Proteasome Subunit 5 (PSMB5)

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NEOPLASIA

Molecular basis of bortezomib resistance: proteasome subunit ␤5(PSMB5) gene mutation and overexpression of PSMB5 protein

*Ruud Oerlemans,1 *Niels E. Franke,2 Yehuda G. Assaraf,3 Jacqueline Cloos,2 Ina van Zantwijk,2 Celia R. Berkers,4 George L. Scheffer,5 Kabir Debipersad,1 Katharina Vojtekova,2 Clara Lemos,6 Joost W. van der Heijden,1 Bauke Ylstra,7 Godefridus J. Peters,6 Gertjan L. Kaspers,2 Ben A. C. Dijkmans,1 Rik J. Scheper,5 and Gerrit Jansen1

Departments of 1Rheumatology and 2Pediatric Oncology/Hematology, VUMC Institute for Cancer & Immunology, VU University Medical Center, Amsterdam, The Netherlands; 3The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel; 4Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands; and Departments of 5Pathology and 6Medical Oncology, and 7Microarray Facility, VUMC Institute for Cancer & Immunology, VU University Medical Center, Amsterdam, The Netherlands

The proteasome inhibitor bortezomib is a trations of bortezomib. Study of the mo- and ALLN, but not to a broad spectrum of novel anticancer drug that has shown lecular mechanism of bortezomib resis- chemotherapeutic drugs, (4) no marked promise in the treatment of refractory tance in these cells revealed (1) an changes in chymotrypsin-like protea- multiple myeloma. However, its clinical Ala49Thr mutation residing in a highly some activity, and (5) restoration of bort- efﬁcacy has been hampered by the emer- conserved bortezomib-binding pocket in ezomib sensitivity in bortezomib-resis- gence of drug-resistance phenomena, the the proteasome ␤5-subunit (PSMB5) pro- tant cells by siRNA-mediated silencing of molecular basis of which remains elu- tein, (2) a dramatic overexpression (up to PSMB5 gene expression. Collectively, sive. Toward this end, we here developed 60-fold) of PSMB5 protein but not of other these ﬁndings establish a novel mecha- high levels (45- to 129-fold) of acquired proteasome subunits including PSMB6, nism of bortezomib resistance associ- resistance to bortezomib in human my- PSMB7, and PSMA7, (3) high levels of ated with the selective overexpression of elomonocytic THP1 cells by exposure to cross-resistance to ␤5 subunit-targeted a mutant PSMB5 protein. (Blood. 2008; stepwise increasing (2.5-200 nM) concen- cytotoxic peptides 4A6, MG132, MG262, 112:2489-2499) Introduction

The ubiquitin proteasome system (UPS) facilitates the degradation of acquired resistance, studies aimed at delineating the mechanism of ubiquitin-tagged intracellular proteins, many of which play a of acquired resistance to the tripeptidyl aldehyde proteasome regulatory role in cell proliferation, cell survival, and signaling inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal) revealed processes.1-3 As such, proteasome inhibitors have been recognized 2 possible molecular mechanisms: (a) enhanced cellular efflux via as a new generation of chemotherapeutic agents and anti- the multidrug resistance (MDR) transporter P-glycoprotein (Pgp; inflammatory drugs.4-13 The boronic dipeptide bortezomib (PS341, ABCC1)25 or multidrug resistance-related protein 1 (MRP1; Velcade) is the first proteasome inhibitor that has been approved for ABCC1)26 and (b) increased detoxification via up-regulated activ- the treatment of refractory multiple myeloma.6,14 Bortezomib is a ity of aldo-keto reductase.27 Recent studies showed that Pgp reversible inhibitor that targets primarily the ␤5-subunit (PSMB5) overexpression conferred low levels (3-fold) of bortezomib resis- subunit/chymotrypsin-like activity of the 26S proteasome and to a tance28 as well as to the irreversible proteasome inhibitor epoxomi- somewhat lesser extent also caspase-like activity harbored by the cin, but cells with 4- to 7-fold acquired resistance to epoxomicin ␤1 (PSMB6) proteasome subunit. At higher concentrations, bor- did not show Pgp overexpression as a molecular basis of resis- tezomib inhibits trypsin-like proteolytic activity facilitated by ␤2 tance.29 Two independent reports analyzing acquired resistance to 15-17 (PSMB7) proteasome subunits. Despite promising clinical the irreversible proteasome inhibitor NLVS (NIP-Leu3-vinyl sul- activity, some patients with multiple myeloma failed to respond to fone) in mouse EL4 thymoma cells showed loss of proteasome bortezomib therapy.18 Moreover, the efficacy for bortezomib may function that was compensated by overexpression of another differ between tumor types.6,19-21 Whether these observations are protease activity, tripeptidylpeptidase II.30-32 However, a more related to common mechanisms of drug resistance frequently seen detailed characterization of these NVLS-resistant EL4 cells re- for anticancer22 or anti-inflammatory drugs23 is largely unknown. vealed that residual proteasome activity was sufficient to sustain However, their characterization is of key importance as it may pave cell function and viability33 so that the actual modality of resistance the way for the overcoming of drug resistance, thereby enhancing in these cells remains to be elucidated. the efficacy of this new class of proteasome-targeted drugs. Altogether, given the lack of a profound understanding of the One mode of primary resistance to bortezomib is conveyed by mechanisms of acquired resistance to bortezomib in mammalian constitutively high levels of heat shock protein 27.24 In the context cells, we set out to characterize human monocytic/macrophage

Submitted August 1, 2007; accepted May 11, 2008. Prepublished online as The online version of this article contains a data supplement. Blood First Edition paper, June 18, 2008; DOI 10.1182/blood-2007-08-104950. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby *R.O. and N.E.F. contributed equally to this work. marked ‘‘advertisement’’ in accordance with 18 USC section 1734. An Inside Blood analysis of this article appears at the front of this issue. © 2008 by The American Society of Hematology

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2490 OERLEMANS et al BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6 cells with high levels of acquired bortezomib resistance after in Supplemental data vitro selection using a protocol of stepwise increases in bortezomib Information on reagents and antibodies, growth inhibition assays, apoptosis concentrations. Our findings establish that bortezomib resistance is assays, immunoblotting (Western blotting and native gels), gel filtration, not conferred by increased expression of MDR efflux pumps. mitochondrial membrane potential assay, gene arrays, immunofluorescence Rather, qualitative and quantitative alterations were observed at the microscopy, reverse-transcription–polymerase chain reaction (RT-PCR)/ level of the ␤5 proteasome subunit; these included a PSMB5 gene siRNA, sequence analysis, and statistics can be found in Document S1 and mutation resulting in a substitution of a crucial amino acid Figures S1-S5, available on the Blood website; see the Supplemental (Ala49Thr) in the bortezomib-binding pocket of the ␤5-subunit. Materials link at the top of the online article. These bortezomib-resistant cells highly overexpressed this mutant ␤5-subunit and displayed a marked cross-resistance to ␤5-targeted cytotoxic peptides but not to other classes of therapeutic drugs. Hence, these findings establish a novel modality or bortezomib Results resistance associated with the selective overexpression of a structur- Establishment of cells with acquired resistance to bortezomib ally altered ␤5-subunit. To explore the molecular basis of acquired resistance to bortezomib, human monocytic/macrophage THP1 cells were exposed Methods in vitro over a period of 6 months to stepwise increasing concentrations of bortezomib representing approximately 1- to Cell culture and development of BTZ-resistant cell lines 50-fold the IC50 (2.5 nM to 200 nM). THP1 cells grown in the presence of 50 nM (THP1/BTZ ), 100 nM (THP1/BTZ ), and Human monocytic/macrophage THP1 cells (ATCC, Manassas, VA) were 50 100 kept as a suspension culture in RPMI-1640 medium supplemented with 5% 200 nM (THP1/BTZ200) bortezomib were used for further charac- fetal calf serum, 20 mM HEPES, 2 mM glutamine, and 100 ␮g/mL terization. Dose-response curves for bortezomib-induced cell growth Ϯ penicillin/streptomycin at 5% CO2 and 37°C. Cell cultures were seeded at a inhibition revealed 45-fold (IC50: 148 54 nM), 79-fold (IC50: density of 3 ϫ 105 cells/mL and refreshed twice weekly. Bortezomib 261 Ϯ 71 nM), and 129-fold (IC50: 426 Ϯ 72 nM) levels of resis- (BTZ)–resistant THP1 cell lines were obtained by stepwise increasing tance in THP/BTZ50, THP1/BTZ100, and THP1/BTZ200 cells, extracellular concentrations of bortezomib over a period of 6 months, respectively, compared with wild-type THP/1 cells (IC50: starting at a concentration of 2.5 nM (IC50 concentration: 3.3 nM) up to a 3.3 Ϯ 0.6 nM; Figure 1A). concentration of 200 nM bortezomib. During this process, cultures were isolated that were grown in the presence of 30 nM (THP/BTZ30), 50 nM Cross-resistance profile of bortezomib-resistant cells to (THP1/BTZ50), 100 nM (THP1/BTZ100), and 200 nM bortezomib (THP1/ proteasome inhibitors and various anticancer/ BTZ200). All bortezomib-resistant cell lines retained parental doubling anti-inflammatory drugs times (28 Ϯ 3 hours) and morphology, and also retained their typical capacity to differentiate into adherent macrophage-like cells upon exposure Bortezomib-resistant cells displayed appreciable cross-resistance to phorbol myristate acetate (PMA). To investigate the stability of the to other known small (3-mer) peptide-based proteasome inhibitors resistance phenotype, an aliquot of THP1/BTZ100 cells was cultured in the (ALLN, MG132, and MG262; Table 1), although to a lower level absence of bortezomib for a period of up to 6 months. These cells will be (6- to 18-fold) than for bortezomib itself. Strikingly, very high further designated as THP1/BTZ(-100) cells. levels of cross-resistance (up to 300-fold) were observed for the 6-mer cytotoxic peptide 4A626 that exerts a specific ␤5-subunit– Proteasome activity in cell lysates related/chymotrypsin-like proteasome inhibitory activity (R.O., Chymotrypsin-like, trypsin-like, and caspase-like proteolytic activities of Y.G.A., R.J.S., G.J., unpublished results). Of note, no appreciable the proteasome were measured in freshly prepared cell lysates as described levels of cross-resistance were observed for a broad spectrum of previously,34,35 with some minor modifications to the protocol. In brief, a chemotherapeutic drugs with distinct mechanisms of action, for total of 5 ϫ 106 untreated or bortezomib-exposed THP1 cells were washed example, methotrexate (folate antagonist), sulfasalazine (inhibitor 3 times with ice-cold PBS and spun down by centrifugation (5 minutes, ␬B kinase/NF␬B inhibitor), 5-fluorouracil (fluoropyrimidine anti- 250g, 4°C). Cell pellets were then resuspended in an ATP-containing lysis metabolite), chloroquine (lysosomotropic drug), bleomycin (DNA- buffer (10 mM Tris-HCl buffer (pH 7.8) containing 5 mM ATP, 0.5 mM interacting agent), gefitinib (epidermal growth factor receptor DTT, and 5 mM MgCl2) and kept on ice for 10 minutes. For complete lysis, tyrosine kinase inhibitor), cisplatin (DNA interchelator), cyclo- cells were sonicated (MSE sonicator, amplitude 7, for 3 ϫ 5 seconds with sporin A (immunosuppressive drug), methylprednisolone (cortico- 20-second time intervals at 4°C) followed by centrifugation (5 minutes, 16 000g, 4°C) to remove cell debris. The supernatant was collected and steroid), geldanamycin (heat shock protein inhibitor), doxorubicin protein concentration was determined using the Bio-Rad protein assay (DNA-interacting drug), and mitoxantrone (topoisomerase inhibi- (Hercules, CA). Fluorogenic peptide substrates to measure the chymotrypsin- tor; Table 1). The latter 2 drugs are bona fide substrates of the like, trypsin-like, and caspase-like activity were Suc-Leu-Leu-Val-Tyr-amc, ATP-driven drug efflux transporters P-glycoprotein (Pgp; ABCB1), Ac-Arg-Leu-Arg-amc, and Z-Leu-Leu-Glu-amc, respectively, all at a multidrug resistance-associated proteins 1-9 (MRP1-9/ABCCs), or 100-␮M final concentration. The substrates were incubated with 20 ␮g total breast cancer resistance protein (BCRP; ABCG2).22 The lack of cell protein extract in the presence or absence of specific inhibitors in a total cross-resistance to these drugs in bortezomib-resistant cells argues assay volume of 200 ␮L. Specific inhibitors for chymotrypsin-like, caspase- against a multidrug resistance (MDR) phenotype. This notion was like, and trypsin-like activity included bortezomib (10 nM), Ac-APnLD-al further corroborated by the observation that parental mRNA levels (25 ␮M), and leupeptin (20 ␮M), respectively. The release of AMC was of Pgp, MRP1-9, and BCRP were retained in bortezomib-resistant monitored online over a 2-hour time period at 37°C with 5-minute intervals. Fluorescence was measured on a Tecan Spectra Fluor apparatus (Giessen, cells (data not shown) and Western blot experiments (Figure S1) The Netherlands) using excitation and emission wavelengths of 360 and showing no differential expression of Pgp, MRP1-6, and BCRP in 465 nm, respectively. Proteolytic activity was calculated from the slopes of bortezomib-resistant cells versus parental THP1/WT cells. Finally, the linear portion of the curves. All results were expressed as percentage bortezomib-resistant cells lacked cross-resistance to AAF-cmk, relative to untreated THP1/WT cells (100%). an inhibitor of the proteolytic system tripeptidylpeptidase II.33 From www.bloodjournal.org by guest on September 10, 2016. For personal use only.

BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6 BORTEZOMIB RESISTANCE & MUTANT PSMB5 UP-REGULATION 2491

Figure 1. Emergence of acquired resistance to bortezomib and impaired bortezomib-induced accumulation of ubiquitinated proteins in bortezomib-resistant cells. (A) Dose-response curve for bortezomib-induced growth inhibition of wild-type (WT) human monocytic/ macrophage THP1 cells and bortezomib (BTZ)–resistant variants THP1/BTZ50, THP1/BTZ100, and THP1/BTZ200, selected for growth in extracellular concentrations of 50 nM, 100 nM, and 200 nM bortezomib, respectively. Results depicted are the mean of 7 to 20 experiments (Ϯ SD). Drug exposure time, 72 hours. (B) Accumula- tion of ubiquitinated proteins in THP1/WT cells and bortezomib-resistant sublines after exposure to bortezomib. Bortezomib-resistant cells were allowed a 4-day drug washout period (control) after which they were exposed for 24 hours to their selective concentrations of bortezomib. THP/WT cells were exposed to 10 nM BTZ for 24 hours. (C) Accumulation of ubiquitinated proteins in THP/WT cells after 24-hour exposure to 10 to 100 nM bortezomib and for THP1/BTZ200 cells after 24-hour exposure to bortezomib concentrations beyond selective concentrations (up to 1000 nM). A representative picture of 2 separate experiments is depicted.

Together, these results demonstrate that cross-resistance of contribute to the bortezomib-resistant phenotype, microarray ana- bortezomib-resistant cells is restricted to (peptide) drugs that lysis was performed to assess differential gene expression in primarily target the proteasome’s ␤5-subunit. control THP1/WT cells versus THP1/BTZ30 cells, THP1/BTZ100 To further characterize the bortezomib-resistant cells, and to cells, and its subline THP1/BTZ(-100) grown in the absence of BTZ gain insight whether multiple mechanisms of resistance may for 6 months. The entire data set of the test series is available at the

Table 1. Growth inhibitory effects of proteasome inhibitors and various anticancer/anti-inﬂammatory drugs for wild-type (WT) and bortezomib (BTZ)–resistant THP1 cells

IC50* Resistance factor†

THP1/WT THP1/BTZ50 THP1/BTZ100 THP1/BTZ200 Proteasome inhibitors Bortezomib, nM 3.3 Ϯ 0.6 45 79 129 MG132, nM 237 Ϯ 54 8.1 11.9 15.8 MG262, nM 2.1 Ϯ 0.6 8.3 10.3 10.8 ALLN, ␮M 3.7 Ϯ 0.4 5.8 10.0 18.1 4A6, ␮M 0.26 Ϯ 0.06 44 117 287 Miscellaneous drugs Methotrexate, nM 8.0 Ϯ 1.4 0.8 1.0 Sulfasalazine, ␮M 275 Ϯ 49 1.3 0.8 Chloroquine, ␮M 56.3‡ 1.1 1.1 Cyclosporin A, ␮M 3.9‡ 0.9 0.8 5-Fluorouracil, ␮M 2.3‡ 0.5 0.4 Methylprednisone, ␮M 0.69‡ 1.1 1.0 Doxorubicin, nM 18.9 Ϯ 6.2 1.3 1.5 Mitoxantrone, nM 2.5 Ϯ 1.3 1.4 1.2 Bleomycin, ␮M 4.9 Ϯ 2.9 0.6 0.5 Cisplatin, ␮M 0.84 Ϯ 0.46 2.1 1.9 Geﬁtinib, ␮M 10.4 Ϯ 4.6 1.3 1.3 Geldanamycin, nM 30.6 Ϯ 13.1 1.0 1.1 AAF-cmk, ␮M 7.3 Ϯ 1.8 1.4 1.9 1.1

Results depicted are the mean of at least 3 to 10 experiments plus or minus SD.

*IC50: drug concentration resulting in 50% growth inhibition compared with control. †Resistance factor: ratio IC50 BTZ-resistant cell line over IC50 THP1/WT cells. ‡Mean of 2 experiments performed in duplicate. From www.bloodjournal.org by guest on September 10, 2016. For personal use only.

2492 OERLEMANS et al BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6

GEO database, accession number GSE11771.36 Preliminary evalu- and/or catalytic activity, the expression pattern of individual ␤1-, ation of expression of genes specifically involved in drug metabo- ␤2-, and ␤5-subunits was determined in wild-type and drug- lism and resistance (n ϭ 101), apoptosis (n ϭ 59), and cell cycling resistant cells grown at their selective bortezomib concentrations. (n ϭ 76) showed no marked and/or robust alterations (Ͻ 2- or Furthermore, to explore the stability of the bortezomib-resistance Ͼ 2-fold) that could be associated with resistance (Figure S2). phenotype, we also examined a subline of THP1/BTZ100 that was Consistently, Western blot analysis for specific proteins in these grown in the absence of bortezomib for 6 months (termed gene categories (eg, Hsp27, Hsp90, XIAP, and p21) showed no THP1/BTZ(-100)). Proteasome ␤1 and ␤2-subunit expression was marked changes in bortezomib-resistant cells compared with only slightly increased (Ͻ 2-fold), whereas proteasome ␣7-subunit bortezomib-sensitive THP1/WT cells (Figure S3). Strikingly, how- expression was not significantly altered in the bortezomib-resistant ever, approximately 40% of genes representing the various protea- cells (Figure 3A). In contrast, ␤5 protein levels, relatively barely some subunits, in particular ␤-subunits, showed a markedly detectable in parental cells, were dramatically increased (up to (Ն 2-fold) increased expression level (Figure S2). Only 2 protea- 60-fold) in bortezomib-resistant cells (Figure 3A). This observa- some subunit-related genes were down-regulated in expression tion was confirmed using 2 different antibodies recognizing (Յ 2-fold): the immunoproteasome subunits ␤5i and ␤1i. Of different epitopes within the ␤5 protein. This dramatic increase in additional interest, no alterations were observed in expression ␤5 expression was proportional to the gradually increasing concen- levels of genes encoding ubiquitin-conjugating enzymes, ubiquitin- trations of bortezomib during the stepwise selection. Notably, over specific proteases, or ubiquitin C-terminal hydrolases. Finally, a concentration range (up to 50 nM bortezomib) where bortezomib up-regulated expression of proteasome subunit genes in bortezomib- primarily inhibits chymotrypsin-like proteasome activity, ␤5 over- resistant cells appeared to be transient as gene expression normalized expression parallels the extent of bortezomib resistance (Figure when THP1/BTZ100 cells were grown in the absence of bortezomib for 3B). Apart from Western blot analysis, we also explored whether 6 months (Figure S2). Together, gene array analyses further support the ␤5 overexpression was associated with the 20S core particle of the notion that alterations in proteasome subunit composition or function proteasome by performing native gel electrophoresis analysis. may be dominantly involved in the bortezomib-resistant phenotype. Results depicted in Figure 3C further demonstrate an increased expression of ␤5 in resistant cells compared with parental cells; but Diminished accumulation of ubiquitinated proteins in all of this increased expression was confined to the 20S proteasome bortezomib-resistant cells complex rather than being present as unassembled “free” subunits. ␣ A characteristic feature of proteasome inhibition is the accumula- As a control, expression of subunit 7 remained unchanged, as did tion of ubiquitinated proteins that provokes loss of mitochondrial the overall proteolytic activity in both parental and resistant cells membrane potential and apoptosis.33,37 Consistently, marked accu- using suc-LLVY-amc as a substrate (Figure 3C). This finding was mulation of ubiquitinated proteins was observed in wild-type cells further corroborated by gel filtration experiments (Figure 3D) that ␤ exposed to bortezomib but not in bortezomib-resistant cells at their demonstrated that elevated expression of 5 was observed in selective concentration of 30 to 200 nM (Figure 1B). However, the bortezomib-resistant cells that eluted from the column solely as a latter cells showed clear accumulation of ubiquitinated proteins if high-molecular-weight fraction of approximately 600 to 700 kDa protein, consistent with the 20S core particle’s molecular weight. In exposed to concentrations of bortezomib above the selective accord with data from Figure 3A,C, ␣7-subunit expression was concentrations (500-1000 nM; Figure 1C). Thus, bortezomib- largely unaltered between parental and bortezomib-resistant cells, resistant cells have retained their capacity to accumulate ubiquiti- although its presence was not exclusively restricted to high- nated proteins, except that this process is initiated at markedly molecular-weight fractions, but also showed up in lower molecular higher concentrations of bortezomib consistent with the resistance weight fractions, possibly even as “free” subunits. Consistent with factor for bortezomib. these experiments, immunohistochemical analysis (Figure S4) also Impaired bortezomib-induced loss of mitochondrial confirmed the colocalization of ␤5 and ␣7 in THP1/BTZ cells. membrane potential and induction of apoptosis in In contrast to ␤5 protein levels, ␤5 mRNA levels in bortezomib- bortezomib-resistant cells resistant cells were only marginally increased at the highest selection dose (Figure 4A), suggesting that the induction of ␤5 The shift in bortezomib-induced accumulation of ubiquitinated protein most likely is effectuated at the posttranscriptional level. As proteins in bortezomib-resistant cells paralleled the shift in bort- for the ␤5 transcript, ␤2 and ␤1 mRNA levels were only modestly ezomib-induced loss of mitochondrial membrane potential (Figure elevated in cells with the highest bortezomib-resistance level 2A) and induction of apoptosis (Figure 2B,C). For comparison, the (Figure 4A). anticancer drug and topoisomerase II inhibitor etoposide (VP16) We next examined the possible role that these quantitative was equally effective in inducing apoptosis in wild-type as well alterations in ␤5-subunit composition may bear on the various as bortezomib-resistant cells (Figure 2D). These results indicate proteolytic activities of the proteasome. Proteasomal proteolytic that bortezomib-resistant cells have retained their capacity to activities represented by chymotrypsin-like activity, caspase-like undergo apoptosis, consistent with the lack of alterations in gene activity, and trypsin-like activity were measured using fluorogenic expression of apoptosis-related genes (Figure S2), but the ability to peptide substrates in cell extracts of parental cells incubated for undergo apoptosis is specifically impaired for proteasome inhibitor 24 hours in the presence or absence of 10 nM bortezomib. type of drugs. Bortezomib-resistant cells were tested after a 4-day drug-free Marked overexpression of proteasome subunit ␤5 but period (control), and after 24-hour incubation at their selective not chymotrypsin-like proteasomal activity in bortezomib concentration (Figure 4B). After 4 days of incubation bortezomib-resistant cells in drug-free medium, bortezomib-resistant cells had a small (1.3- to 1.4-fold) increase in chymotrypsin-like activity compared with Because the cross-resistance data (Table 1) were consistent with wild-type cells. Under bortezomib-selective conditions (30, 100, or specific alterations at the level of proteasome subunit expression 200 nM), residual chymotrypsin-like activity was reduced to 8% to From www.bloodjournal.org by guest on September 10, 2016. For personal use only.

BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6 BORTEZOMIB RESISTANCE & MUTANT PSMB5 UP-REGULATION 2493

Figure 2. Differential induction of apoptosis by bortezomib and VP16 in bortezomib-resistant cells.

(A) Loss of mitochondrial membrane potential (⌬␺m) after 24-hour exposure of THP1/WT cells and THP1/BTZ200 cells to a concentration range of bortezomib. (B) Induc- tion of apoptosis (annexin V–positive cells) in THP1/WT cells and THP1/BTZ200 cells after 24-hour exposure to a concentration range of bortezomib. (C) A representative ﬂow cytometric picture of apoptosis induction (annexin V/7-AAD staining) after 24-hour incubation of THP1/WT cells and THP1/BTZ200 cells with 100 nM BTZ. (D) A representative ﬂow cytometric picture of apoptosis induction (annexin-V/7-AAD staining) after 48-hour incubation of THP1/WT cells and THP1/BTZ200 cells with 1 ␮M VP-16/etoposide. All results present the means (Ϯ SD) for 3 independent experiments.

10% of drug-free controls, as observed for parental cells exposed to Stability of the bortezomib-resistance phenotype 10 nM bortezomib. After drug washout of bortezomib-resistant cells, caspase-like proteasome activity was signiﬁcantly increased To investigate the stability of the resistance phenotype and ␤ (1.8- to 2.3-fold; P Ͻ .001) over parental cells. In the presence of expression of 5-subunit in the absence of bortezomib, resistant bortezomib, caspase-like activity was reduced to 26% to 35% of cells were transferred to bortezomib-free medium for a period of ␤ wild-type levels. Finally, basal trypsin-like activity was 1.4- to 7 days and showed a gradual decline of 5 expression to 40% of the 1.7-fold (P Ͻ .01) increased in bortezomib-resistant cells com- original levels (Figure 5A); whereas, no substantial alterations ␤ pared with wild-type cells. In the presence of bortezomib, trypsin- were observed in 5-subunit mRNA levels (Figure 5B). Interest- ␤ like activity was stimulated by another 20% to 40% both in ingly, however, Figure 3A demonstrated that 5-subunit expression bortezomib-resistant cell lines and wild-type cells (Figure 4B). was further reduced in THP1/BTZ(-100) cells that were cultured in These results indicate that the qualitative characteristics of protea- bortezomib-free medium for 6 months, although they had retained somal inhibition by bortezomib were retained in bortezomib- a more than 35-fold resistance level for bortezomib (not shown). resistant cells, whereas quantitatively, a marked up-regulation of This prompted us to investigate whether ␤5-subunit expression ␤5-subunit protein did not lead to a ␤5-subunit–related increase in could be rapidly reinduced upon bortezomib reexposure. Indeed, chymotrypsin-like proteasome activity. when THP1/WT, THP1/BTZ(-100), and THP1/BTZ100 cells were ϫ Finally, probing of active proteasome subunits was performed exposed to a range of bortezomib concentrations (1-3 IC50) for 38 ␤ using the bodipyFL-Ahx3L3VS afﬁnity labeling reagent. Labeling 24 hours, a marked and selective induction of 5-subunit expression of constitutive ␤5 and its immunoproteasome counterpart ␤5i was was noted for THP1/BTZ(-100) cells and to a lesser extent for readily observed in parental THP1 cells (Figure 4C), but markedly THP1/BTZ100 cells (Figure 5C,D). Fine-tuning the 0- to 24-hour inhibited at relatively low levels of bortezomib resistance in time course of ␤5 induction revealed that within 8 to 16 hours after ␤ THP1/BTZ30 cells. Interestingly, probing of active ␤5 and ␤5i was bortezomib exposure, 5-subunit expression was markedly in- partly restored in THP1 cells with higher levels of bortezomib duced in THP1/BTZ(-100) cells (Figure 5E) and was accompanied by ␤ resistance including THP1/BTZ50 and THP1/BTZ100 cells, thus a 2- to 3-fold induction of 5 mRNA levels (Figure 5F). Collec- underscoring the fact that proteasome activity is retained. tively, these results suggest that once bortezomib resistance has From www.bloodjournal.org by guest on September 10, 2016. For personal use only.

2494 OERLEMANS et al BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6

Figure 3. Selective induction of proteasome ␤5- subunits in bortezomib-resistant cells. (A) Protein expression of proteasome ␤5-, ␤2-, and ␤1-subunits and ␣7-subunits in wild-type and bortezomib-resistant

THP1 sublines. THP1/BTZ(-100) represents a subline of THP1/BTZ100 that was grown in the absence of bortezomib for 6 months. Note: 2 different sources of ␤5 antibodies were used: Biomol (PW8895; Plymouth Meeting, PA) and 20S X from Novus Biologicals (Little- ton, CO), the latter indicated by an asterisk (*). (B) Induction of proteasome subunits ␤5, ␤2, and ␤1in relation to resistance factors to bortezomib for the selected panel of bortezomib-resistant THP1 cells. Densitometry results are presented as the mean (Ϯ SD) of 4 separate experiments. (C) Native gel electrophoresis of crude cell extracts of THP1/WT cells and THP1/

BTZ100 cells subsequently analyzed for ␤5- and ␣7- subunit expression (left panels) and catalytic activity for the substrate Suc-LLVY-AMC (right panel). (D) Gel ﬁltration of crude extracts of THP1/WT cells and THP1/

BTZ100 cells via a high-performance liquid chromatogra- phy (HPLC)–linked Superdex 200 HR 10/30 column (Supelco, Bellefonte, CA). Proteins were eluted by washing the column with 20 mM Tris-HCl, pH 7.5, 5 mM ATP, and 120 mM NaCl at a ﬂow rate of 0.4 mL/min. Fractions were collected every minute and subject to Western blot analysis for ␤5 and ␣7. The column was calibrated with a mixture of puriﬁed proteins in the MW range of 16.6 kDa to 669 kDa.

been provoked after chronic exposure, the drug resistance pheno- based silencing of ␤5 expression were explored in THP1/WT, type not only can remain dormant for long-term periods when cells THP1/BTZ(-100), and THP1/BTZ200 cells. In THP1/BTZ(-100) cells, are not exposed to bortezomib, but it would also be rapidly revived the ␤5-siRNA–dependent prevention of ␤5-subunit up-regulation upon reexposure to bortezomib. after bortezomib exposure (Figure 7A) was accompanied by both a significantly higher fraction of apoptotic cells (Figure 7B) as well Identification of a mutation in the PSMB5 gene in as increased bortezomib sensitivity (Figure 7C). For THP1/BTZ200 THP1/BTZ cells cells with constitutively high levels of ␤5 expression (Figure 3A), Given the stable and long-term retention of the bortezomib- ␤5 siRNA–dependent silencing also repressed ␤5 expression resistance phenotype, along with the dominant involvement of the (Figure 7A), but not to a level that compromised bortezomib ␤5-subunit, we explored whether a genetic alteration in the PSMB5 resistance (results not shown). Collectively, these results further gene could have contributed to the onset of acquired resistance to underscore the involvement of proteasome ␤5-subunit overexpres- bortezomib. To this end, we sequenced part of exon 2 of the PSMB5 sion in conferring bortezomib resistance and provide a targeted gene that encodes for the highly conserved binding pocket region strategy to reverse bortezomib resistance. for proteasome inhibitors within the ␤5 protein. In THP1 cells, selected for a very low level of acquired resistance to bortezomib, that is, 7 nM (THP1/BTZ7), no genetic alterations could be Discussion observed (Figure 6). However, in THP1 cells displaying higher levels of bortezomib resistance including THP1/BTZ30 and THP1/ This study provides several lines of evidence for the proteasome BTZ100 cells, a single G to A nucleotide shift was identified at involvement, that of the ␤5-subunit in particular, in the acquisition position 322 of the PSMB5 gene. In the mature and functional of bortezomib resistance in THP1 cells: (1) a mutation in the ␤5-subunit protein, this mutation introduces an Ala to Thr substitu- PSMB5 gene involving an Ala49Thr substitution in the highly tion at amino acid 49 (Figure 6). We further established that the conserved substrate/inhibitor binding domain of the ␤5-subunit, (2) Ala49Thr mutation was retained in THP1/BTZ(-100) cells (Figure 6). selective overexpression of the mutant ␤5-subunit protein paral- Based on the fact that Ala49 resides in the highly conserved leled bortezomib-resistance levels in THP1/BTZ cells, (3) rapid substrate/inhibitor binding domain of the ␤5-subunit, this homozy- and marked induction of ␤5 in THP1/BTZ(-100) cells after exposure gous mutation is likely to contribute to the bortezomib-resistance to bortezomib, (4) siRNA-guided silencing of ␤5-subunit gene phenotype. expression restored bortezomib sensitivity and induced apoptosis, SiRNA-dependent silencing of ␤5 induction in bortezomib-resistant and (5) bortezomib-resistant cells displayed marked cross- cells reverses bortezomib sensitivity and induces apoptosis resistance to other peptide-based proteasome inhibitors, in particular those that specifically target the ␤5-subunit. Finally, based on data in Figure 5, we determined whether Crystallography data from yeast and mammalian protea- prevention of ␤5-subunit induction would lead to restoration of somes39,40 indicated that Ala49 is implicated in the efficient binding bortezomib sensitivity. To this end, the consequences of siRNA- of bortezomib in the substrate/inhibitor binding pocket of the From www.bloodjournal.org by guest on September 10, 2016. For personal use only.

BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6 BORTEZOMIB RESISTANCE & MUTANT PSMB5 UP-REGULATION 2495

Figure 4. Proteasome ␤5-, ␤2-, and ␤1-subunit mRNA levels and proteasome subunit–related catalytic activity in wild-type and bortezomib-resistant cells. (A) mRNA levels for proteasome ␤5-, ␤2-, and ␤1-subunits in selected variants of bortezomib-resistant THP1 cells relative to THP1/WT cells. mRNA levels were quantified using ␤-glucuronidase (GUS) as reference gene and depicted relative to THP1/WT cells. (B) Chymotrypsin- like, caspase-like, and trypsin-like proteasomal activities assayed with specific fluorogenic peptide substrates in cell extracts of THP1/WT, THP1/BTZ30, THP1/BTZ100, and THP1/BTZ200 cells after a 4-day drug washout period (control) and after 24-hour incubation with 10 nM bortezomib (for THP/WT) and selective concentrations of 30 nM, 100 nM, and 200 nM bortezomib for the indicated bortezomib-resistant THP1 sublines. Controls for selective inhibition of caspase-like activity and trypsin-like activity included Ac-APnLD (25 ␮M) and leupeptin (20 ␮M), respectively. All results represent the mean (Ϯ SD) of 3 independent experiments. (C) Activity labeling of constitutive and immunoproteasome ␤-subunits in intact THP1/WT and selected bortezomib-resistant THP/BTZ cells using bodipyFL-Ahx3L3VS affinity probe.

␤5-subunit. Moreover, Ala49 is a highly conserved residue among reactions42 could be obtained from gene array analysis of CytP450 many prokaryotic and mammalian species. As such, substitution of enzymes. A putative role for MDR proteins in bortezomib-resistant Ala49 for Thr containing a neutral polar side chain is likely to have cells could be ruled out both by the lack of cross-resistance to consequences for efficiency of (reversible) binding of bortezomib prototypical MDR substrates (doxorubicin and mitoxantrone) and and other peptide-based proteasome inhibitors. Prototypically this by the lack of up-regulation of MDR proteins typically involved in may be exemplified by the 6-mer peptide 4A6 that selectively drug resistance. Consistently, Minderman et al28 and our laboratory targets ␤5 (but not subunits ␤1 and ␤2) for which up to more than (R.O., R.J.S., G.J., unpublished observations, April 2008) showed 250-fold level of cross-resistance was observed in the bortezomib- that low levels (Ͻ 3-fold) of resistance to bortezomib were resistant cells. The finding that cross-resistance levels for ALLN, detected only in mammalian cells expressing high levels of Pgp, MG132, and MG262 were lower than the resistance factors for and not by other MDR drug efflux transporters such as MRP1-6 or bortezomib may be consistent with the fact that these proteasome BCRP. In fact, none of these MDR transporters were found to be inhibitors have additional targets apart from ␤5, ␤2, and ␤1 overexpressed in bortezomib-resistant THP1 cells. Furthermore, proteasome subunits, for example, lysosomal proteases, which may up-regulation of another proteolytic system, tripeptidylpeptidase II, not be affected in bortezomib-resistant cells.9,15,20,38 The potential to compensate for proteasome inhibition by irreversible protea- impact of Ala49 mutations in conferring bortezomib resistance may some inhibitors,30-32 was ruled out based on the lack of cross- be further supported by recent preliminary observations by Lu et resistance to the TPP II inhibitor, AAF-cmk.33 Finally, potential al41 showing the same PSMB5 mutation in bortezomib-resistant loss of bortezomib activity after short drug exposures due to human lymphoblastic Jurkat T cells. constitutive or transient induction of heat shock proteins (Hsp’s)24,43 In keeping with the notion of a prominent role of the ␤5 was excluded in the present chronic exposure model as judged from proteasome subunit in conferring high levels of resistance to the lack of cross-resistance to the potent Hsp inhibitor geldanamy- bortezomib, other types of molecular mechanisms previously cin44 and unaltered hsp27 expression in bortezomib-resistant cells reported to influence bortezomib activity did not seem to apply for (Figure S3). bortezomib-resistant THP1 cells. No evidence for extensive metab- Up-regulation of target enzymes is a common mode of resis- olism (oxidative deboronation) of bortezomib by cytochrome P450 tance to several types of chemotherapeutic drugs.45,46 In this From www.bloodjournal.org by guest on September 10, 2016. For personal use only.

2496 OERLEMANS et al BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6

Figure 5. Dynamics of proteasome subunit expression in bortezomib-resistant cells after exposure and removal of bortezomib. (A) Effect of 1- to 7-day removal

of bortezomib-selective pressure from THP1/BTZ100 cells on proteasome subunit ␤5 protein expression and (B) ␤5 proteasome subunit mRNA expression. Results are the means (Ϯ SD) of 3 separate experiments. (C) Protein expression of ␤1-, ␤2-, ␤5- and ␣/␤-core proteasome

subunits in THP1/WT cells, THP1/BTZ(-100) cells, and THP1/BTZ200 cells after 4-day bortezomib washout (control/0) and 24-hour exposure to the indicated concentrations of bortezomib. (D) Protein expression of ␤1, ␤2, ␤5- and ␣/␤-core proteasome subunits for THP1/WT cells,

THP1/BTZ(-100) cells, and THP1/BTZ200 cells after a 4-day bortezomib washout period (control) and after 24-hour incubation with the indicated concentrations of bortezomib. Results of scanning of protein band intensities are presented as the mean Ϯ SD of 3 separate experiments. (E) Effect of short-term (0-24 hours) exposure

of THP1/BTZ(-100) cells to 100 nM bortezomib on proteasome subunit ␤5 protein expression and (F) ␤5 proteasome subunit mRNA expression. Results are the means of duplicate experiments (E) and 3 separate experiments (Ϯ SD; F).

context, effects on proteasomal ␤5-subunits as the primary target of a mutant ␤5 protein serves as a compensatory mechanism to for bortezomib may not be unexpected. In fact, some recent studies retain sufﬁcient chymotrypsin-like activity. An enhanced expres- revealed an overall induction of proteasome ␤-subunits (␤5, ␤2, sion of 20S proteasome core particle-associated ␤5 in drug- and ␤1) as an initial adaptive response to low-dose bortezomib resistant cells (Figure 3C,D) may be consistent with this notion. exposure.20,21,38 Results from the present study indicate that in cells Also data from proteasome activity labeling experiments (Figure with established acquired resistance to bortezomib after chronic 4C) indicate that at relatively low levels of bortezomib resistance exposure to stepwise increasing doses of bortezomib, up-regulation (THP1/BTZ30) the bodipyFL-Ahx3L3VS probe showed attenuated From www.bloodjournal.org by guest on September 10, 2016. For personal use only.

BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6 BORTEZOMIB RESISTANCE & MUTANT PSMB5 UP-REGULATION 2497

Figure 6. Analysis of PSMB5 gene mutations in bortezomib-resistant THP1 cells. Sequencing of PSMB5 gene exon 2 in THP1/WT cells and various BTZ-resistant sublines:

THP1/BTZ7, THP1/BTZ30, THP1/BTZ100, and THP1/BTZ(-100). Depicted is the single nucleotide shift (G3A) at nucleotide position 322 in THP1/BTZ30, THP1/BTZ100, and THP1/BTZ(-100) along with the corresponding change in a single amino acid substitution (Ala49Thr) within the mature PSMB5/␤5 protein.

binding/labeling to the mutant ␤5-subunit, but upon further up- which primarily ␤5-associated chymotrypsin-like proteasome activ- regulation of mutated protein in THP1/BTZ50 and THP1/BTZ100 ity, and to a lesser extent also ␤1-associated caspaselike activity, cells, labeling with the probe can increase concomitantly. It is not will be essentially abolished. Consistently, beyond concentrations readily clear whether the up-regulation of constitutive ␤5in of 50 nM bortezomib, induction of ␤5 expression is leveling off, as bortezomib-resistant cells (Figure 3A,C,D) is accompanied by a from this point inhibition of proteasome subunit ␤2-associated concomitant decrease in immunoproteasome ␤5 expression as trypsinlike activity will be initiated.16,17 suggested by gene array data (Figure S2). This status may be The present ﬁnding that the marked overexpression of ␤5 normalized to control levels in the absence of bortezomib, as protein in bortezomib-resistant cells is not paralleled by induction illustrated for THP1/BTZ(-100) cells (Figure 3A), but rapidly of ␤5 mRNA levels points to a posttranscriptional regulatory restored upon rechallenging with bortezomib (Figure 5C). The mechanism. The nature of possible posttranscriptional effects in up-regulation of ␤5 was most pronounced in cells exposed to bortezomib-resistant cells is presently unclear but may possibly bortezomib concentrations up to 50 nM, a concentration range at include alterations in proteasome homeostasis facilitated by an

Figure 7. SiRNA-mediated silencing of proteasome ␤5-subunit induction restores bortezomib sensitivity and induces apoptosis in bortezomib-resistant cells. (A) ␤5-siRNA–induced silencing of ␤5-protein expression

in THP1/BTZ(-100) and THP1/BTZ200 cells after 24-hour preincubation with transfection medium (DF2), control siRNA constructs (nontarget/GAPDH), and a ␤5-specific siRNA construct (transduction efficiency: Ͼ 85% based on siGloϩ cells by flow cytometry). After transfection, cells were incubated (24-48 hours) with the indicated concentrations of bortezomib. Protein expressions of ␣/␤-core and ␤-actin are shown as controls. A representative picture of 3 separate experiments is depicted. (B) Bortezomib-induced

apoptosis in THP1/WT, THP1/BTZ(-100), and THP1/BTZ200 cells after siRNA-induced prevention of induction of ␤5-subunit expression as described in panel A. Bor- tezomib exposure time: 24 hours. Bortezomib concentra-

tions used: 25 nM for THP1/WT and THP1/BTZ(-100) cells. Means of 4 separate experiments (Ϯ SD) are shown. *P Ͻ .01. (C) Bortezomib-induced cell growth inhibition

of THP1/BTZ(-100) cells after prevention of induction of ␤5-subunit expression by siRNA silencing as described in panel A and 24-hour exposure to 25 nM bortezomib. Means of 3 separate experiments (Ϯ SD) are shown. *P Ͻ .01. From www.bloodjournal.org by guest on September 10, 2016. For personal use only.

2498 OERLEMANS et al BLOOD, 15 SEPTEMBER 2008 ⅐ VOLUME 112, NUMBER 6 autoregulatory mechanism that mediates the differential polyubiq- It may be anticipated that the molecular mechanism of resis- uitination and degradation of multiple proteasome subunits, includ- tance reported in the current study specifically applies for bor- ing ␤5.47 Hence, impaired polyubiquitination under bortezomib- tezomib or other proteasome inhibitors that selectively target the selective pressure (Figure 1B) may differentially attenuate ␤5 ␤5-subunit of the proteasome. With the emergence of second- degradation to yield a significant increase in ␤5 protein levels. generation proteasome inhibitors targeting (ir)reversibly other Under bortezomib-selective conditions, THP1/BTZ cells did proteasome subunits or multiple all ␤-subunits,13,19,53-55 it will be not display any accumulation of ubiquitinated proteins (Figure 1B) important to address whether they are able to circumvent bor- or induction of stress-induced proteins (Figure S3), prototypical for tezomib resistance, or that similar mechanisms of resistance are the action of proteasome inhibitors. Beyond the effect of the mutant operative as described herein for bortezomib. If so, this would ␤5 protein, as well as its overexpression, it may be anticipated that warrant the design of strategies to prevent selective up-regulation the up to 2-fold elevated bortezomib-stimulated trypsin-like activ- of specific proteasome subunits and thereby enhance the therapeu- ity (Figure 4B) may compensate for the inhibition of chymotrypsin- tic efficacy of this novel class of therapeutic agents. like and caspase-like activities by sustaining proteasome activity above critical catalytic levels. Consequently, such a condition alone, or in combination with activities of deubiquitinating en- Acknowledgments zymes,48 for example, Poh149 as a possible candidate based on preliminary gene array studies (Figure S3), could also contribute to Prof M. H. Glickman (Department of Biology, Technion, Haifa, prevent the accumulation of toxic polyubiquitinated proteins, Israel), Dr H. Ovaa (Netherlands Cancer Institute, Amsterdam, The Netherlands), and Dr Frank Kruyt (Department of Medical Oncol- which may otherwise trigger loss of mitochondrial membrane ogy, VU University Medical Center, Amsterdam, The Netherlands) potential and apoptosis. are acknowledged for helpful discussions. Dr R. Honeywell We recently obtained evidence that a similar molecular basis of (Department of Medical Oncology, VU University Medical Center) selective ␤5 induction after chronic exposure to bortezomib as is acknowledged for excellent technical assistance. described herein for THP1 cells also applies for bortezomib- This study is supported by a grant from the Dutch Arthritis resistant variants of human T-lymphocytic CCRF-CEM cells50 Association (Amsterdam, The Netherlands; grant NRF-03-I-40 to (Figure S5). Interestingly, when human 8226 myeloma cells were G.J.), Cancer Center Amsterdam (Amsterdam, The Netherlands; exposed according to the same strategy, only moderate (Յ 5-fold) STR Grant to J.C.), and ZonMW (The Netherlands Organization resistance levels were observed.50 These results suggest that the for Health Research and Development, The Hague, The Nether- onset of bortezomib resistance may differ significantly between lands; N.E.F.). hematologic tumor cell lineages. A possible lower propensity for multiple myeloma cells to acquire resistance to bortezomib may be consistent with the lack of detailed reports on this issue. Beyond Authorship this, in the case of multiple myeloma, host microenvironments may also play an important role in the efficacy of proteasome target- Contribution: R.O., N.E.F., Y.G.A., J.C., G.L.K., B.A.C.D., R.J.S., 12 ing. With the accumulating knowledge of potential mechanisms and G.J. designed research, analyzed and interpreted data, and of resistance to bortezomib, more direct screening for these wrote the paper; R.O., N.E.F., I.Z., G.L.S., K.D., C.L., J.W.H., parameters should be the subject of future clinically directed C.R.B., G.J.P., and G.J. performed experimental research and laboratory studies. Screening for PSMB5 gene mutations in an analytical techniques; N.E.F., J.C., G.J., and B.Y. performed isolated case of a bortezomib nonresponsive multiple myeloma microarray analysis; K.V. and N.E.F. performed sequencing experi- patient did not provide evidence for mutations.51 However, based ments; R.O., N.E.F., J.C., and G.J. collected data; and R.O. and G.J. on the present observation that PSMB5 gene mutations may be performed statistical analysis. provoked after exposure to clinically achievable concentrations of Conflict-of-interest disclosure: The authors declare no compet- bortezomib in the range of 7 to 30 nM (Figure 6) screening for ing financial interests. PSMB5 mutations should be reconsidered. In the context of Correspondence: Gerrit Jansen, Department of Rheumatology, proteasome activity and proteasome subunit composition, the Rm 3A64, VU Institute for Cancer & Immunology, VU University recent identification and validation of specific proteasome-targeted Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The probes38,52 may facilitate analyses on limited cell numbers. Netherlands; e-mail: [email protected]. References

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2008 112: 2489-2499 doi:10.1182/blood-2007-08-104950 originally published online June 18, 2008

Molecular basis of bortezomib resistance: proteasome subunit β5 ( PSMB5) gene mutation and overexpression of PSMB5 protein

Ruud Oerlemans, Niels E. Franke, Yehuda G. Assaraf, Jacqueline Cloos, Ina van Zantwijk, Celia R. Berkers, George L. Scheffer, Kabir Debipersad, Katharina Vojtekova, Clara Lemos, Joost W. van der Heijden, Bauke Ylstra, Godefridus J. Peters, Gertjan L. Kaspers, Ben A. C. Dijkmans, Rik J. Scheper and Gerrit Jansen

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