Proteasome proteolytic profile is linked to Bcr-Abl expression

Experimental Hematology 2009;37:357–366

Proteasome proteolytic profile is linked to Bcr-Abl expression Lisa J. Crawforda, Phlip Windruma, Laura Magilla, Junia V. Melob, Lynn McCalluma, Mary F. McMullina, Huib Ovaac, Brian Walkerd, and Alexandra E. Irvinea a

Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, UK; bDepartment of Haematology, Imperial College, London, UK; cNetherlands Cancer Institute, Division of Cellular Biochemistry, Amsterdam, The Netherlands; dDepartment of Pharmacy, Queen’s University Belfast, Belfast, UK (Received 25 April 2008; revised 10 November 2008; accepted 12 November 2008)

Objective. We have previously demonstrated that proteasome activity is higher in bone marrow from patients with chronic myeloid leukemia (CML) than normal controls. This study investigates whether there is any relationship between Bcr-Abl expression and proteasome activity. Materials and Methods. Fluorogenic substrate assays and an activity-based probe were used to profile proteasome activity in CML cell-line models and the effect of the proteasome inhibitor BzLLLCOCHO on these cell-line models and primary CML cells was investigated. Results. We have demonstrated that oncogenic transformation by BCR-ABL is associated with an increase in proteasome proteolytic activity. Furthermore, small interfering RNA targeted against BCR-ABL reduces proteasome activity. In addition, we have found that Bcr-AblLpositive cells are more sensitive than Bcr-AblLnegative cells to induction of apoptosis by the proteasome inhibitor BzLLLCOCHO, and that sequential addition of imatinib followed by BzLLLCOCHO has an additive effect on the induction of apoptosis in BcrAblLpositive cells. Finally, we demonstrate that cell lines that become resistant to imatinib remain sensitive to proteasome inhibition. Conclusion. This is the first time that a direct relationship has been demonstrated between BCR-ABL transformation and the enzymatic activity of the proteasome. Our results suggest that the proteasome might provide a useful therapeutic target in CML, particularly in those patients who have developed resistance to conventional treatment. Ó 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Chronic myeloid leukemia (CML) is characterized by a translocation involving chromosomes 9 and 22. The resulting fusion gene encodes a chimeric protein, Bcr-Abl, which is a constitutively activated tyrosine kinase that plays an essential role in the molecular pathology of CML [1]. The drug imatinib mesylate (STI571; Gleevec, Novartis, Basel, Switzerland) is designed to inhibit the Bcr-Abl tyrosine kinase and is currently the first-line treatment for CML. Although imatinib induces durable responses, a number of patients develop resistance [2]. Second-generation tyrosine kinase inhibitors, such as dasatinib and nilotinib, have become clinically available and demonstrate encouraging efficacy in imatinib-resistant patients [3]. However, a number of patients still remain insensitive to this treatment. ConseOffprint requests to: Alexandria E. Irvine, Ph.D., Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Ground Floor, 97 Lisburn Road, Belfast BT9 7BL, UK; E-mail: [email protected]

quently, there is a need to find new therapeutic strategies to treat these patients. Understanding the process of leukemogenesis driven by Bcr-Abl is essential to identifying new molecular targets for drug development. The proteasome is a proteolytic complex found in the nucleus and cytoplasm of all eukaryotic cells. It plays a crucial role in regulating cellular processes, such as cell cycle, apoptosis, and signal transduction, through controlled degradation of intracellular proteins. For this reason, it would not appear to be an obvious therapeutic target. Nonetheless, proteasome inhibitors were shown to potently induce apoptosis in tumorigenic cell lines in vitro and in human xenograft models of various tumor types in vivo [4–10]. The first clinically available proteasome inhibitor, Bortezomib (PS-341, VELCADE, Millenium Pharmaceuticals, Cambridge, MA, USA), is now established as an effective treatment for relapsed and refractory multiple myeloma and shows potential in other hematological

0301-472X/09 $–see front matter. Copyright Ó 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2008.11.004

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malignancies, particularly when combined with other therapeutic agents [11–16]. There is some evidence to support a role for targeting the proteasome in CML. In vitro studies found that leukemic stem cells are significantly more sensitive to apoptosis by proteasome inhibitors than normal hematopoietic stem cells [17]. Sublethal doses of Bortezomib have been shown to interact synergistically with histone deacetylase inhibitors [18], Flavopiridol [19], and Adaphostin [20] to induce apoptosis of Bcr-Ablpositive cells, including those that have become imatinib-resistant. In addition, we have shown that proteasome activity is higher in bone marrow mononuclear cells from CML patients than normal controls, suggesting that CML cells may be more susceptible to proteasome inhibition [21]. However, a direct relationship between Bcr-Ablinduced transformation and modulation of proteolytic activity of the proteasome has not been demonstrated thus far. The proteasome consists of a 20S central catalytic core with two 19S regulatory cap structures at either end. The core structure is made up of four heptameric rings, two outer a rings and two inner b rings. The enzymatic activities of the proteasome are associated with three distinct b subunits and are classified into three major categories, based upon preference to cleave a peptide bond after a particular amino acid residue. These activities are chymotrypsin-like (CT-L), with a preference for hydrophobic residues; trypsin-like (T-L), with a preference for basic residues; and post-glutamyl peptidyl hydrolysing (PGPH), with a preference for acidic residues and are localized to subunits b5, b2, and b1, respectively [22,23]. These activities do not function as separate entities and each b-subunit active site is formed only through interactions with other adjacent inactive b subunits [24]. Many proteasome inhibitors, including Bortezomib, are specifically targeted to the CT-L activity of the proteasome, which is believed to be rate-limiting [5,25], however, there is emerging evidence to show that all three catalytic sites make a significant contribution to protein degradation [26]. In addition, it has recently been shown that expression levels of individual proteasome activities influence the sensitivity of hematological malignancies to Bortezomib [27,28]. In this study, we have used a cell-line model to demonstrate that BCR-ABL transformation increases all three catalytic activities of the proteasome, and small interfering RNA (siRNA) targeted against BCR-ABL reduces these catalytic activities. We also show that Bcr-Ablpositive cells are more sensitive than Bcr-Ablnegative cells to induction of apoptosis by the proteasome inhibitor BzLLLCOCHO, and that sequential addition of imatinib followed by BzLLLCOCHO has an additive effect on induction of apoptosis of Bcr-Ablpositive cells. Furthermore, we found that cell lines that had become resistant to imatinib were as sensitive to proteasome inhibition as their imatinib-sensitive counterparts. This is the first time

that a direct relationship has been demonstrated between BCR-ABL transformation and enzymatic activity of the proteasome. This study provides evidence to suggest that the proteasome might be a useful therapeutic target in CML.

Materials and methods Cell lines and culture conditions ts-Bcr-Abl FDCP-Mix cells were a gift from A. D. Whetton (Manchester, UK). FDCP-Mix cells were transfected with a myeloproliferative sarcoma virusbased defective retroviral vector pM5-neo carrying a p210 ts BCR-ABL cDNA [29]. The p210 ts BCR-ABL cDNA encodes for a temperature-sensitive kinase mutant of p210 Bcr-Abl. Expression is unaffected by temperature, but the tyrosine kinase activity of Bcr-Abl is active at 32 C (permissive) and inactive at 39 C (restricted). Mock-transfected FDCP-Mix cells were used as a negative control. Cells were maintained in Fischer’s medium (Invitrogen Ltd, Paisley, UK) supplemented with selected batches of horse serum (20% v/v) and murine interleukin-3 (5% v/v). To allow for selection, G418 (Calbiochem, Merck Biosciences Ltd, Nottingham, UK) was added at 50 mg/mL. Cells were maintained at 32 C or 39 C in 5% CO2. The K562 cell line is a human CML cell line and was obtained from DSMZ (Deutsche Sammlung von Mikrorganismen und Zellkulturen, GmbH, Braunschweig, Germany). K562 cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco BRL, Paisley, UK). LAMA84 imatinib-resistant and -sensitive clones (LAMA84-r and LAMA84-s) and KCL22 imatinib-resistant and -sensitive clones (KCL22-r and KCL22-s) are human CML cell lines. LAMA84-r cells have increased copy numbers of BCR-ABL and the multidrug resistance p-glycoprotein. The mechanism of resistance of KCL22-r cells is independent of BCR-ABL expression [30]. Imatinib-resistant cells were grown in RPMI-1640 plus 10% fetal calf serum, supplemented with 1 mM imatinib. Parental-sensitive cell lines were maintained in parallel cultures without imatinib. The HL60 cell line is a human acute myeloid leukemia cell line and was obtained from the European Collection of Cell Cultures (Salisbury, UK). HL60 cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum. Primary CML samples and normal controls Bone marrow aspirate samples were obtained from healthy donors and patients with CML at diagnosis. All human samples were obtained with ethical approval from the Research Ethics Committee Northern Ireland, and those involved gave their informed consent for participation in accordance with the Declaration of Helsinki. Aspirates were collected in RPMI-1640 supplemented with 10% fetal calf serum and containing 100 IU preservative-free heparin (Leo Laboratories, Princes Risborough, UK). Mononuclear cells were separated over Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) by centrifugation at 400g for 20 minutes. Compounds Imatinib, a gift from Novartis (Basel, Switzerland), was prepared as a 100-mM stock solution in sterile phosphate-buffered saline. The proteasome inhibitor BzLLLCOCHO was synthesized as

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described previously and was stored as a 10-mM stock solution in dimethyl sulfoxide [31]. Cell viability and apoptosis assays Cell viability was determined using Trypan blue dye exclusion, and the induction of apoptosis was assessed by Hoechst 33342/ propidium iodide and Mitosensor stains. The Hoechst 33342/propidium iodide stains were used as an assessment of membrane integrity and nuclear morphology, respectively. Hoechst 33342 stock solution (1 mg/mL in dimethyl sulfoxide) was added to approximately 1  106 cells in culture media (1 mL per 200 mL) and cells incubated at 37 C for 20 minutes. Cells were pelleted and resuspended in 10 mL of 10 mg/mL propidium iodide in phosphate-buffered saline. Apoptosis was assessed using a fluorescent microscope with a DAPI band-pass filter (40 magnification). Apoptosis was additionally assessed using the ApoAlert Mitochondrial Membrane Sensor Kit (Clontech UK, Hampshire, UK). This kit is based on the collapse in the mitochondrial inner transmembrane potential (Djm) following induction of apoptosis [32]. This change in Djm facilitates release of caspase-activating proteins from the mitochondria into the cytosol. The cationic dye, Mitosensor, is taken up by the mitochondria of nonapoptotic cells where it forms aggregates that fluoresce red; due to the altered transmembrane potential apoptotic cells cannot take up the dye and fluoresce green. The kit was used according to manufacturer’s instructions. Apoptosis was assessed under a fluorescent microscope using a band-pass filter to detect fluorescein and rhodamine (40 magnification). Cell viability and the degree of apoptosis were assessed every 24 hours over a 72-hour time course. For both fluorescent techniques, 200 cells were typically scored per sample. Proteasome extraction Cells were pelleted by centrifugation and resuspended in adenosine triphosphate (ATP)/dithiothreitol (DTT) lysis buffer (10 mM Tris-HCl [pH 7.8], 0.5 mM DTT, 5 mM ATP, and 5 mM MgCl2] at a ratio of 1 mL per 1  107 cells. The cell suspension was left on ice for 10 minutes, followed by sonication for 15 seconds to ensure complete cell lysis. The suspension was then centrifuged at 400g for 10 minutes at 4 C and the resulting supernatant, containing proteasomes, was stored at 80 C, following the addition of 20% glycerol. Protein concentrations of the samples were measured using the Bradford dye binding assay (Perbio Science, Northumberland, UK) with bovine serum albumin as a standard. Enzyme assay The fluorogenic substrates N-Succinyl-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich, Dorset, UK), Z-Ala-Arg-Arg-AMC and Z-LeuLeu-Glu-AMC (Calbiochem, Merck Biosciences Ltd, Nottingham, UK) were used to measure the CT-L, T-L, and PGPH proteasome activities, respectively. Assays were carried out in a 200-mL reaction volume containing 50 mg proteasome extract, 5 mM ethylene diamine tetraacetic acid, and 50 mM fluorogenic substrate in ATP/ DTT lysis buffer at 37 C. Proteasome activity was determined as the rate of cleavage of the fluorescent substrate over 35 minutes and activity was expressed as arbitrary fluorescence units. Fluorescence was measured on a Cytofluor Series 400 multiwell plate reader (Applied Biosystems, Warrington, UK) at excitation and emission wavelengths of 395 nm and 460 nm, respectively.

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Western blot analysis Cells were pelleted by centrifugation and lysed in radioimmunoprecipitation assay buffer. For analysis of proteasome subunits, cell lysates corresponding to 50 mg protein were incubated with 50 mM proteasome active site-directed probe, DansylAhx3L3VS [33], for 1 hour at 37 C. For immunoprecipitation, cell lysates were incubated with protein A/G agarose (Santa Cruz Biotechnology Inc, Heidelberg, Germany). Equal amounts of protein were denatured by boiling at 95 C for 5 minutes, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% Bis-Tris gels (NuPAGE; NOVEX Invitrogen Ltd, Paisley, UK) and subsequently transferred to a polyvinylidene fluoride membrane. Immunoblotting was carried out using antibodies against dansyl-sulfonamidohexanoyl (Molecular Probes, Invitrogen Ltd), ubiquitin (Santa Cruz Biotechnology Inc), c-ABL, p27, and p-CRKL (Cell Signaling Technology, Hertfordshire, UK); equivalent protein loading was controlled by monitoring glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression using an anti-GAPDH antibody (Abcam, Cambridge, UK). Blots were scanned into the AutoChemi System (UVP, Cambridge, UK) and densitometry was performed using LabWorks 4.5 image acquisition and analysis software. Inhibition of BCR-ABL gene expression by siRNA K562 cells were nucleofected following manufacturer’s instructions using the Cell Line Nucleofector Kit V, program T-03 (Amaxa GmbH, Cologne, Germany) and either siRNA directed against BCR-ABL, a scrambled siRNA sequence or a nonsilencing fluorescently labeled siRNA (Amaxa GmbH) to monitor transfection efficiency. siRNA directed against the fusion sequence of BCR-ABL was used to specifically reduce gene expression (GCA GAG UUC AAA AGC CCU U) [34] and a scrambled siRNA sequence was used as control (UUG UAC GGC AUC AGC GUU ATT). Real-time polymerase chain reaction was performed, as described previously [35], to determine the reduction in BCR-ABL mRNA and Western blotting was carried out to confirm reduction of Bcr-Abl at the protein level. Analysis of drug combinations The median effect method of Chou-Talaly was employed to analyze the effect of imatinib in combination with the proteasome inhibitor, BzLLLCOCHO, using Calcusyn Software (Biosoft, Cambridge, UK) [36]. The values used for the analysis were the degree of apoptosis induced by the different combinations of imatinib and BzLLLCOCHO, as measured by Hoechst 33342/propidium iodide and Mitosensor. The ts Bcr-Abl FDCP-Mix cells were treated with the two compounds at constant-ratio combinations. These ratios were selected based on the IC50 values previously determined for imatinib (1 mM) and BzLLLCOCHO (1 mM).

Results BCR-ABL expression increases proteasome activity The ts-Bcr-Abl FDCP-Mix cells were compared to mocktransfected FDCP-Mix cells to test the hypothesis that leukemic cells exhibit higher proteasome activity than nonleukemic cells [37]. Proteasome activity levels were assessed with fluorogenic substrate assays and an

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activity-based probe, and both methods demonstrated that Bcr-Ablpositive cells have higher levels of proteasome activity than Bcr-Ablnegative cells. Using the fluorogenic assay measurements, we found significantly higher turnover of the fluorescent substrates for CT-L (p 5 0.002) and PGPH (p 5 0.04) activities in Bcr-Ablpositive cells (Fig. 1A). Analysis with the activity-based probe, DansylAhx3L3VS [33], demonstrated a significant increase in labeling of the probe to b5 (CT-L; p 5 0.02) and b2 (T-L; p 5 0.04) subunits in the Bcr-Ablpositive cells (Fig. 1B).

decrease in all three proteasome proteolytic activities (Fig. 2B; p ! 0.05 for CT-L, T-L, and PGPH activities) as assessed by the fluorogenic activity assay. Western blotting using the activity-based probe also showed a significant decrease in T-L (p 5 0.002) and PGPH (p 5 0.002) activities, but found no significant change in CT-L activity. No effect was observed on treatment with the scrambled control sequence. These findings demonstrate that oncogenic transformation by BCR-ABL results in increased proteasome activity and this can be reversed by reducing BCR-ABL expression.

Knockdown of BCR-ABL expression reduces proteasome activity K562 cells were treated with siRNA targeted against BCRABL to determine if there was a direct relationship between BCR-ABL expression and proteasome activity. Real-time polymerase chain reaction analysis demonstrated that siRNA caused a mean reduction of BCR-ABL expression of 8262 copies after 8 hours (9550 copies in control vs 1288 copies in siRNA) and this was confirmed at the protein level by Western blot analysis (Fig. 2A). Downregulation of BCR-ABL was associated with a significant

Proteasome inhibition preferentially induces apoptosis of CML cells BzLLLCOCHO (1 mM) was found to selectively induce apoptosis in Bcr-Ablpositive cells (ts-Bcr-Abl FDCPMix and primary CML cells) compared to Bcr-Ablnegative cells (mock-transfected FDCP-Mix cells and normal mononuclear cells). This effect was most pronounced at 72 hours posttreatment, as measured by both Mitosensor and Hoechst. At this time point, ts-Bcr-Abl FDCP-Mix cells displayed 56.5% 6 6.4% apoptosis compared to 16.5% 6 13.4% apoptosis in mock-transfected

Figure 1. Bcr-Ablpositive cells contain significantly greater proteasome activity than Bcr-Ablnegative cells. (A,B) All three proteolytic activities (chymotrypsin-like [CT-L], trypsin-like [T-L], and post-glutamyl peptidyl hydrolyzing [PGPH]) of the proteasome were found to be higher in ts-Bcr-Abl FDCP-Mix cells grown at the permissive temperature (Bcr-Ablþ) than mock transfected cells (Bcr-Abl-) grown at the same temperature. (A) Proteasome activity was measured using fluorogenic substrate assays. Results are shown as rate of substrate turnover; CT-L and PGPH activities were found to be significantly increased (p 5 0.002 and 0.04, respectively). (B) Proteasome activity was evaluated by Western blot analysis using activity based probe DansylAhx3L3VS. Densitometry was carried out and results are expressed as percentage of mock transfected (Bcr-Abl-) control and corrected to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. CT-L and T-L activities were found to be significantly increased (p 5 0.02 and p 5 0.04, respectively).

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Figure 2. Treatment of K562 cells with small interfering RNA (siRNA) directed against BCR-ABL decreases proteasome activity. (A) Western blot and realtime polymerase chain reaction analysis demonstrate a decrease in Bcr-Abl expression at the protein level using siRNA directed against BCR-ABL. ABL is used a loading control. (B,C) Transfection of K562 cells with siRNA targeted against BCR-ABL reduced all three proteolytic activities of the proteasome. No significant effect was observed when cells were transfected with a control scrambled siRNA. (B) Proteasome activity measured as rate of turnover of fluorescent substrates; chymotrypsin-like (CT-L), trypsin-like (T-L), and post-glutamyl peptidyl hydrolyzing (PGPH) activities were each found to be significantly decreased (p 5 0.006, p 5 0.003, and p 5 0.001, respectively). (C) Western blot analysis was performed to evaluate proteasome activity using the activity-based probe DansylAhx3L3VS. Densitometry results are expressed as percentage of control and corrected to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. T-L and PGPH activities were found to be significantly decreased (p 5 0.003 and p 5 0.002, respectively).

FDCP-Mix cells (Fig. 3A) and primary CML cells exhibited 39.7% 6 9.6% apoptosis compared to 18.1% 6 5.4% apoptosis in the normal controls (Fig. 3B). These observations are consistent with the higher proteasome activity in Bcr-Ablpositive cells rendering the cells more susceptible to induction of apoptosis by proteasome inhibition. Studies on the effect of imatinib and BzLLLCOCHO established that both drugs alone exert a dose-dependent effect on ts-Bcr-Abl FDCP-Mix cells. Additionally, both drugs demonstrate selectivity for Bcr-Ablpositive cells rather than Bcr-Ablnegative cells. The effect on cell viability and induction of apoptosis by a combination of the drugs was then examined. The simultaneous addition of imatinib and BzLLLCOCHO caused an antagonistic effect on the degree of apoptosis as compared to either of the two drugs administered alone. The effect was observed over all drug combinations and at each of the time points (Table 1, eg, IC50 both

drugs, 72 hours, Combination Index O1, denoting strong antagonism). The effect of sequential addition of imatinib and BzLLLCOCHO on ts-FDCP-Mix cells was studied by adding one drug at t 5 0 and then adding the second drug after 12 hours; both drugs were added at their IC50 values (1 mM). By 36 hours, there was a synergistic effect on induction of apoptosis when imatinib was added 12 hours prior to addition of BzLLLCOCHO (Fig. 3C, Combination Index 5 0.340). When BzLLLCOCHO was added first, the degree of antagonism was similar to that observed when the drugs were added simultaneously (Combination Index 5 3.86, denoting strong antagonism). A similar trend was observed with sequential addition of imatinib and BzLLLCOCHO on primary CML cells, however, the effects were significantly less pronounced (Fig. 3D). By 36 hours, there was a slight additive effect on induction of apoptosis when imatinib was added 12 hours prior to

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Figure 3. The proteasome inhibitor BzLLLCOCHO (BzLLL) selectively induces apoptosis of Bcr-Ablpositive cells. (A,B) Mitosensor assays were carried out at 24-hour intervals on Bcr-Ablþ and Bcr-Abl- cells cultured with or without the proteasome inhibitor BzLLL (1 mM). (A) There was a significantly higher level of apoptosis after 72 hours in ts-Bcr-Abl FDCP-Mix cells (Bcr-Ablþ) than in mock-transfected cells (Bcr-Abl-), p 5 0.03. (B) There was a significantly higher level of apoptosis after 72 hours in primary chronic myeloid leukemia (CML) cells (Bcr-Ablþ) than in normal mononuclear cells (Bcr-Abl-). (C,D) Mitosensor assays of apoptosis were carried out at 12-hour intervals on Bcr-Ablþ cultured in the presence of BzLLL (1 mM), imatinib (1 mM), or a combination of both drugs added simultaneously or sequentially. (C) Using ts-Bcr-Abl FDCP-Mix cells, the highest level of apoptosis was observed in cells treated initially with imatinib followed by BzLLL. This combination was synergistic only when the drugs were added in this order. (D) Using primary CML cells, the highest level of apoptosis was observed in cells treated initially with imatinib followed by BzLLL, this combination was additive. (E) P-CRKL activity was evaluated in Bcr-Ablþ cells treated with BzLLL (1 mM), imatinib (1 mM), or a combination of both drugs added simultaneously or sequentially by Western blot analysis, 36 hours posttreatment. Densitometry was carried out and results are expressed as a percentage of untreated control and corrected to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control.

addition of BzLLLCOCHO (Combination Index 5 1.09). Treatment with BzLLLCOCHO first resulted in moderate antagonism (Combination Index 5 1.55). The effect of the treatments on Bcr-Abl kinase activity was determined by Western blotting for levels of phospho-CRKL (p-CRKL) 36 hours posttreatment (Fig. 3E). Densitometry

analysis (n 5 3) revealed that BzLLLCOCHO treatment alone had a minimal effect on p-CRKL activity (90.3% 6 13.6% of control), however, all three combinations of BzLLLCOCHO and imatinib resulted in greater abrogation of p-CRKL activity (36% 6 9.7%; 45% 6 9.4%; 40.6% 6 9.5% of control) than imatinib alone (63% 6 11.9% of

L.J. Crawford et al./ Experimental Hematology 2009;37:357–366 Table 1. Chou-Talalay analysis of the effect of drug combinations on ts-Bcr-Abl FDCP-Mix cells Imatinib (mM)

BzLLLCOCHO (mM)

CI value

Effect

0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0

2.146 4.037 1.738 1.725 5.477 3.973 2.843 4.930 9.550

Antagonism Strong antagonism Antagonism Antagonism Strong antagonism Antagonism Antagonism Strong antagonism Strong antagonism

0.5 0.5 0.5 1.0 1.0 1.0 2.0 2.0 2.0 CI 5 Combination index.

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control). This suggests that treatment with BzLLLCOCHO may enhance the sensitivity of the Bcr-Abl kinase to imatinib. Cells that have become resistant to imatinib remain sensitive to proteasome inhibition Proteasome activity levels were measured in two human Bcr-Ablpositive cell lines with imatinib-sensitive and -resistant clones (KCL22-s and KCL22-r; LAMA84-s, and LAMA84-r) and a Bcr-Ablnegative acute myeloid leukemia cell line (HL60). KCL22-r cells displayed higher levels of all three catalytic activites compared to KCL22-s cells, although this increase did not reach statistical significance (Fig. 4A; p O 0.05). LAMA84-r cells showed

Figure 4. Cells that have become resistant to imatinib display altered proteasome activity profiles and remain sensitive to proteasome inhibition. (A,B) Proteasome activity was measured in CML (Bcr-Ablþ) cell lines that are sensitive and resistant to imatinib treatment, using fluorogenic substrate assays. (A) Catalytic activities of the proteasome were higher in KCL22 cells resistant to imatinib treatment (KCL22-r) than their sensitive counterparts (KCL22-s), however, the differences did not reach statistical significance. (B) In LAMA84-r cells, chymotrypsin-like (CT-L) activity was higher than in LAMA84-s cells (p ! 0.05). In contrast, trypsin-like (T-L), and post-glutamyl peptide hydrolyzing (PGPH) activities were higher in the LAMA84-s cells than in the resistant cell line. (C) Proteasome activity was measured in AML HL60 cell line. HL60 cells display a different proteolytic profile to KCL22 and LAMA84 cells. (DF) Cell lines were treated with 1 mM BzLLLCOCHO (BzLLL); apoptosis was measured at 24-hour intervals using Mitosensor. (D) There is no significant difference in percentage apoptotic cells from KCL22-s and KCL22-r cell population over 72 hours. (E) There is no significant difference in percentage apoptotic cells from LAMA84-s and LAMA84-r cell population over 72 hours. (F) KCL22-s and LAMA84-s cells are significantly more sensitive to apoptosis than HL60 cells at 72 hours posttreatment; p ! 0.01. (G) KCL22-s, KCL22-r, LAMA84-s, LAMA84-r, and HL60 cells were treated with 1 mM BzLLL. Western blot analysis shows increasing accumulation of polyubiquitinated proteins of varying molecular weights over 72 hours, confirming that there is inhibition of proteasome activity. (H) KCL22-s and LAMA84-s cells were treated with 1 mM BzLLL for 24 hours. Coimmunoprecipitation of p-27 and ubiquitin demonstrate an accumulation of polyubiquitinated p27 in BzLLL-treated samples. (I) Western blot analysis demonstrates that LAMA84-r cells express a higher level of Bcr-Abl than LAMA84-s, KCL22-s, and KCL22-r cells; HL60 cells do not express Bcr-Abl. Both imatinib-sensitive and -resistant cell lines express a similar level of p-CRKL. IB 5 immunoblotting; IP 5 immunoprecipitation; ub 5 ubiquitin.

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significantly increased CT-L like activity and reduced levels of T-L and PGPH activities compared to LAMA84-s cells (Fig. 4B; p ! 0.05). The T-L and PGPH activities account for a large proportion of total proteasome activity in the CML cell lines. In contrast, HL60 cells display a different profile, whereby the CT-L activity is predominant (Fig. 4C). These results suggest that proteolytic profiles are altered between disease types and also in sensitive and resistant disease. The ability of BzLLLCOCHO to induce apoptosis in the cell lines was evaluated using Mitosensor and Hoeschst/ propidium iodide staining. Cells were incubated with 1 mM BzLLLCOCHO and apoptosis was measured at 24-hour intervals. Inhibition of proteasome activity by BzLLLCOCHO induced apoptosis in all cell lines. Cells that had become resistant to imatinib were equally as sensitive to proteasome inhibition as their imatinib-sensitive counterparts (Fig. 4D and E). Bcr-Ablpositive cell lines (KCL22-s and LAMA84-s) were found to be significantly more sensitive to induction of apoptosis by BzLLLCOCHO at 72 hours than a Bcr-Ablnegative cell line (HL60). To demonstrate that BzLLLCOCHO inhibits proteasome activity in each of the cell lines, BzLLLCOCHO-treated cells were analyzed by Western blotting for accumulation of polyubiquitinated proteins (Fig. 4G). Furthermore, because BCR-ABL is thought to partly regulate cell cycle in CML cells by inducing proteasome-mediated degradation of the cyclin-dependent kinase inhibitor p27 [38], we looked specifically for accumulation of p27 following treatment with BzLLLCOCHO. Figure 4H shows accumulation of polyubiquitinated p27 in KCL22-s and LAMA84-s cells that have been treated with BzLLLCOCHO. Finally, we compared the levels of Bcr-Abl and Bcr-Abl activity (p-CRKL) in the imatinib-sensitive and -resistant cells by Western blotting (Fig. 4I). There was no apparent difference in levels of Bcr-Abl between KCL22-s and KCL22-r cells, however, levels of Bcr-Abl are significantly increased in LAMA84-r compared to LAMA84-s cells. Imatinib resistance in the LAMA84-r cell line is due to increased expression of BCR-ABL and p-glycoprotein [30], therefore, this result would be expected. The observed increase in CT-L activity in LAMA84-r cells may be a direct consequence of increased BCR-ABL expression. Surprisingly, the increase in Bcr-Abl expression in LAMA84-r cells did not translate into a corresponding increase in p-CRKL levels, there was no difference observed in p-CRKL levels between any of the cell lines.

Discussion We have used cell-line models to demonstrate that BCRABL transformation increases all three catalytic activities of the proteasome and that siRNA targeted against BCRABL reduces these catalytic activities. Bcr-Ablpositive cells were more sensitive to induction of apoptosis by

proteasome inhibition than Bcr-Ablnegative cells, and this effect was increased by pretreatment with imatinib. Furthermore, cell lines that are resistant to imatinib remain sensitive to proteasome inhibition. This is the first time that a direct relationship has been demonstrated between oncogenic transformation and the catalytic activity of the proteasome. Immunocytochemical studies have previously demonstrated higher proteasome expression levels in leukemic cell lines than normal mononuclear cells, but did not examine functional proteasome activity in comparable normal and transformed cells [37]. Similarly, proteasome activity has been measured in several tumor cell lines, but has not been examined in relation to the normal cell counterpart. Oncogenic transformation of other cell types has previously been associated with an increase in sensitivity to proteasome inhibitors, although no direct link to the enzymatic activity of the proteasome has been demonstrated. Lymphoblasts transformed by c-myc, fibroblasts transformed with ras and myc alone, and a Burkitt’s lymphoma line that overexpresses c-Myc were found to be up to 40 times more sensitive to induction of apoptosis by proteasome inhibitors than nontransformed fibroblasts and normal human lymphoblasts [8]. SV-40transformed fibroblasts have also been shown to be more sensitive to proteasome inhibitors than nontransformed cells, and it has been suggested that this may be due to the increased expression of p27KIP1 in the transformed cells [39]. Oncogenic transformation by BCR-ABL results in the increased degradation of a number of important proteasomal substrates, including IkB, p27 KIP1, and Bim [40], which contribute to growth and survival in CML cells. This may account, in part, for the increase in proteasome activity seen in Bcr-Ablpositive cells and also the increased sensitivity of these cells as proteasome inhibition would abrogate these effects and promote cell-cycle arrest and apoptosis. In addition, there has been some evidence from in vitro studies to suggest that proteasome inhibitors used alone can induce apoptosis in CML cells by preventing proteasome-mediated degradation of natural inhibitors of BcrAbl [41,42]. The proteasome inhibitor Bortezomib has also been reported to synergistically interact with Flavopiridol, histone deacetylase inhibitors, and Adaphostin to induce apoptosis in Bcr-Ablpositive cells [18–20]. Imatinib was designed to specifically target the ATP binding site of the Bcr-Abl tyrosine kinase [43]. Imatinib has now been studied in vitro in combination with numerous antileukemic agents to determine if this can enhance its efficacy [18,44–46]. We examined the effect of combining imatinib and our proteasome inhibitor BzLLLCOCHO in vitro. Sequential addition of imatinib followed by proteasome inhibitor resulted in an additive effect of apoptosis in Bcr-Ablpositive cells; reversing this order or simultaneous addition of these compounds was antagonistic. Studies of the crystal structure of the c-Abl kinase domain

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in complex with imatinib have shown that a specific inactive conformation of Abl is required for the drug to bind [47,48]. Treatment of Bcr-Ablpositive cells with imatinib first allows this drug to interact with Bcr-Abl and prevent further downstream signaling. This should allow the proteasome inhibitor to act optimally, resulting in synergy. Interestingly, we found that all combinations of BzLLLCOCHO and imatinib result in a greater reduction of p-CRKL (a measure of Bcr-Abl kinase activity) than imatinib alone, suggesting that proteasome inhibition may sensitize BcrAblpositive cells to the kinase inhibitory effects of imatinib. Gatto et al. [49] found the sequential combination of Bortezomib followed by imatinib to be synergistic and proapoptotic, while concomitant exposure to the drugs was antagonistic. The cell lines used in these studies were derived from patients with CML in myeloid blast crisis. Our studies were carried out using the ts-Bcr-Abl FDCPMix cell line model and primary CML cells, reflecting chronic-phase disease, and we used the inhibitor BzLLLCOCHO, which we have previously shown to have different specificities to Bortezomib [50]. This may partly account for the differences in our observations. These findings emphasize the need for greater understanding of the mechanistic basis for the action of these compounds to ensure they elicit optimum efficacy in vivo. Resistance to imatinib therapy is now a recognized clinical problem. The mechanisms of resistance are not fully understood, but include cells with mutations in the Abl kinase domain, cells with upregulated Bcr-Abl expression and reduced cellular drug intake [51]. We used two cellline models to investigate proteasome function in imatinib-resistant CML cells. LAMA84-r cells overexpress BCR-ABL and the multidrug resistance p-glycoprotein, while the mechanism of resistance of KCL22-r cells is independent of BCR-ABL expression [30]. Increased expression of BCR-ABL in LAMA84-r cells was associated with significantly increased CT-L activity. These data are consistent with our observations that Bcr-Abl expression directly modulates proteasome activity. KCL22-r cells showed a different proteolytic profile with an increase in all three enzyme activities. In both model systems, cell lines that had become resistant to imatinib remained sensitive to proteasome inhibition and were significantly more sensitive to apoptosis than the acute myeloid leukemia cell line, HL60, which does not express Bcr-Abl. This is in agreement with the hypothesis that oncogenic transformation by BCR-ABL sensitizes cells to induction of apoptosis by proteasome inhibition and also suggests that the proteasome may provide a useful therapeutic target in imatinib-resistant patients. Bortezomib, a first-generation proteasome inhibitor targeting the CT-L activity of the proteasome, is now established as a treatment for relapsed and refractory multiple myeloma [11] and has demonstrated efficacy in phase

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I and II clinical trials for a number of other malignancies [12–15]. Second-generation compounds, such as NPI0052 [52] and carfilzomib [53], are currently in early clinical trials and may allow greater selectivity and specificity for the component activities of the proteasome. The present findings provide a rational basis to examine the potential of these proteasome inhibitors to treat CML.

Acknowledgments We wish to thank A. D. Whetton for providing the ts-Bcr-Abl FDCP-Mix cell line, Novartis for imatinib, the consultant hematologists at Belfast City Hospital for allowing access to their patients, S. Price, A. Jordan, and W. H. Lu for skilled technical assistance and Northern Ireland Leukaemia Research Fund for supporting this work.

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