Proteasome proteolytic profile is linked to Bcr-Abl expression.

Abstract

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.
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.
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-Abl-positive cells are more sensitive than Bcr-Abl-negative 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 Bcr-Abl-positive cells. Finally, we demonstrate that cell lines that become resistant to imatinib remain sensitive to proteasome inhibition.
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.

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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
aCentre 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 protea-
some 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 tar-
geted 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 imati-
nib followed by BzLLLCOCHO has an additive effect on the induction of apoptosis in Bcr-
AblLpositive 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.
Hematology and Stem Cells.Published by Elsevier Inc.
? 2009 ISEH - Society for
Chronicmyeloidleukemia(CML)ischaracterizedbyatrans-
location 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 essen-
tial 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
efficacyinimatinib-resistantpatients[3].However,anumber
of patients still remain insensitive to this treatment. Conse-
quently, there is a need to find new therapeutic strategies to
treat these patients. Understanding the process of leukemo-
genesis 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,andsignal
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 inhib-
itor, Bortezomib (PS-341, VELCADE, Millenium Pharma-
ceuticals, Cambridge, MA, USA), is now established as an
effective treatment for relapsed and refractory multiple
myeloma and shows potential in other hematological
transduction,through
Offprint 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: s.irvine@qub.ac.uk
0301-472X/09 $–see front matter. Copyright ? 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.
doi: 10.1016/j.exphem.2008.11.004
Experimental Hematology 2009;37:357–366

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malignancies, particularly when combined with other ther-
apeutic 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-Abl?positive cells, including those that
have become imatinib-resistant. In addition, we have shown
that proteasome activity is higher in bone marrow mononu-
clear cells from CML patients than normal controls, sug-
gesting that CML cells may be more susceptible to
proteasome inhibition [21]. However, a direct relationship
between Bcr-Abl?induced 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 activi-
ties 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 chymo-
trypsin-like (CT-L), with a preference for hydrophobic resi-
dues; 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 activ-
ities 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 inhib-
itors, 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 hemato-
logical malignancies to Bortezomib [27,28].
In this study, we have used a cell-line model to demon-
strate 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-Abl?positive
cells are more sensitive than Bcr-Abl?negative 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-Abl?positive cells. Further-
more, 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 myelo-
proliferative sarcoma virus?based 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 main-
tained in Fischer’s medium (Invitrogen Ltd, Paisley, UK) supple-
mented 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 Zell-
kulturen, 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-glyco-
protein. The mechanism of resistance of KCL22-r cells is indepen-
dent 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 Euro-
pean 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 ob-
tained 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/pro-
pidium 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 ? 106cells 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 phos-
phate-buffered saline. Apoptosis was assessed using a fluorescent
microscope with a DAPI band-pass filter (40? magnification).
Apoptosis was additionally assessed using the ApoAlert Mito-
chondrial Membrane Sensor Kit (Clontech UK, Hampshire, UK).
This kit is based on the collapse in the mitochondrial inner trans-
membrane 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 man-
ufacturer’s instructions. Apoptosis was assessed under a fluores-
cent 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 adeno-
sinetriphosphate (ATP)/dithiothreitol
(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 ? 107cells. The cell suspen-
sion 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 super-
natant, 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.
(DTT) lysis buffer
Enzyme assay
The fluorogenic substrates N-Succinyl-Leu-Leu-Val-Tyr-AMC
(Sigma-Aldrich, Dorset, UK), Z-Ala-Arg-Arg-AMC and Z-Leu-
Leu-Glu-AMC (Calbiochem, Merck Biosciences Ltd, Nottingham,
UK) were used to measure the CT-L, T-L, and PGPH proteasome
activities,respectively.Assayswerecarriedoutina200-mLreaction
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. Fluores-
cence 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.
Western blot analysis
Cells were pelleted by centrifugation and lysed in radioimmuno-
precipitation 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 Biotech-
nology 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, Invitro-
gen 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 instruc-
tions 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 transfec-
tion 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 per-
formed, 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/pro-
pidium iodide and Mitosensor. The ts Bcr-Abl FDCP-Mix cells
were treated with the two compounds at constant-ratio combina-
tions. These ratios were selected based on the IC50values previ-
ously 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 mock-
transfected FDCP-Mix cells to test the hypothesis that
leukemic cells exhibit higher proteasome activity than
nonleukemiccells[37].Proteasome
were assessed with fluorogenic substrate assays and an
activity levels
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activity-based probe, and both methods demonstrated that
Bcr-Abl?positive cells have higher levels of proteasome
activity than Bcr-Abl?negative 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-Abl?positive cells
(Fig. 1A). Analysis with the activity-based probe, Dansy-
lAhx3L3VS [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-Abl?positive cells
(Fig. 1B).
Knockdown of BCR-ABL
expression reduces proteasome activity
K562 cells were treated with siRNA targeted against BCR-
ABL 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). Downregu-
lation of BCR-ABL was associated with a significant
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 blot-
ting using the activity-based probe also showed a significant
decrease in T-L (p 5 0.002) and PGPH (p 5 0.002) activ-
ities, but found no significant change in CT-L activity. No
effect was observed on treatment with the scrambled
control sequence. These findings demonstrate that onco-
genic transformation by BCR-ABL results in increased
proteasome activity and this can be reversed by reducing
BCR-ABL expression.
Proteasome inhibition
preferentially induces apoptosis of CML cells
BzLLLCOCHO (1 mM) was found to selectively induce
apoptosis in Bcr-Abl?positive cells (ts-Bcr-Abl FDCP-
Mix and primary CML cells) compared to Bcr-Abl?nega-
tive 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
to16.5%
6
13.4%apoptosis inmock-transfected
Figure 1. Bcr-Abl?positive cells contain significantly greater proteasome activity than Bcr-Abl?negative 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 signif-
icantly 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 glyceral-
dehyde 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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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 observa-
tions are consistent with the higher proteasome activity in
Bcr-Abl?positive cells rendering the cells more susceptible
to induction of apoptosis by proteasome inhibition.
Studies on the effect of imatinib and BzLLLCOCHO es-
tablished that both drugs alone exert a dose-dependent
effect on ts-Bcr-Abl FDCP-Mix cells. Additionally, both
drugs demonstrate selectivity for Bcr-Abl?positive cells
rather than Bcr-Abl?negative 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 BzLLLCO-
CHO caused an antagonistic effect on the degree of
apoptosis as compared to either of the two drugs adminis-
tered alone. The effect was observed over all drug combina-
tions and at each of the time points (Table 1, eg, IC50both
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 add-
ing 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 withsequential
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
addition of imatiniband
Figure 2. Treatment of K562 cells with small interfering RNA (siRNA) directed against BCR-ABL decreases proteasome activity. (A) Western blot and real-
time 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 fluo-
rescent substrates; chymotrypsin-like (CT-L), trypsin-like (T-L), and post-glutamyl peptidyl hydrolyzing (PGPH) activities were each found to be signifi-
cantly 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 dehy-
drogenase (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).
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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
(p-CRKL) 36 hours posttreatment (Fig. 3E). Densitometry
levelsof phospho-CRKL
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
Figure 3. The proteasome inhibitor BzLLLCOCHO (BzLLL) selectively induces apoptosis of Bcr-Abl?positive 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 signif-
icantly 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-Ablþcells 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.
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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-Abl?positive cell lines with imatinib-sensitive and
-resistant clones (KCL22-s and KCL22-r; LAMA84-s,
and LAMA84-r) and a Bcr-Abl?negative 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 signif-
icance (Fig. 4A; p O 0.05). LAMA84-r cells showed
Table 1. Chou-Talalay analysis of the effect of drug combinations on
ts-Bcr-Abl FDCP-Mix cells
Imatinib (mM) BzLLLCOCHO (mM)CI valueEffect
0.5
0.5
0.5
1.0
1.0
1.0
2.0
2.0
2.0
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
CI 5 Combination index.
Figure 4. Cells that have become resistant to imatinib display altered proteasome activity profiles and remain sensitive to proteasome inhibition. (A,B) Pro-
teasome 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.
(D?F) Cell lines were treated with 1 mM BzLLLCOCHO (BzLLL); apoptosis was measured at 24-hour intervals using Mitosensor. (D) There is no signif-
icant 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-sensi-
tive 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 sensi-
tive to proteasome inhibition as their imatinib-sensitive
counterparts (Fig. 4D and E). Bcr-Abl?positive 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-Abl?negative 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 degrada-
tion of the cyclin-dependent kinase inhibitor p27 [38], we
looked specifically for accumulation of p27 following treat-
ment 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 resis-
tance in the LAMA84-r cell line is due to increased expres-
sion 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 BCR-
ABL transformation increases all three catalytic activities
of the proteasome and that siRNA targeted against BCR-
ABL reduces these catalytic activities. Bcr-Abl?positive
cells were more sensitive to induction of apoptosis by
proteasome inhibition than Bcr-Abl?negative 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 onco-
genic transformation and the catalytic activity of the
proteasome.
Immunocytochemical studies have previously demon-
strated 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 coun-
terpart. 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 demon-
strated. 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-40?transformed
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 p27KIP1in the transformed cells [39]. Onco-
genic transformation by BCR-ABL results in the increased
degradation of a number of important proteasomal
substrates, including IkB, p27KIP1, 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-Abl?positive cells and also the increased sensi-
tivity of these cells as proteasome inhibition would abro-
gate 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 pro-
teasome-mediated degradation of natural inhibitors of Bcr-
Abl [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-Abl?positive 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-Abl?positive cells; reversing this order or simulta-
neous addition of these compounds was antagonistic.
Studies of the crystal structure of the c-Abl kinase domain
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Page 9

in complex with imatinib have shown that a specific inac-
tive conformation of Abl is required for the drug to bind
[47,48]. Treatment of Bcr-Abl?positive cells with imatinib
first allows this drug to interact with Bcr-Abl and prevent
further downstream signaling. This should allow the protea-
some inhibitor to act optimally, resulting in synergy. Inter-
estingly, 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 Bcr-
Abl?positive 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 FDCP-
Mix cell line model and primary CML cells, reflecting
chronic-phasedisease,and
BzLLLCOCHO, which we have previously shown to have
different specificities to Bortezomib [50]. This may partly
account for the differences in our observations. These find-
ings 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 clin-
ical 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 cell-
line modelsto investigate
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 sensi-
tive 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 transfor-
mation 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 tar-
geting the CT-L activity of the proteasome, is now estab-
lished as a treatment for relapsed and refractory multiple
myeloma [11] and has demonstrated efficacy in phase
we used theinhibitor
proteasomefunctionin
I and II clinical trials for a number of other malignancies
[12–15]. Second-generation compounds, such as NPI-
0052 [52] and carfilzomib [53], are currently in early clin-
ical 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 hematol-
ogists 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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