jpet fast forward. published on january 10, 2012 as...

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.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 10, 2012 as DOI: 10.1124/jpet.111.187542

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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: [email protected]

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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Reference List

Agarwal V, Mittal S K and Misra R (2009) Expression of Multidrug Resistance-1 Protein

Correlates With Disease Activity Rather Than the Refractoriness to Methotrexate

Therapy in Rheumatoid Arthritis. Clin Rheumatol 28:427-433.

Bennett MK and Kirk C J (2008) Development of Proteasome Inhibitors in Oncology and

Autoimmune Diseases. Curr Opin Drug Discov Devel 11:616-625.

Borgnia MJ, Eytan G D and Assaraf Y G (1996) Competition of Hydrophobic Peptides,

Cytotoxic Drugs, and Chemosensitizers on a Common P-Glycoprotein Pharmacophore

As Revealed by Its ATPase Activity. J Biol Chem 271:3163-3171.

Bram EE, Adar Y, Mesika N, Sabisz M, Skladanowski A and Assaraf Y G (2009)

Structural Determinants of Imidazoacridinones Facilitating Antitumor Activity Are Crucial

for Substrate Recognition by ABCG2. Mol Pharmacol 75:1149-1159.

Chauhan D, Singh A V, Aujay M, Kirk C J, Bandi M, Ciccarelli B, Raje N, Richardson P

and Anderson K C (2010) A Novel Orally Active Proteasome Inhibitor ONX 0912

Triggers in Vitro and in Vivo Cytotoxicity in Multiple Myeloma. Blood 116:4906-4915.

Demo SD, Kirk C J, Aujay M A, Buchholz T J, Dajee M, Ho M N, Jiang J, Laidig G J,

Lewis E R, Parlati F, Shenk K D, Smyth M S, Sun C M, Vallone M K, Woo T M,

Molineaux C J and Bennett M K (2007) Antitumor Activity of PR-171, a Novel

Irreversible Inhibitor of the Proteasome. Cancer Res 67:6383-6391.


at ASPE



ownloaded from


JPET # 187542

25

Dick LR and Fleming P E (2010) Building on Bortezomib: Second-Generation

Proteasome Inhibitors As Anti-Cancer Therapy. Drug Discov Today 15:243-249.

Elliott PJ, Zollner T M and Boehncke W H (2003) Proteasome Inhibition: a New Anti-

Inflammatory Strategy. J Mol Med 81:235-245.

Eytan GD, Borgnia M J, Regev R and Assaraf Y G (1994) Transport of Polypeptide

Ionophores into Proteoliposomes Reconstituted With Rat Liver P-Glycoprotein. J Biol

Chem 269:26058-26065.

Gillet JP, Efferth T and Remacle J (2007) Chemotherapy-Induced Resistance by ATP-

Binding Cassette Transporter Genes. Biochim Biophys Acta 1775:237-262.

Groll M, Berkers C R, Ploegh H L and Ovaa H (2006) Crystal Structure of the Boronic

Acid-Based Proteasome Inhibitor Bortezomib in Complex With the Yeast 20S

Proteasome. Structure 14:451-456.

Gutman D, Morales A A and Boise L H (2009) Acquisition of a Multidrug-Resistant

Phenotype With a Proteasome Inhibitor in Multiple Myeloma. Leukemia 23:2181-2183.

Hershko A and Ciechanover A (1998) The Ubiquitin System. Annu Rev Biochem

67:425-479.

Kisselev AF, Callard A and Goldberg A L (2006) Importance of the Different Proteolytic

Sites of the Proteasome and the Efficacy of Inhibitors Varies With the Protein Substrate.

J Biol Chem 281:8582-8590.


at ASPE



ownloaded from


JPET # 187542

26

Kloetzel PM (2004) The Proteasome and MHC Class I Antigen Processing. Biochim

Biophys Acta 1695:225-233.

Lee SW, Kim J H, Park Y B and Lee S K (2009) Bortezomib Attenuates Murine

Collagen-Induced Arthritis. Ann Rheum Dis 68:1761-1767.

Li X, Wood T E, Sprangers R, Jansen G, Franke N E, Mao X, Wang X, Zhang Y,

Verbrugge S E, Adomat H, Li Z H, Trudel S, Chen C, Religa T L, Jamal N, Messner H,

Cloos J, Rose D R, Navon A, Guns E, Batey R A, Kay L E and Schimmer A D (2010)

Effect of Noncompetitive Proteasome Inhibition on Bortezomib Resistance. J Natl

Cancer Inst 102:1069-1082.

Lu S, Yang J, Chen Z, Gong S, Zhou H, Xu X and Wang J (2009) Different Mutants of

PSMB5 Confer Varying Bortezomib Resistance in T Lymphoblastic

Lymphoma/Leukemia Cells Derived From the Jurkat Cell Line. Exp Hematol 37:831-837.

Minderman H, Zhou Y, O'Loughlin K L and Baer M R (2007) Bortezomib Activity and in

Vitro Interactions With Anthracyclines and Cytarabine in Acute Myeloid Leukemia Cells

Are Independent of Multidrug Resistance Mechanisms and P53 Status. Cancer

Chemother Pharmacol 60:245-255.

Muchamuel T, Basler M, Aujay M A, Suzuki E, Kalim K W, Lauer C, Sylvain C, Ring E R,

Shields J, Jiang J, Shwonek P, Parlati F, Demo S D, Bennett M K, Kirk C J and

Groettrup M (2009) A Selective Inhibitor of the Immunoproteasome Subunit LMP7

Blocks Cytokine Production and Attenuates Progression of Experimental Arthritis. Nat

Med 15:781-787.


at ASPE



ownloaded from


JPET # 187542

27

Neubert K, Meister S, Moser K, Weisel F, Maseda D, Amann K, Wiethe C, Winkler T H,

Kalden J R, Manz R A and Voll R E (2008) The Proteasome Inhibitor Bortezomib

Depletes Plasma Cells and Protects Mice With Lupus-Like Disease From Nephritis. Nat

Med 14:748-755.

Oerlemans R, Franke N E, Assaraf Y G, Cloos J, van Z, I, Berkers C R, Scheffer G L,

Debipersad K, Vojtekova K, Lemos C, van der Heijden J W, Ylstra B, Peters G J,

Kaspers G L, Dijkmans B A, Scheper R J and Jansen G (2008) Molecular Basis of

Bortezomib Resistance: Proteasome Subunit Beta5 (PSMB5) Gene Mutation and

Overexpression of PSMB5 Protein. Blood 112:2489-2499.

Oerlemans R, van der H J, Vink J, Dijkmans B A, Kaspers G J, Lems W F, Scheffer G L,

Ifergan I, Scheper R J, Cloos J, Assaraf Y G and Jansen G (2006) Acquired Resistance

to Chloroquine in Human CEM T Cells Is Mediated by Multidrug Resistance-Associated

Protein 1 and Provokes High Levels of Cross-Resistance to Glucocorticoids. Arthritis

Rheum 54:557-568.

Ogiso Y, Tomida A and Tsuruo T (2002) Nuclear Localization of Proteasomes

Participates in Stress-Inducible Resistance of Solid Tumor Cells to Topoisomerase II-

Directed Drugs. Cancer Res 62:5008-5012.

Orlowski RZ and Kuhn D J (2008) Proteasome Inhibitors in Cancer Therapy: Lessons

From the First Decade. Clin Cancer Res 14:1649-1657.

Parlati F, Lee S J, Aujay M, Suzuki E, Levitsky K, Lorens J B, Micklem D R, Ruurs P,

Sylvain C, Lu Y, Shenk K D and Bennett M K (2009) Carfilzomib Can Induce Tumor Cell


at ASPE



ownloaded from


JPET # 187542

28

Death Through Selective Inhibition of the Chymotrypsin-Like Activity of the Proteasome.

Blood 114:3439-3447.

Ri M, Iida S, Nakashima T, Miyazaki H, Mori F, Ito A, Inagaki A, Kusumoto S, Ishida T,

Komatsu H, Shiotsu Y and Ueda R (2010) Bortezomib-Resistant Myeloma Cell Lines: a

Role for Mutated PSMB5 in Preventing the Accumulation of Unfolded Proteins and Fatal

ER Stress. Leukemia 24:1506-1512.

Ruschak AM, Slassi M, Kay L E and Schimmer A D (2011) Novel Proteasome Inhibitors

to Overcome Bortezomib Resistance. J Natl Cancer Inst 103:1007-1017.

Scheffer GL, Kool M, Heijn M, de H M, Pijnenborg A C, Wijnholds J, van H A, de Jong M

C, Hooijberg J H, Mol C A, van der L M, de Vree J M, van d, V, Elferink R P, Borst P and

Scheper R J (2000) Specific Detection of Multidrug Resistance Proteins MRP1, MRP2,

MRP3, MRP5, and MDR3 P-Glycoprotein With a Panel of Monoclonal Antibodies.

Cancer Res 60:5269-5277.

Seifert U, Bialy L P, Ebstein F, Bech-Otschir D, Voigt A, Schroter F, Prozorovski T,

Lange N, Steffen J, Rieger M, Kuckelkorn U, Aktas O, Kloetzel P M and Kruger E (2010)

Immunoproteasomes Preserve Protein Homeostasis Upon Interferon-Induced Oxidative

Stress. Cell 142:613-624.

Shafran A, Ifergan I, Bram E, Jansen G, Kathmann I, Peters G J, Robey R W, Bates S E

and Assaraf Y G (2005) ABCG2 Harboring the Gly482 Mutation Confers High-Level

Resistance to Various Hydrophilic Antifolates. Cancer Res 65:8414-8422.


at ASPE



ownloaded from


JPET # 187542

29

Sharma RC, Inoue S, Roitelman J, Schimke R T and Simoni R D (1992) Peptide

Transport by the Multidrug Resistance Pump. J Biol Chem 267:5731-5734.

Taylor CW, Dalton W S, Parrish P R, Gleason M C, Bellamy W T, Thompson F H, Roe D

J and Trent J M (1991) Different Mechanisms of Decreased Drug Accumulation in

Doxorubicin and Mitoxantrone Resistant Variants of the MCF7 Human Breast Cancer

Cell Line. Br J Cancer 63:923-929.

Toornvliet R, van Berckel B N, Luurtsema G, Lubberink M, Geldof A A, Bosch T M,

Oerlemans R, Lammertsma A A and Franssen E J (2006) Effect of Age on Functional P-

Glycoprotein in the Blood-Brain Barrier Measured by Use of (R)-[(11)C]Verapamil and

Positron Emission Tomography. Clin Pharmacol Ther 79:540-548.

van de Ven, Oerlemans R, van der Heijden J W, Scheffer G L, de Gruijl T D, Jansen G

and Scheper R J (2009) ABC Drug Transporters and Immunity: Novel Therapeutic

Targets in Autoimmunity and Cancer. J Leukoc Biol 86:1075-1087.

van der Heijden JW, de Jong M C, Dijkmans B A, Lems W F, Oerlemans R, Kathmann I,

Schalkwijk C G, Scheffer G L, Scheper R J and Jansen G (2004) Development of

Sulfasalazine Resistance in Human T Cells Induces Expression of the Multidrug

Resistance Transporter ABCG2 (BCRP) and Augmented Production of TNFalpha. Ann

Rheum Dis 63:138-143.

van der Heijden JW, Oerlemans R, Lems W F, Scheper R J, Dijkmans B A and Jansen

G (2009) The Proteasome Inhibitor Bortezomib Inhibits the Release of NFkappaB-


at ASPE



ownloaded from


JPET # 187542

30

Inducible Cytokines and Induces Apoptosis of Activated T Cells From Rheumatoid

Arthritis Patients. Clin Exp Rheumatol 27:92-98.

Wielinga PR, Reid G, Challa E E, van d H, I, van D L, de H M, Mol C, Kuil A J,

Groeneveld E, Schuetz J D, Brouwer C, De Abreu R A, Wijnholds J, Beijnen J H and

Borst P (2002) Thiopurine Metabolism and Identification of the Thiopurine Metabolites

Transported by MRP4 and MRP5 Overexpressed in Human Embryonic Kidney Cells.

Mol Pharmacol 62:1321-1331.

Zamora JM, Pearce H L and Beck W T (1988) Physical-Chemical Properties Shared by

Compounds That Modulate Multidrug Resistance in Human Leukemic Cells. Mol

Pharmacol 33:454-462.

Zhou HJ, Aujay M A, Bennett M K, Dajee M, Demo S D, Fang Y, Ho M N, Jiang J, Kirk C

J, Laidig G J, Lewis E R, Lu Y, Muchamuel T, Parlati F, Ring E, Shenk K D, Shields J,

Shwonek P J, Stanton T, Sun C M, Sylvain C, Woo T M and Yang J (2009) Design and

Synthesis of an Orally Bioavailable and Selective Peptide Epoxyketone Proteasome

Inhibitor (PR-047). J Med Chem 52:3028-3038.


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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: [email protected]


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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






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.

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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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


1.1

1.0

28.9

3.6 15 ± 1

19 ± 4

13 ± 1

13 ± 1


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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