at Ser235/236, as well as at Ser240/244, in cells treated with imatinib and/or CGP57380. In whole-cell lysates, we found that either imatinib or CGP57380 treatment of Ba/F3-Bcr-Abl cells decreased rpS6 phosphorylation at Ser235/236 and Ser240/244 and that the combination was required to maximally inhibit phosphorylation at both sites (Fig. 3B). Similar results were obtained for K562 cells, as well as for purified CD34+ cells from a patient in myeloid blast crisis. As expected, an examination of the m7-GTP pull-down assay (which also contains the PIC and the rpS6 protein [9, 25]) revealed that treatment with either drug also decreased phosphorylation of cap-bound rpS6 at Ser235/236 and Ser240/244 (Fig. 3C). In addition, we found that either imatinib or CGP57380 treatment resulted in a slight decrease in the amount of cap-bound rpS6 but that the effects on the phosphorylation of cap-bound rpS6 were more prominent.

These results demonstrate that CGP57380's effect on polysome assembly is associated with the inhibition of rpS6 phosphorylation at Ser235/236. Since single-agent CGP57380 therapy is able to impair polysome assembly without significantly influencing eIF4F formation (Fig. 1A and 3A), these data indicate that phosphorylation of cap-bound rpS6 may also be important for efficient functioning of the PIC, following its induction.

CGP57380 inhibits phosphorylation of multiple substrates that regulate cap-dependent translation within the MAPK and mTOR signaling pathways. To date, CGP57380 has been employed as an inhibitor of the Mnk kinases (11, 18). However, the results shown in Fig. 3B demonstrated that CGP57380 was also functioning to inhibit rpS6 phosphorylation. In addition, we noticed that there was a high degree of similarity between the molecular structure of CGP57380 and that of another compound, BI-D1870, that was recently shown to be a specific inhibitor of the p90 S6 kinase (Rsk) (Fig. 4A) (29). Both compounds consist of fluoroanilines connected to a heterobicyclic core. Indeed, since it has very few functional groups, CGP57380 might be expected to be more promiscuous than other N-arylaniline kinase inhibitors (like BI-D1870 or gefitinib). These observations suggested that CGP57380 might actually be functioning as a multikinase inhibitor. We therefore tested the ability of CGP57380 to inhibit substrates of the major signaling pathways known to regulate cap-dependent translation, including those of the mTORC1, Rsk, and Mnk kinases (Fig. 4B). Using LY294002, rapamycin, and BI-D1870 as positive controls for PI3K, mTORC1, and Rsk inhibition, respectively, we found that CGP57380 was also able to inhibit a similar range of substrates including S6K1 and rpS6 at both Ser235/236 and Ser240/244 (Fig. 4B). In addition, unlike the other inhibitors in the panel, CGP57380 was also able to inhibit Mnk kinase activity, as determined by eIF4E phosphorylation (34). Interestingly, CGP57380 was able to differentiate between two mTOR substrates, S6K1 and 4E-BP1, suggesting that their phosphorylation might be differentially regulated (Fig. 4C). Together, these results demonstrate that CGP57380 inhibits the phosphorylation of a broad range of kinase substrates. CGP57380 is, thus, functioning as a multikinase inhibitor, a property that is consistent with its chemical structure.


Figure 4
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  		FIG. 4. CGP57380 inhibits phosphorylation of multiple substrates in the MAPK and mTOR signaling pathways that regulate cap-dependent translation. (A) Chemical structures of CGP57380 and BI-D1870. (B) Signaling diagram of known regulators of cap-dependent translation in the MAPK and mTOR pathways. Gray boxes indicate substrates that were evaluated by the immunoblotting shown in panel C. (C) Ba/F3-Bcr-Abl and K562 cells were treated with DMSO (D), 10 µM LY294002 (LY), 10 ng/ml rapamycin (R), 10 µM CGP57380 (C), 2 µM imatinib (IM), or 20 µM of BI-D1870 (BI) for 2 (Ba/F3-Bcr-Abl) and 8 (K562) h. Western analyses were performed with cell lysates using antibodies to phosphorylated 4E-BP1, S6K1, rpS6, and eIF4E. Loading was assessed using antibodies for total 4E-BP1, S6K1, rpS6, eIF4E, and β-actin. Results are representative of three independent experiments.

CGP57380 induces the activation of ERK in Bcr-Abl-containing cells. Another feature of the Rsk inhibitor BI-D1870 is its ability to activate the ERK pathway via a negative-feedback loop (29). Because activation of the MAPK pathway is implicated in the pathogenesis of many human cancers, including CML (6), we were prompted to investigate this possibility. Here, we found that CGP57380 treatment did indeed result in increased ERK and Mnk phosphorylation in a dose- and time-dependent manner in K562 cells (Fig. 5A). Activation of this loop was first evident at 2.5 µM CGP57380 (Fig. 5A, left panel) and was associated with an early transient phosphorylation of both ERK and Mnk, beginning within 30 min of treatment, followed by a second late phosphorylation beginning at 24 h and persisting for 48 to 72 h (Fig. 5A, right panel). Consistent with an increase in Mnk kinase activity, we also observed recrudescent eIF4E phosphorylation, although levels of phosphorylation did not return to baseline. Interestingly, by repeating the studies of the Ba/F3 system, we also show that certain features of the negative-feedback loop were dependent on the presence of Bcr-Abl. These features included the early CGP57380-mediated ERK phosphorylation, as well as sustained Mnk phosphorylation, since these effects were seen with Ba/F3-Bcr-Abl cells but not with parental cells (Fig. 5B). To confirm that the feedback loop was present in primary cells, similar experiments were conducted in primary BP CML cells. Here, we also observed the phenomenon of CGP57380-mediated ERK and Mnk activation (Fig. 5C).


Figure 5
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  		FIG. 5. CGP57380 induces the activation of ERK and Mnk in Bcr-Abl-containing cells. (A, left panel) K562 cells were exposed to increasing doses of CGP57380, after which immunoblots of cell lysates were probed with antibodies to the phosphorylated and total forms of ERK, Mnk, and eIF4E. Antibody to β-actin was used as a loading control. (Right panel) K562 cells were exposed to 10 µM CGP57380 over a time course, and cell lysates were analyzed as described in the legend to panel A, above. (B) Parental Ba/F3 and Ba/F3-Bcr-Abl cells were exposed to 10 µM CGP57380 over a time course, and cell lysates were analyzed as described in the legend above. (C) Primary CML cells from a patient in blast phase (BP-1) were treated with DMSO, 2 µM imatinib, 10 µM CGP57380, or both for 24 h. Immunoblots of cell lysates were probed as described in the legend to panel A. (D) K562 cells were incubated with a panel of pharmacologic inhibitors or DMSO (D) for 24 h, as follows: 200 nM imatinib (IM/I), 10 µM U0126 (U), 10 µM SB203580 (S), 10 µM CGP57380 (C), or combinations of inhibitors. Immunoblots of cell lysates were then probed as described above. Results are representative of at least four independent experiments.

Because of the marked phosphorylation and activation of Mnk that were observed after CGP57380 exposure, we determined if the feedback loop was mediated by MEK/ERK and/or p38 MAPK activation, since both of these pathways are known to phosphorylate and activate Mnk (35). We therefore treated K562 cells with either the MEK inhibitor U0126 or the p38 MAPK inhibitor SB203580 and performed immunoblotting of cell lysates. In these experiments, we found that U0126 was able to completely inhibit ERK phosphorylation and to attenuate Mnk and eIF4E phosphorylation (Fig. 5D, compare lane 3 to lane 1), while SB203580 actually increased ERK phosphorylation and had little effect on either Mnk or eIF4E phosphorylation (Fig. 5D, compare lane 4 to lane 1). In CGP57380-treated cells, MEK inhibition prevented ERK, Mnk, and eIF4E phosphorylation (Fig. 5D, compare lane 7 to lane 1), while in SB203580-treated cells, ERK/Mnk/eIF4E phosphorylation persisted (Fig. 5D, compare lane 8 to lane 1). Also, consistent with a role for Bcr-Abl in mediating this phenomenon, imatinib prevented the CGP57380-ERK/Mnk activation (Fig. 5D, compare lane 6 to 5). Taken together, these results demonstrate that CGP57380 induces a negative-feedback loop via MEK that regulates ERK and Mnk activation in Bcr-Abl-containing cells.

Inhibition of the MEK/ERK feedback loop enhances activity of CGP57380 and imatinib against Bcr-Abl-containing cells and imatinib-resistant cell lines. Prior work with CML has shown that the unintended activation of mitogenic pathways by kinase inhibitors, including activation by imatinib itself, can be turned to therapeutic advantage through the inhibition of the pathways concerned (5, 39). It was of interest, therefore, to determine if the cytotoxic effect of CGP57380 might be enhanced by concurrent MEK inhibition. Accordingly, U0126 was added to the imatinib/CGP57380 combination, and the three-drug combination was tested against parental Ba/F3, Ba/F3-Bcr-Abl, and K562 cells, using the MTS assay. The addition of U0126 to the imatinib/CGP57380 combination greatly enhanced the effect of the combination on Bcr-Abl-bearing cells, while it left parental Ba/F3 cells unaffected (Fig. 6A). Remarkably, the addition of the MEK inhibitor U0126 sensitized Ba/F3-Bcr-Abl and K562 cells to noncytotoxic doses of imatinib (250 nM and 50 nM, respectively). Immunoblot analysis of identically treated cells demonstrated that the three-drug combination also augmented the effect of any two-drug combination in activating caspase-3, as well as in decreasing the overexpression of the antiapoptotic protein Bcl-XL, in Ba/F3-Bcr-Abl cells but not in parental cells (Fig. 6B).


Figure 6
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  		FIG. 6. Overcoming imatinib resistance by using the MEK inhibitor UO126 and CGP57380. (A) Parental Ba/F3, Ba/F3-Bcr-Abl, and K562 cells were exposed to increasing doses of imatinib (IM), with or without 10 µM CGP57380 (CGP) and with or without 10 µM CGP57380 plus 10 µM UO126 (C+U). At 72 h, cell viability was assessed using the MTS assay and plotted as a percentage of DMSO-treated cells. Bars are standard deviations (SDs) calculated from three independent experiments. The effects of single-agent treatment with DMSO, CGP57380, and UO126 are also displayed as bars in each of the cell lines tested. (B) Parental Ba/F3 and Ba/F3-Bcr-Abl cells were exposed to 10 µM CGP573802 (C), 10 µM UO126 (U), 2 µM imatinib (IM/I), or combinations of inhibitors for 24 h and harvested. Immunoblots of cell lysates were then probed with antibody to Bcl-XL, activated caspase-3 (A-Cas-3), and β-actin. (C) Ba/F3-Bcr-Abl cells expressing "wild-type" Bcr-Abl (WT) or imatinib-resistant Bcr-Abl (T315I, E255K, M351T), as well as those overexpressing "wild-type" Bcr-Abl (BAR) were exposed to various inhibitors/inhibitor combinations, and viability was assessed with the MTS assay as described in the legend to panel A. Bars are SDs calculated from three independent experiments. (D) Summary of the findings in this study. Bcr-Abl-dependent signaling promotes translation initiation and polysome assembly and thereby translation of genes associated with survival and cell cycle progression. Bcr-Abl promotes translation initiation via activating the MAPK and PI3K pathways, which, through their effects on rpS6 and eIF4E, results in the assembly of eIF4F and the PIC. Small-molecule inhibitors of polysome assembly, including CGP57380, enhance the activity of imatinib against CML cells but can induce cytoprotective responses, e.g., MAPK activation. Pharmacologic interruption of these cytoprotective responses, e.g., with U0126, further enhances the activity of the CGP57380/imatinib combination against CML cells, including imatinib-resistant variants.

Because of this encouraging find, we went on to determine the efficacy of this combination against cells bearing imatinib-resistant mutants of Bcr-Abl, as well as those overexpressing "wild-type" Bcr-Abl, which together represent the majority of cases of clinical imatinib resistance (8, 31). We found that the imatinib-CGP57380 combination decreased the viability of all resistant cells compared to that of either agent alone. In each case, we also found an additional effect with the triple combination (Fig. 6C).


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DISCUSSION
 
The role of aberrant cap-dependent translation in cancer has been the subject of several recent reviews (15, 27). The importance of cap-dependent translation has also been highlighted by the potential for therapeutic targeting, using novel compounds (3, 10, 13, 17, 28), including a small molecule inhibitor of the eIF4E-eIF4G interaction which was found to have activity against Bcr-Abl-positive cell lines (17). Indeed, FDA approval of temsirolimus, an inhibitor of the translation regulator mTOR, underlines the therapeutic potential of such drugs. However, the discovery of compounds which can target normal cellular processes with acceptable therapeutic indices remains a challenging task. Nevertheless, as a result of our prior work describing a direct effect of Bcr-Abl in the formation of the cap-binding complex, eIF4F, and mRNA translation, we began a series of experiments to screen for small molecules that could inhibit the process of translation initiation in a Bcr-Abl-specific manner. Here, we describe the activity of one such compound, CGP57380, against CML cells and uncover its mechanism of action.

First, using polysomal mRNA profiling, we confirmed that a novel function of Bcr-Abl is to promote polysome assembly, as would be predicted by the ability of Bcr-Abl to induce eIF4F formation (23). An important point to note in these experiments is that very brief (4-h) exposures to the drug were sufficient to elicit gross changes in the polysome profile, thus ruling out the possibility that the inhibition of translation was due to either imatinib-mediated caspase-3 activation and/or cell cycle arrest, both of which occur several hours after the polysome changes shown in Fig. 1 (23). Next, we showed that CGP57380 was also able to inhibit polysome assembly as a single agent and, furthermore, that it could act cooperatively with imatinib in this manner. Although the effect of CGP5780 on polysomal mRNA assembly had been observed previously (18), the mechanism underlying this phenomenon had been unclear, given the inability of CGP57380 to inhibit 4E-BP1 phosphorylation and/or inhibit eIF4F induction (18). Our finding that CGP57380 also functions to inhibit rpS6 phosphorylation may provide an explanation for this phenomenon (Fig. 3 and 4), particularly in light of recent work by Roux et al. (25). In that study, the authors showed that phosphorylation at Ser235/236 is largely Rsk kinase-dependent and that phosphorylation at Ser235/236 promotes the recruitment of rpS6 to the 7-methylguanosine-cap complex (25). Interestingly, they also show that Rsk activity stimulates cap-dependent translation and polysome formation, although they did not directly examine the effect of Rsk on the cap-binding complex. Our results complement the results of Roux et al. in that the current data suggest a role for rpS6 phosphorylation in modulating the function of the cap-binding complex. Thus, although we did detect a slight decrease in the amount of cap-bound rpS6 following CGP57380 treatment (Fig. 3C), there was no effect of CGP57380 on the induction of eIF4F (Fig. 3A). Despite this lack of activity against eIF4F, CGP57380 impaired polysome assembly to the same degree as imatinib (Fig. 1), suggesting an effect on eIF4F function. Indeed, a role for rpS6 phosphorylation in eIF4F function would explain the cooperative effect of imatinib and CGP57380 to inhibit polysome assembly, since both drugs were required to maximally inhibit rpS6 phosphorylation (Fig. 3B). Because CGP57380 also inhibits rpS6 phosphorylation at Ser240/244, our findings also provide a rationale for investigating the role of phosphorylation at Ser240/244 in polysome assembly. Because both Ser235/236 and Ser240/244 are preferentially phosphorylated by two different kinases, Rsk and p70S6K1, respectively (25), it will be important to define the contribution that each residue makes to polysome assembly and cap-dependent translation, since it may be possible to selectively target these kinases in the clinic (29).

The results of inhibiting polysome assembly in CML cells are decreased proliferation and survival (Fig. 2). For either of these cellular processes, modulation of polysome assembly presumably decreases the translation of genes that positively regulate proliferation and survival. In the case of the decreased G1/S transition that was observed following drug treatment, this is likely a result of the decreased expression of the D-type cyclins and CDK4 (Fig. 2B), which together control this checkpoint. Prior work from our group and others has demonstrated that in the Ba/F3 system, Bcr-Abl regulates both cyclin D2 and D3 expression but does so at the transcriptional and translational levels, respectively (20, 23). In this respect, the ability of a single agent, CGP57380, to inhibit cyclin D3 expression while leaving cyclin D2 unaffected (Fig. 2B) is consistent with the greater dependence of cyclin D3 expression on translation versus transcription, although this needs to be shown formally. At a global level, both imatinib and CGP57380 decrease the polysome density of mRNAs and, thus, the translational efficiency of mRNA (Fig. 1). This result implies differential effects of these drugs on efficiently versus inefficiently translated mRNAs. Factors within the 5' untranslated region (UTR) of mRNAs that influence translational efficiency include the degree of secondary structure and the length of the 5' UTR, with mRNAs possessing higher levels of secondary structure having a greater requirement for eIF4F function than those with less structure (33). More specifically, the degree of secondary structure proximal to the 5' cap (within 37 nucleotides of the cap) seems to be particularly critical to the binding of translation initiation factors (21). This fact, coupled with our findings that CGP57380 modulates functioning of the cap-binding complex (Fig. 3), suggests that an analysis of the secondary structure of the first 30 to 40 nucleotides of genes that are prevented from being recruited to polysomes by CGP57380 might yield interesting information regarding the existence of motifs in the proximal 5' UTR of mRNAs that play an important role in the regulation of mRNA translation.

CGP57380 was initially isolated from a screen for Mnk kinase inhibitors (11) and has been employed by several groups to investigate the interplay between eIF4E phosphorylation and translational regulation. Our results now reveal that CGP57380 inhibits the phosphorylation of multiple substrates of the MAPK/Mnk/Rsk and PI3K/mTOR/S6K1 axes in vivo. Importantly, since this work was performed, Bain et al. have shown that CGP57380 is indeed able to inhibit multiple kinases in vitro, including Rsk1, with the same potency with which it inhibits the Mnk kinases (2). CGP57380 thus joins the ranks of several small-molecule multikinase inhibitors that include a number of FDA-approved cancer therapies: imatinib (38), sorafenib, and sunitinib. Recent reviews of these drugs have highlighted their abilities to target multiple kinases and have underlined the theoretical advantages of multitargeting (7, 37), including a broader spectrum of activity to more effectively combat the multifaceted nature of tumor growth, a reduced likelihood of drug resistance, improved patient compliance, and a reduction in the drug interactions and toxicities associated with monopharmacy versus polypharmacy. An important disadvantage is that the search for biomarkers for effective clinical decision making will be more complicated, and, perhaps more importantly, the critical molecular targets that fuel the cancer will be more difficult to uncover. In this respect, while we have shown that CGP57380 impairs polysome assembly and targets multiple components of the translation machinery, we cannot exclude an effect mediated primarily via one particular protein, e.g., inhibition of rpS6 phosphorylation by Rsk1. Indeed, since Rsk1 has a documented role in cap-dependent translation regulation (25), our initial efforts at further elucidating the mechanism of action of CGP57380 in CML will be focused on this kinase.

The interactions between CGP57380 and imatinib were also noteworthy in several respects, including their synergism and ability to overcome imatinib resistance, as well as effects on MAPK signaling. The increased MAPK activity in CGP57380-treated CML cells suggests that this may represent a cytoprotective mechanism by which Bcr-Abl-containing cells respond to the stress of CGP57380 exposure. Indeed, pharmacologic interruption of this response is associated with enhanced killing of CML cells by the imatinib/CGP57380 combination, including the potentiation of the lethal effects of extremely low concentrations (50 nM) of imatinib. Such activity may be particularly useful in the instance of suboptimal imatinib responses associated with low intracellular imatinib concentrations and reduced OCT-1-mediated drug influx (36). We also found that cells with various forms of imatinib-resistant mutations were sensitive to the combination. The response of cells with the M351T and E255K mutations and Bcr-Abl amplification was now comparable to sensitive cells treated with imatinib alone. It is also noteworthy that cells with the T315I mutation, which confers extreme imatinib resistance, responded with a 71% decrease in cell viability (Fig. 6C).

In conclusion, our studies demonstrate that pharmacologic inhibitors of polysome assembly represent a novel group of compounds which may be useful in the therapy for CML, including overcoming resistance to tyrosine kinase inhibitors (Fig. 6D). Such an approach may also be useful against other cancers associated with dysregulated translation.


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ACKNOWLEDGMENTS
 
We thank Bert Semler for helpful advice regarding obtaining and analyzing polysomal mRNA profiles, David Van Vranken for discussions regarding the chemical structure of CGP57380, and Parimal Sarayu for calculating the areas under the curves for the polysome profiles.

This work was supported by Public Health Service grants RO1CA107041, R21CA112936, and R33CA105514 from the National Cancer Institute.


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FOOTNOTES
 
* Corresponding author. Present address: Duke-NUS Graduate Medical School, 2 Jalan Bukit Merah, Singapore 169547. Phone: 65 6516 7763. Fax: 65 6534 8632. E-mail: sintiong.ong{at}duke-nus.edu.sg Back

{triangledown} Published ahead of print on 11 August 2008. Back

{dagger} Present address: Paichai Bio-Diagnostic Fusion Technology Center, Dept. of Life Science and Technology, Pai Chai University, Daejeon, Republic of Korea 302-735. Back


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Molecular and Cellular Biology, October 2008, p. 6496-6509, Vol. 28, No. 20
0270-7306/08/$08.00+0     doi:10.1128/MCB.00477-08
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