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JPET #187542
Inactivating PSMB5 mutations and P-glycoprotein (MDR1/ ABCB1) mediate
resistance to proteasome inhibitors: ex vivo efficacy of (immuno) proteasome
inhibitors in mononuclear blood cells from rheumatoid arthritis patients
Sue Ellen Verbrugge, Yehuda G. Assaraf, Ben A.C. Dijkmans, George L. Scheffer,
Marjon Al, Debby den Uyl, Ruud Oerlemans, Elena T. Chan, Christopher J. Kirk,
Godefridus J. Peters, Joost W. van der Heijden, Tanja D. de Gruijl, Rik J. Scheper, and
Gerrit Jansen
Department of Rheumatology (S.E.V, B.A.C.D., M.A., D.d.U., R.O., J.vd H., G.J.);
Pathology (G.L.S., R.J.S.); and Medical Oncology (G.J.P., T.D.d.G.) VU University
Medical Center, Amsterdam, The Netherlands; The Fred Wyszkowski Cancer Research
Laboratory, Department of Biology, The Technion-Israel Institute of Technology, Haifa,
Israel (Y.G.A.) ; and Onyx Pharmaceuticals, South San Francisco, CA, U.S.A (E.T.C.,
C.J.K.)
JPET Fast Forward. Published on January 10, 2012 as DOI:10.1124/jpet.111.187542
Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.
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a) Running title: Resistance to epoxyketone-based proteasome inhibitors
b) Corresponding author:
Dr. Gerrit Jansen, PhD, Department of Rheumatology
VU Institute for Cancer & Immunology, Rm CCA 2.46
De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands
Tel: +31-20-4446685, Fax: +31-20-4442138
E-mail: g.jansen@vumc.nl
c) Number of …. text pages: 33
tables: 3
figures: 5
references: 40
words in the Abstract: 250
words in the Introduction: 655
words in the Discussion: 1332
d) Abbreviations
PSMB5: Proteasome subunit, beta type 5
BTZ: Bortezomib, Velcade®, [(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-
ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid
CFZ: Carfilzomib, PR-171, (S)-4-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-
methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-
((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide
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ONX-0912: PR-047, N-((S)-3-methoxy-1-(((S)-3-methoxy-1-(((S)-1-((R)-2-
methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-
yl)amino)-1-oxopropan-2-yl)-2-methylthiazole-5-carboxamide
ONX-0914: PR-957, (S)-3-(4-methoxyphenyl)-N-((S)-1-((S)-2-methyloxiran-2-
yl)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)
propanamido)propanamide
PI: Proteasome inhibitor
ABC: ATP-Binding Cassette
MDR: Multidrug resistance
MRP: Multidrug resistance-associated protein
MRP1; Multidrug Resistance-associated Protein 1 (ABCC1)
Pgp: P-glycoprotein(ABCB1; ATP Binding Cassette protein B1)
BCRP; Breast Cancer Resistance Protein (ABCG2)
TNFα: Tumor necrosis factor α
RA: Rheumatoid Arthritis
Vlb; Vinblastin
MXR; Mitoxantrone
e) Section: Drug discovery and translational medicine
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ABSTRACT
Bortezomib (BTZ), a registered proteasome inhibitor (PI) for multiple myeloma, has also
been proposed as a potential anti-rheumatic agent. Its reported side effects however,
make it unappealing for long-term administration and resistance may also develop. To
overcome this, second generation PIs became available. Here, we investigated whether
a novel class of peptide epoxyketone-based PIs including carfilzomib, ONX 0912 and
ONX 0914, might escape two established BTZ-resistance mechanisms: (a) mutation(s)
in the proteasome β5 subunit (PSMB5) targeted by these PIs, and (b) drug efflux
mediated by ABC transporters. THP1 myeloid sublines with acquired resistance to BTZ
(54-235 fold) due to mutations in PSMB5, displayed marked cross-resistance but less
pronounced to carfilzomib (9-32 fold), ONX 0912 (39-62 fold) and ONX 0914 (27-97
fold). As for ABC transporter mediated efflux: lymphoid CEM/VLB cells with P-
glycoprotein (Pgp/MDR1) overexpression exhibited substantial resistance to carfilzomib
(114-fold), ONX 0912 (23-fold), ONX 0914 (162-fold) whereas less resistance to BTZ
(4.5-fold) was observed. Consistently, the β5 subunit-associated chymotrypsin-like
proteasome activity was significantly less inhibited in these CEM/VLB cells. Ex vivo
analysis of peripheral blood mononuclear cells (PBMCs) from therapy naive rheumatoid
arthritis patients revealed that, although basal Pgp levels were low, it compromised the
inhibitory effect of carfilzomib and ONX 0914. However, the use of P121, a Pgp
transport inhibitor, restored parental cell inhibitory levels both in CEM/VLB cells as well
as in PBMCs. These results indicate that the pharmacologic activity of these PIs may be
hindered by drug resistance mechanisms involving PSMB5 mutations and PI extrusion
via Pgp.
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INTRODUCTION
The ubiquitin-proteasome system (UPS) plays a central role in maintaining cellular
homeostasis by controlling the timely breakdown of many key proteins. This includes the
regulation of cell cycle, activation of transcription factors (e.g. NF-κB) and induction of
apoptosis (Hershko and Ciechanover, 1998). As such, the UPS has been recognized as
an attractive target for cancer therapeutic intervention by proteasome inhibitors (PIs).
Currently available PIs target at least one of three β-subunits within the 20S core of the
proteasome that harbor catalytic activity; the β5 subunit (chymotrypsin-like activity), the
β1 subunit (caspase-like activity) and the β2 subunit (trypsin-like activity) (Kisselev et
al., 2006). Upon stimulation by pro-inflammatory stimuli, e.g. interferon-γ or TNF-α,
these constitutive proteasome subunits can be replaced by immunoproteasome subunits
β5i (LMP7), β2i (MECL1) and β1i (LMP2). This is followed by their assembly into
immunoproteasomes that have specialized functions in facilitating antigen presentation
via MHC-I (Kloetzel, 2004), (Hershko and Ciechanover, 1998), or, as indicated recently
(Seifert et al., 2010) to preserve protein homeostasis after interferon-γ-induced oxidative
stress.
Bortezomib (BTZ), a boron-containing dipeptide which primarily targets the β5 subunit of
the proteasome, was the first clinically approved proteasome inhibitor for the treatment
of therapy-refractory multiple myeloma (Orlowski and Kuhn, 2008). Emergence of
resistance to this drug, (Oerlemans et al., 2008), (Ruschak et al., 2011) as well as side
effects like peripheral neuropathy (Orlowski and Kuhn, 2008), initiated the search for
novel generation PIs that would be devoid of these limitations. These efforts have
resulted in a second generation of PIs that selectively target either β-subunits within the
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constitutive proteasome (Demo et al., 2007), (Zhou et al., 2009), (Chauhan et al., 2010),
(Ruschak et al., 2011) or the immunoproteasome (Muchamuel et al., 2009).
Beyond the successful application of PIs in cancer chemotherapy, there is a growing
body of data that these agents may also hold promise as drugs for the treatment of
(chronic) autoimmune diseases, e.g. rheumatoid arthritis and ulcerative colitis (Elliott et
al., 2003), (Bennett and Kirk, 2008). In fact, both BTZ and second generation PIs
displayed clear therapeutic efficacy in animal models of rheumatoid arthritis or lupus-like
disease (Lee et al., 2009), (Neubert et al., 2008), (Muchamuel et al., 2009). These
effects are in part mediated by their ability to suppress the production of NF-κB-inducible
proinflammatory cytokines from immune competent cells (van der Heijden et al., 2009).
Whenever it comes to clinical application of PIs for the treatment of autoimmune
diseases, the issue of retaining long term efficacy is particularly important as this type of
patients would face chronic drug treatment during which, drug resistance modalities may
emerge. Little is known about the underlying mechanisms of intrinsic or acquired
resistance to second generation PIs. For a prototypical epoxyketone-based proteasome
inhibitor like epoxomicin, it has been reported that cellular extrusion via the ABC
transporter ABCB1 (Pgp/MDR1) could confer drug resistance (Gutman et al., 2009).
Whether or not cellular extrusion by ABC pumps other than ABCB1 (Pgp/MDR1) can
also contribute to these second generation expoxyketone-based PIs is yet unknown.
In the present study we explored whether mutations in the PSMB5 gene, encoding for
the β5 subunit of the proteasome, and cellular extrusion via drug efflux transporters
(Gillet et al., 2007), (van de Ven et al., 2009) may interfere with the efficacy of 3 second
generation epoxyketone-based PIs i.e. Carfilzomib (formerly named PR-171) (Demo et
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al., 2007), ONX 0912 (formerly named PR-047) (Zhou et al., 2009;Chauhan et al.,
2010), and the immunoproteasome inhibitor ONX 0914 (formerly named PR-957)
(Muchamuel et al., 2009). Our data provide evidence that only ABCB1 (Pgp/MDR1), but
none of the other ABC transporters, harbors the ability to extrude these drugs and can
confer drug resistance in a cell line model with high ABCB1(Pgp/MDR1)-overexpression.
However, in an ex vivo setting with peripheral blood mononuclear cells (PMBCs) from
healthy controls and rheumatoid arthritis patients, basal ABCB1(Pgp/MDR1) activities
were modest and still allowed for the retention of proficient proteasome inhibitory
capacity by the 3 epoxyketone-based drugs.
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MATERIALS AND METHODS
Proteasome inhibitors and other chemicals
Carfilzomib, ONX 0912, ONX 0914 were provided by Onyx Pharmaceuticals, Inc. (South
San Francisco, USA). Bortezomib (BTZ)/Velcade™ was kindly provided by Millennium
Pharmaceuticals (Cambridge, USA). The chemical structures of these compounds are
depicted in Figure 1. The P-glycoprotein inhibitor Reversin-121 (P121) was obtained
from Alexis Benelux, The Netherlands. Syto16 was purchased from Invitrogen, Breda,
The Netherlands.
Cell lines
Human monocytic-macrophage THP1 cells with various levels of acquired resistance to
BTZ were selected as described previously (Oerlemans et al., 2008). Multiple human
cell lines were selected based on their known expression of ABC transporters; P-
glycoprotein/MDR1 (CEM/VLB) (Zamora et al., 1988), Multidrug Resistance-associated
Protein 1 (CEM/CHQ, 2008/MRP1) (Oerlemans et al., 2006), (Scheffer et al., 2000),
MRP2 (2008/MRP2) (Scheffer et al., 2000), MRP3 (2008/MRP3) (Scheffer et al., 2000),
MRP4 (HEK293/MRP4) (Wielinga et al., 2002), MRP5 (HEK293/MRP5) (Wielinga et al.,
2002), and BCRP (CEM/SSZ & MCF7/MR) (van der Heijden et al., 2004), (Taylor et al.,
1991).
Cell cultures were maintained in RPMI-1640 medium supplemented with 10% FCS, 2
mM L-glutamine and 1000U/ml penicillin/streptomycin. Cells were routinely cultured at
an initial density of 3 x 105 cells/ml (for suspension cells) and 5 x 103 cells/cm2 for
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adherent cells, all in 25-cm2 culture flasks (Greiner Bio-One, Frickenhansen, Germany)
at 37°C in a humidified 5% CO2 atmosphere.
Patients/healthy controls and isolation of blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls (n= 22)
and newly diagnosed untreated RA patients. Samples for this study were collected
within the COBRA-light study, a randomized trial comparing two combination schedules
in early rheumatoid arthritis. Informed consent was obtained from all patients and the
study was approved by the Ethical Review Board of the VU medical center. Blood was
collected at baseline prior to initiation of the therapy protocol with methotrexate and
prednisolone. Patients eligible to be included in this study fulfilled the following criteria:
RA diagnosis according to the ACR 1987 criteria, disease duration > two years, age >
18 years, active RA with > 6 swollen joints, >6 painful joints and an erythrocyte
sedimentation rate of > 28 mm of VAS (global health) >2. PBMCs were isolated by
Ficoll-Paque (Amersham Biosciences, Amersham, UK) density gradient centrifugation
(15 minutes at 1000 x g at room temperature) according to manufacturer’s instructions.
The interphase was collected and washed three times with phosphate-buffered saline
(PBS) supplemented with 1% BSA. PBMCs were then counted and resuspended in
RPMI-1640 culture medium supplemented with 10% FCS.
Assessment of cell growth inhibition by proteasome inhibitors
The growth inhibitory effects of BTZ, carfilzomib, ONX-0912, ONX-0914 were analyzed
by methods essentially as previously described (Oerlemans et al., 2006), (Shafran et al.,
2005). Briefly, for suspension cell cultures, 1.25 x 105 cells/ml were plated in individual
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wells of a 24-well cell culture plates containing an increasing concentration of each
proteasome inhibitor. For adherent cells, a plating density of 1 x 104/cm2 was used and
drugs were added 24 hours after plating. Inhibition of cell growth was determined after
72 hours drug incubation by counting viable cells (for adherent cells after trypsinization)
on the basis of trypan blue exclusion. Eight drug concentrations (in duplicate) were used
covering a 100-200 fold concentration range. The proteasome inhibitor concentration
required to inhibit cell growth by 50% compared with control growth is defined as the
IC50 concentration.
Intact cell-based assay for chymotrypsin-like, caspase-like and trypsin-like
proteasome activities
An intact cell-based assay to measure basal and proteasome inhibitor induced
suppression of each of 3 types of proteasome activities; chymotrypsin-, caspase-, and
trypsin-like activity was performed using Proteasome-GloTM assay kit (Promega,
Madison, WI, USA) according to manufacturer’s instructions. Prior to determination of
proteasome activity, cells were incubated with the proteasome inhibitors for 1 hour at
37°C in a flat, white-bottomed 96-well plate (Thermo Scientific, NUNC, Roskilde,
Denmark) at a density of 15,000 cells per well in 50 µl. After drug exposure, a
luminogenic substrate specific for chymotrypsin-like, caspase-like or trypsin-like
protease activity was added to the intact cell suspension. After 10 min incubation at
37°C, luminescence was determined with an Infinite 200 pro microplate reader (Tecan,
Giessen, The Netherlands). Negative controls consisted of wells with medium without
cells. The potency of proteasome inhibitors is indicated by the drug concentration that
elicits 50% inhibition of luminescence signal.
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Proteasome subunit determination with the ProCISE assay
The ProCISE (proteasome constitutive/immunoproteasome subunit enzyme linked
generated and immunosorbent) assay was performed on untreated cell lysates as was
previously described (Parlati et al., 2009). Luminescence was measured using a
GENios-Basic plate reader (Tecan Austria, GmBH). Data was statistically analysed with
XLfit Excel Add-In (ID Business Solutions Limited).
Assessment of Pgp/MDR1 functional activity by flow cytometry
Measurement of Pgp functional activity was performed essentially as described
previously by Toornvliet et al. (Toornvliet et al., 2006). The assay was validated using
the Pgp- overexpressing human cell line CEM/VLB. In brief, cells were incubated in a
total volume of 500 µl at a cell density of 3 x 105 cells/ml for 60 minutes in a 37°C water
bath in the presence of a fluorescent chromophore and Pgp substrate Syto16 (10 nM) in
the absence or presence of the specific Pgp transport inhibitor P121 (final concentration:
7.5 µM). After incubation, cells were washed twice with ice-cold PBS supplemented with
0.1% BSA and kept on ice protected from light for 30 min. Flow cytometric analysis was
performed using a FACS Scan (Becton Dickinson) with a 530nm (FL1) laser. Cell quest
software (Becton Dickinson) was used for data acquisition and analysis. Pgp transporter
activity was expressed as Activity Index (AI) described by the following formula:
cells loadednon antagonist of absence in the level ceFluorescenMean
cells loadednon antagonist of presence in the level ceFluorescenMean
An index of > 1.10 (i.e. 10% above background, set to 1) is representative for functional
Pgp activity.
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Statistical analysis
For comparison between groups a two sided paired Student’s t-test was used.
Differences were considered to be significant when p < 0.05.
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RESULTS
Bortezomib-resistant THP1 cells with acquired mutations in PSMB5 display cross-
resistance to epoxyketone-based proteasome inhibitors.
Human THP1 cells with acquired resistance to BTZ cells due to a mutation residing in a
highly conserved BTZ-binding pocket of the β5 subunit of the proteasome (PSMB5)
protein (Oerlemans et al., 2008) were evaluated for cross-resistance to epoxyketone-
based PIs. Two BTZ-resistant THP1 selectants were studied; one subline (THP1/BTZ50)
isolated after stepwise selection up to 50 nM BTZ that had a single Ala49Thr mutation in
the PSMB5 protein, exhibited 54-fold resistance to BTZ. The other subline
(THP1/BTZ500) that was established by gradual selection up to 500 nM BTZ, harbored
two PSMB5 mutations (Ala49Thr and Met45Ile) and was 235-fold BTZ resistant (Table
1). Both THP1/BTZ50 and THP1/BTZ500 cells displayed marked cross-resistance to the
epoxyketone-based PIs ONX 0912 (39-fold and 62-fold, respectively) and ONX 0914
(27-fold and 97-fold, respectively). Cross-resistance levels to carfilzomib were moderate
in THP1/BTZ50 cells (9-fold), but elevated (32-fold) in THP1/BTZ500 cells, but still 10-
fold less than for BTZ (Table 1A). Part of the cross-resistance may be associated with a
2-3 fold increase in constitutive proteasome subunits β5, β2 and β1 expression (Table
1B). Together, these results suggest that mutations in PSMB5 induced by BTZ lead to
drug resistance to different classes of PIs which bind both the β5 and the β5i (Zhou et
al., 2009), (Chauhan et al., 2010), (Muchamuel et al., 2009) subunit of the proteasome.
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Role of drug efflux transporter (Pgp/MDR1) in resistance to epoxyketone-based
PIs.
Previous evidence indicated that epoxomicin, a prototypical epoxyketone-based PI, is a
substrate for the multidrug efflux transporter P-glycoprotein (Pgp)/MDR1/ABCB1
(Gutman et al., 2009). We explored whether or not carfilzomib, ONX 0912 and ONX
0914 could also serve as substrates for Pgp/MDR1/ABCB1 and other drug efflux
transporters of the ABC-superfamily including multidrug resistance-associated protein 1-
5 (MRP1-5/ABCC1-5) and breast cancer resistance protein (BCRP/ABCG2). To this end
we examined the growth inhibitory effects of BTZ, carfilzomib, ONX 0912 and ONX 0914
in a panel of human cell lines differentially overexpressing these MDR efflux pumps
(Table 2). The basal growth inhibitory potency was tested in a panel of 4 parental cell
lines (CEM, 2008, HEK293 and MCF7) in which the greatest sensitivity was displayed
for BTZ and carfilzomib, followed by ONX 0912 and the immunoproteasome inhibitor
ONX 0914. For BTZ, only human CEM T cell leukemia cells (CEM/VLB) overexpressing
Pgp displayed a modest level (4.5 fold) of cross-resistance. In contrast, CEM/VLB cells
exhibited marked levels of resistance to ONX 0914 (162-fold), carfilzomib (114-fold) and
ONX 0912 (23-fold), compared to parental cells. Notably, no appreciable level of
resistance to BTZ, carfilzomib, ONX 0912 and ONX 0914 was noted for cell lines
overexpressing MRP1-5 (Table 2). Interestingly, MCF7/MR cells with BCRP/ABCG2
overexpression displayed ~3-fold greater sensitivity to carfilzomib, ONX 0912 and ONX
0914, than their parental MCF7 cells. However, this effect was not related to BCRP
overexpression as Ko143, a potent transport inhibitor of BCRP activity, failed to alter this
increased sensitivity (results not shown). These results demonstrate that Pgp, but not
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MRP1-5 or BCRP, may contribute to drug efflux and consequent resistance to
epoxyketone-based PIs and BTZ.
P-glycoprotein blockade overcomes resistance to epoxyketone-based PIs by
retention of inhibition of proteasome activity.
To confirm the potential role of Pgp in conferring resistance to BTZ, carfilzomib, ONX
0912 and ONX 0914, we examined the growth inhibitory effects of these PIs after
blockade of Pgp efflux activity with the specific inhibitor P121/Reversin. Indeed, blocking
Pgp activity in CEM/VLB cells nearly fully restored parental CEM cell sensitivity levels to
PIs (Figure 2).
We next assessed whether or not drug extrusion by Pgp would also diminish the ability
of the proteasome inhibitors to inhibit chymotrypsin-like proteasome activity. As
expected, markedly higher concentrations of carfilzomib (22.3-fold), ONX 0912 (11.0-
fold), ONX 0914 (26.3-fold) and BTZ (1.4-fold) were required for 50% inhibition of
intracellular chymotrypsin-like proteasome activity in CEM/VLB cells (Table 3).
Furthermore, blocking Pgp function by P121 restored the capacity of carfilzomib, ONX
0912, ONX 0914 and BTZ to inhibit chymotrypsin-like proteasome activity at inhibitory
concentrations obtained with parental CEM cells. Together, these data underscore a
role for Pgp, both in attenuating the potency of proteasome inhibition and conferring
resistance to carfilzomib, ONX 0912 and ONX 0914. BTZ function, in comparison to the
epoxyketone-based PIs, is the least dependent on Pgp. Nevertheless, this effect could
be readily counteracted by blocking Pgp function.
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Pgp activity in PBMCs from controls and RA patients modestly contributes to
diminished proteasome inhibitory capacity of epoxyketone-based PIs.
Since high levels of Pgp overexpression underlie the marked resistance to epoxyketone-
based PIs in CEM/VLB cells, we assessed whether or not basal physiological levels of
Pgp activity in peripheral blood mononuclear cells (PBMCs) would also compromise the
efficacy of these PIs in comparison to BTZ. To this end, we first determined Pgp efflux
activity in parental CEM/WT vs. CEM/VLB cells and compared it with Pgp activity in
PBMCs cells from healthy controls and RA patients. CEM/VLB cells displayed a Pgp
activity index of 17.3 ± 6.2 which is > 80-fold higher than CEM/WT cells (activity index:
1.18 ± 0.13), which is just above a functionally detectable activity index of 1.10 ± 0.1.
Functional Pgp activity in PBMCs from RA patients and controls were slightly higher
(activity index: 1.55 ± 0.36 and 2.08 ± 0.69) than in parental CEM/WT cells, but were still
> 30-fold lower than in Pgp-overexpressing CEM/VLB cells (Figure 3). We next
determined the effect of Pgp blockade by P121 on the efficiency of carfilzomib, ONX
0912, ONX 0914 and BTZ to inhibit chymotrypsin-like activity in PBMCs. Both
carfilzomib- and ONX 0914-induced inhibition of chymotrypsin-like activity was
significantly enhanced (2-fold and 1.3-fold, respectively, ∗p < 0.05) after blocking Pgp
efflux activity in PBMCs from RA patients (Figure 4A). Similar findings were observed in
the control PBMCs (Figure 4A) where the Pgp function blockage only seem to affect
inhibitory capacity of carfilzomib and ONX 0914 (1.9-fold and 1.4-fold, respectively, ∗∗∗p
= 0.001; ∗∗p = 0.007). In contrast, both in RA as in control PBMCs, no significant
changes were observed in the inhibitory potential of ONX 0912 and BTZ after Pgp
blockade. It is noteworthy that the concentration of carfilzomib, ONX 0912, ONX 0914
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and BTZ needed for 50% inhibition of the chymotrypsin-like proteasome activity in RA
PBMCs is higher compared to control PBMCs. This effect seems to be due to a higher
chymotrypsin-like activity found in RA PBMCs as compared to healthy controls (Figure
4B).
These results suggest that further enhancement of inhibition of chymotrypsin-like activity
by carfilzomib and ONX 0914 may be achieved by abolishing the Pgp-mediated cellular
efflux of these PIs.
Potency of carfilzomib, ONX912, ONX914 and bortezomib to inhibit chymotrypsin-
like proteasome activity in PBMCs from controls and RA patients.
To further explore the mechanism by which the epoxyketone-based PIs function in
PBMCs we examined whether or not, apart from chymotrypsin-like activity, these PIs
could also inhibit the caspase-like and trypsin-like proteasome activities in PBMCs from
healthy controls and RA patients. Notably, chymotrypsin-like activity was preferentially
inhibited by all 3 epoxyketone-based PIs as well as by BTZ (Figure 5). In a small series
of PBMCs from healthy controls (n=2) and RA patients (n=4), we noted that the relative
potency (referring to drug concentrations required for 50% proteasome activity inhibition)
to inhibit caspase-like and trypsin-like activity relative to chymotrypsin-like activity (set to
1) was 0.17 and 0.06 for bortezomib; 0.1 and 0.3 for carfilzomib; 0.02 and 0.06 for ONX
0912, and 0.02 and 0.09 for ONX 0914, respectively (not shown).
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DISCUSSION
Here we demonstrate that bortezomib shows a different resistance profile than the
epoxyketone-based PIs carfilzomib, ONX 0912 and ONX 0914. An established
resistance mechanism to bortezomib due to mutations in the PSMB5 gene encoding the
β5 subunit of the proteasome results in cross-resistance to the novel compounds, albeit
at a lower level. However, the novel PIs display resistance due to drug efflux via Pgp, in
a human CEM cell line (CEM/VLB) with high Pgp overexpression. In PBMCs from
healthy controls and RA patients with basal levels of Pgp, blocking Pgp drug efflux
enhanced proteasome inhibition.
Notwithstanding the established therapeutic activity of BTZ (Velcade) as the first
approved PI, emergence of drug resistance phenomena and side effects of BTZ such as
neuropathy, called for the design and (pre)clinical evaluation of second generation PIs
that can overcome these drawbacks (Dick and Fleming, 2010). Epoxyketone-based PIs
like carfilzomib, ONX 0912 and ONX 0914 differ from BTZ in their mode of action by the
fact that they bind irreversibly to active site amino acid Thr1 within the binding pocket of
the β5-subunit of the proteasome. In contrast, BTZ binds reversibly to this active site
(Groll et al., 2006), (Demo et al., 2007). ONX 0914 was designed as a more selective
inhibitor of the immunoproteasome over the constitutive proteasome (Muchamuel et al.,
2009). We (Oerlemans et al., 2008) and others have previously shown that acquired
BTZ resistance led to single amino acid substitutions in the highly conserved substrate-
and BTZ-binding pocket of the β5-subunit (Ri et al., 2010), (Lu et al., 2009). These
mutations in the PSMB5 gene conferred resistance to the parent drug BTZ as well as
cross-resistance to other peptide aldehyde-based PIs including ALLN, MG132, MG262
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and a hexapeptide 4A6 (Oerlemans et al., 2008). Therefore we investigated whether the
epoxyketone-based PIs like carfilzomib, ONX 0912 and ONX 0914 confer cross-
resistance in BTZ-resistant cell lines with PSMB5 mutations: THP1/BTZ 50 cells
(Ala49Thr) and the highly BTZ-resistant THP1/BTZ500 cells (Ala49Thr and Met45Ile
mutation). Notably, marked cross-resistance was observed for these drugs in cell lines
harboring β5-subunit mutations, although to a lesser extent, thus suggesting that these
acquired mutations confer upon cells reduced binding capacity for a broad spectrum of
PIs. It should be mentioned, however, that to date, β5-subunit mutations have not been
identified yet in clinical specimens which are refractory to BTZ treatment; hence, the
clinical relevance of the in vitro PSMB5 mutation data in multiple BTZ-resistant cell line
models (Ri et al., 2010), (Lu et al., 2009), (Li et al., 2010) is still not clear.
Inherent or acquired drug-induced overexpression of ABC drug efflux transporters is a
common resistance modality that can result in the multidrug resistance phenotype (Gillet
et al., 2007). Pgp/MDR1 is the best known family member of MDR efflux transporters
facilitating not only cellular extrusion of neutral/hydrophobic small molecule drugs but
also several types of peptides (cyclic/linear and neutral/hydrophobic), including
leupeptin, pepstatin A, dolastatin 10, as well as PIs such as the tripeptide N-acetyl-
leucyl-leucyl-norleucinal and expoxomicin (Eytan et al., 1994), (Borgnia et al., 1996),
(Sharma et al., 1992), (Gutman et al., 2009). Therefore it may not seem surprising that
peptide epxoyketone-based PIs may be recognized by Pgp as transport substrates. This
was established by several lines of evidence; (a) Marked cross-resistance of Pgp-
overexpressing cells to these PIs, (b) requirement for higher proteasome inhibitor
concentrations in Pgp-overexpressing cells compared to parental cells to inhibit
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intracellular chymotrypsin-like proteasome activity, and (c) full reversal of both cross-
resistance and chymotrypsin-like proteasome inhibition after Pgp blockade with the
established Pgp transport inhibitor, P121. BTZ, a boron-containing peptide proved to be
a poor Pgp-substrate, consistent with previous studies (Minderman et al., 2007;
Oerlemans et al., 2008).
The notion that the orally active proteasome inhibitor ONX0912 was also
recognized as a Pgp transport substrate, albeit less prominently than carfilzomib and
ONX 0914, may be of relevance for its bioavailability, which was found to be 39% in
rodents and dogs (Zhou et al., 2009), given the fact that intestinal Pgp is a major
determinant of drug efflux and absorption (van de Ven et al., 2009). Hence, evading the
Pgp-dependent mucosal epithelium barrier may further enhance ONX 0912
bioavailability. The present study also provided evidence that carfilzomib, ONX 0912
and ONX 0914 were not substrates for other multidrug efflux transporters including
MRP1-5 and BCRP. Interestingly, BCRP-overexpressing MCF7/MR cells even displayed
3-4 fold greater sensitivity for BTZ, carfilzomib, ONX 0912 and ONX 0914. This
hypersensitivity is likely due to an intrinsic property of MCF7/MR cells acquired after
establishment of resistance to the topoisomerase inhibitor mitoxantrone as Ogiso et al.
(Ogiso et al., 2002) reported that under these conditions, increased accumulation of the
proteasome occurs at the nucleus with concomitantly decreased proteasome levels in
the cytoplasm. This might have a more prominent inhibition of cytoplasmic proteasome
activity hence resulting in hypersensitivity to PIs.
In order to explore the potential in vivo impact of Pgp in drug resistance to peptide
epoxyketone-based PIs, we assessed the efficiency of proteasome inhibition in PBMCs
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isolated from RA patients and healthy controls. We (van der Heijden et al., 2009) and
others (Agarwal et al., 2009) reported that Pgp expression on lymphocytes from RA
patients correlated with disease activity. Here we showed that the low basal Pgp activity
in ex vivo isolated PBMCs partially reduced the potent (i.e. nanomolar) inhibition of the
chymotrypsin-like proteasome activity by carfilzomib, ONX 0912, ONX 0914 and BTZ.
However, for carfilzomib and ONX 0914, the two PIs that displayed the highest cross-
resistance levels in Pgp-overexpressing CEM cells, up to 2-fold further enhancement of
chymotrypsin-like proteasome activity inhibition could be achieved by blocking Pgp-
mediated drug efflux, thereby underscoring the role of Pgp in resistance to these PIs. It
is of interest to note that the ranking of potency of chymotrypsin-like proteasome
inhibition in PBMCs from RA patients and healthy controls (carfilzomib > ONX 0914 >
ONX 0912) is different from their ranking for growth inhibitory potency (carfilzomib >
ONX 0912 > ONX 0914) observed for 4 different parental cell lines (CEM, HEK293,
A2008 and MCF7 cells). This result may be due to higher immunoproteasome content in
ex vivo isolated PBMCs than in cell line cultures in vitro. As such, cell line models may
underestimate the full potential of immunoproteasome targeted drugs like ONX914,
which may be more prominently displayed in immunocompetent cells in vivo.
In conclusion, single amino acid substitutions in the substrate/inhibitor binding site of the
proteasome β5 subunit as well as overexpression of the drug efflux transporter Pgp
were identified as modalities conferring resistance to the peptide epoxyketone-based
(immuno)proteasome inhibitors carfilzomib, ONX 0912 and ONX 0914. However,
targeting of immune cells harboring basal levels of Pgp did not show cross resistance to
ONX 0912 while carfilzomib was effected only to a small extent. Hence, to identify the
structural elements within peptide epoxyketone-based chemical structures that render
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them Pgp substrates, may be an important strategy to evade Pgp-mediated drug efflux
and to overcome drug resistance. In fact, we have recently shown that small chemical
modifications can generate analogues of a class of topoisomerase drug inhibitors, i.e.
imidazoacridinones, which were no longer substrates for the drug efflux transporter
BCRP (Bram et al., 2009). Indeed, also Zhou et al (Zhou et al., 2009) showed that
modifications in the N-cap of the peptide epoxyketone backbone, in particular
introducing (5-Me)-3-isoxazole or 2-(S)-tetrahydrofuran, retained chymotrypsin-like
proteasome activity while Pgp substrate activity was abolished. Such considerations in
future drug design may be crucial for the rational overcoming of established modalities
of MDR.
Taken together, our data provide evidence that cellular extrusion via the drug efflux
transporter ABCB1 (Pgp/MDR1), but not by other ABC transporters, can facilitate
resistance to peptide epoxyketone-based proteasome inhibitors in Pgp-overexpressing
model systems. In PBMCs of controls and RA patients, the presence of basal Pgp
activity only modestly influenced the proteasome inhibitory potential by the epoxyketone
based drugs. Blocking of Pgp activity could further potentiate their activity, though
caution for increased toxicity may also need to be exercised when these combinations
would be used in the clinic.
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Authorship Contributions
Participated in research design: Verbrugge, Assaraf, de Gruijl and Jansen
Conducted experiments: Verbrugge, Al, Chan, Oerlemans and Jansen
Contributed new reagents or analytic tools: Scheffer, den Uyl, Oerlemans, van der
Heijden, Chan, Kirk, Peters and Jansen
Performed data analysis: Verbrugge, Chan and Jansen
Wrote or contributed to the writing of the manuscript: Verbrugge, Assaraf, Jansen,
Dijkmans, Scheffer, Kirk, Peters, de Gruijl and Scheper.
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Footnotes
This study is supported by Cancer Center Amsterdam (CCA)/VU- Institute of Cancer and
Immunology (V-ICI) [Grant 07/36]
Y.G. Assaraf is recipient of an Award Visiting Professor Fellowships from the Royal
Netherlands Academy of Arts and Sciences (KNAW) and the Netherlands Organization
for Scientific Research (NWO).
Address correspondence to:
Dr. Gerrit Jansen, PhD
Department of Rheumatology
VU Institute for Cancer & Immunology, Rm CCA 2.46
De Boelelaan 1117
1081 HV Amsterdam
The Netherlands
T: +31-20-4446685
F: +31-20-4442138
E: g.jansen@vumc.nl
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Legends for figures
Figure 1: Chemical structures of proteasome inhibitors.
Chemical structures of the peptide epoxyketone-based compounds carfilzomib, ONX
0912 and ONX 0914, and the boronic acid peptide bortezomib.
Figure 2: Reversal of resistance to carfilzomib, ONX 0912 and ONX 0914 by P121-
mediated blocking of Pgp.
Pgp-overexpressing CEM/VLB cells were incubated with or without the Pgp-blocking
peptide P121 (7.5 μM) and proteasome inhibitors for 72 hours. Depicted is the change in
resistance factor which is the ratio of drug concentration required to inhibit cell growth of
CEM/VLB by 50% over CEM/WT cells. Results are the mean ± S.D. of at least 3
experiments.
Figure 3: Pgp functional activity in PBMCs from healthy controls and RA patients
vs CEM/VLB cells.
Cells were incubated with the chromophore Syto16 for 1 hour in the absence or
presence of 7.5 μM of the Pgp-blocking peptide P121. The Pgp activity index is
presented as ratio of Syto16 retention in cells incubated with P121 over cells without
P121. Results for PBMCs are means ± SD of 4-8 experiments.
Figure 4: Effect of Pgp blocking on inhibition of chymotrypsin-like (ChT-L)
proteasome activity in PBMCs from RA patients.
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A: PBMCs from RA patients were incubated for 1 hour with various concentrations of
carfilzomib, ONX 0912, ONX 0914 and bortezomib in the absence or presence of the
Pgp-blocking peptide P121 (7.5 μM) and then analyzed for inhibition of chymotrypsin-
like proteasome activity. Results depict drug concentrations required to inhibit
chymotrypsin-like proteasome activity for 50% (IC50). Note: at the indicated
concentration, P121 had no effect on chymotrypsin-like proteasome activity (not shown).
B: Chymotrypsin-like proteasome activity measured in PBMCs from RA patients and
healthy controls after isolation.
Figure 5: Inhibitory effects of carfilzomib, ONX 0912, ONX 0914 and bortezomib on
chymotrypsin-like, caspase-like and trypsin-like proteasome activity in PBMCs
from healthy controls and RA patients.
PBMCs from healthy controls and RA patients were incubated for 1 hour with various
concentrations of carfilzomib, ONX 0912, ONX 0914 and bortezomib and then analyzed
for inhibition of chymotrypsin-like (ChT-L), caspase-like (CL) and trypsin-like (TL)
proteasome activity. Results depict individual (●) and group mean ( ⎯⎯) drug
concentrations required to inhibit each of these proteasome activities by 50% (IC50). ↑
indicates that IC50 value was not reached at the highest drug concentration tested.
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3.5 ± 1.7 11.7 ± 1.8 2.6 ± 1.7 11.1 ± 3.8 5.7 ± 1.9 THP1/BTZ500
4.9 ± 2.9 10.4 ± 1.6 2.9 ± 1.4 10.6 ± 1.9 THP1/BTZ50
5.7 ± 1.8 5.6 ± 0.7 3.1 ± 0.7 5.7 ± 0.7 2.5 ± 1.3 THP1/WT
ng subunit/ ug total protein
Cell line
7.3 ± 2.4
3.3 ± 0.7
1.0 ± 0.5 0.5 ± 0.2
ng subunit/ ug total protein
ng subunit/ ug total protein
ng subunit/ ug total protein
ng subunit/ ug total protein
ng subunit/ ug total protein
Beta5 LMP7 Beta1
823 ± 65 (235) 10092 ± 1642 (97) 4395 ± 458 (62) 501 ± 69 (32) Ala49Thr & Met45lle THP1/BTZ500
218 ± 39 (54) 2741 ± 428 (27) 2686 ± 318 (39) 160 ± 4 (9) Ala49Thr THP1/BTZ50
3.5 ± 0.7 (1) 99 ± 11 (1) 69 ± 9 (1) 16.4 ± 3.9 (1) none THP1/WT
IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM) ONX 0914 ONX 0912 Carfilzomib Bortezomib Cell line PSMB5 mutation
(amino acid shift)
Beta2 MECL1 LMP2
Table 1A: Effect of PSMB5 mutations on growth inhibitory effects of Bortezomib and epoxyketone-based proteasome inhibitors
Data presented are the mean of 3-6 separate experiments ± SD after 72h drug exposure. Values between brackets depict Resistance Factor (ratio IC50 selected cell line over parental (WT) cell line) Table 1B: Effect of PSMB5 mutations on proteasome subunit composition
Data presented are the mean of 3 separate experiments ± SD.
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Table 2: Effect of Multidrug Resistance (MDR) drug efflux transporter expression on growth inhibitory effect of Bortezomib and expoxyketone-based proteasome inhibitors
Data presented are the mean of 3-6 separate experiments ± SD. Values between brackets depict the Resistance Factor (ratio of IC50 values of selected cell line over parental (WT) cell line Abbreviations: Pgp: P-glycoprotein/MDR1, MRP: Multidrug Resistance-related Protein, BCRP: Breast Cancer Resistance Protein
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29 ± 4 + P121
148 ± 34 no blocker CEM/VLB
6 ± 1 + P121
7 ± 1 no blocker CEM/WT
IC50 (nM)
ONX 0914 ONX 0912 Carfilzomib Cell line
Resistance Factor
1.0
1.0
24.3
4.1 93 ± 49
693 ± 247
54 ± 18
63 ± 24
IC50 (nM) Resistance Factor
1.0
1.0
7.7
1.0 181 ± 77
1438 ± 453
51 ± 7
55 ± 7
IC50 (nM) Resistance Factor
1.1
1.0
28.9
3.6 15 ± 1
19 ± 4
13 ± 1
13 ± 1
IC50 (nM) Resistance Factor
1.0
1.0
1.4
1.1
Bortezomib
Table 3: Inhibition of chymotrypsin-like (ChT-L) proteasome activity in CEM/WT cells vs. Pgp/MDR1-overexpressing CEM/VLB cells in the absence or presence of the Pgp-blocker P121
IC50 values (in nM) depict drug concentrations establishing 50% inhibition of ChT-L activity after 1 hour drug exposure. Data presented are the mean of 3 separate experiments ± SD. Resistance Factor depicts the ratio of IC50 values of selected cell line over parental (WT) cell line.
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