Bortezomib is an effective agent for treating multiple myeloma (MM). To investigate the underlying mechanisms associated with acquired resistance to this agent, we established two bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, the 50% inhibitory concentration values of which were respectively 24.7- and 16.6-fold higher than their parental cell lines. No activation of caspase and BH3-only proteins such as Noxa was noted in bortezomib-resistant cells after exposure to the drug. The accumulation of polyubiquitinated proteins was reduced in bortezomib-resistant cells compared with the parental cells, associated with avoidance of catastrophic ER stress as assessed by downregulation of CHOP expression. These resistant MM cells have a unique point mutation, G322A, in the gene encoding the proteasome β5 subunit (PSMB5), likely resulting in conformational changes to the bortezomib-binding pocket of this subunit. KMS-11 parental cells transfected to express mutated PSMB5 also showed reduced bortezomib-induced apoptosis compared with those expressing wild-type PSMB5 or the parental cells. Expression of mutated PSMB5 was associated with the prevention of the accumulation of unfolded proteins. Thus, a fraction of MM cells may acquire bortezomib resistance by suppressing apoptotic signals through the inhibition of unfolded protein accumulation and subsequent excessive ER stress by a mutation of the PSMB5 gene.
Bortezomib, a proteasome inhibitor, is widely used in the treatment of multiple myeloma (MM), resulting in remarkable response rates in both relapsed/refractory MM and newly diagnosed MM.1, 2 However, bortezomib treatment often achieves only very short-duration responses and drug resistance rapidly develops.3, 4 Therefore, understanding the mechanisms underlying this drug resistance is necessary to develop novel approaches to overcome this problem. Bortezomib was originally developed as a proteasome inhibitor, which blocked the degradation of ubiquitinated IκBα, a negative regulator of the canonical nuclear factor (NF)-κB pathway, and prevented its translocation into the nucleus.5 However, several investigators have proposed additional mechanisms for its antitumor effects, especially focusing on the expression of BH3-only proteins, including Noxa, Bid, puma and Bik,6, 7, 8, 9 and on misfolded protein accumulation followed by endoplasmic reticulum(ER) stress-associated apoptosis.10, 11 When proteasome function is inhibited, damaged proteins including unfolded or oxidatively modified proteins accumulate in the intracellular environment, which causes ER overload, well recognized as an ER stress.12, 13 This in turn induces cellular protective responses, so-called ‘unfolded protein responses’ (UPR) that promote refolding or elimination of unfolded proteins, but can ultimately trigger apoptosis by activating CHOP, caspase-4 and caspase-12 if the accumulation of damaged protein is excessive.14 Administration of low doses of proteasome inhibitors can disrupt this mechanism, protecting cells from the effects of damaged protein accumulation, particularly effectively in cells such as MM and pancreatic tumors, which actively secrete proteins.15 For this reason, modifications to the mechanism for disposal of misfolded proteins and avoidance of catastrophic ER stress caused by their accumulation may be one of the means by which MM cells acquire bortezomib resistance.
Several investigators have reported on the mechanisms of bortezomib tolerance in different tumor cell lines induced by continuous exposure to stepwise-increasing doses of bortezomib. Lu et al.16 and Oerlemans et al.17 have proposed either mutation of the gene for the proteasome β5 subunit (PSMB5) (a single G to A nucleotide shift at the position 322) or overexpression of this protein as possible mechanisms associated with bortezomib resistance in the T-lymphoblastic/leukemia cell line JURKATB, and the monocytic/macrophage cell line, THP1/BTZ, respectively. In another study, Rückrich et al.18 proposed that the suppression of protein biosynthesis contributes to the adaptation to impaired proteasome activity in myeloid leukemia HL60a cells, which have acquired resistance to bortezomib. These investigators also established a bortezomib-resistant MM cell line, designated AMO-1a, but did not report any details with regard to resistance mechanisms. To the best of our knowledge, there have been no published studies on the mechanisms responsible for bortezomib resistance in MM cells. Here, we established two bortezomib-resistant MM cell lines, which tolerated the drug even at doses 10-fold higher than the 50% inhibitory concentration (IC50) values for parental cells. Using these new resistant lines, we investigated the alteration of PSMB5, misfolded protein accumulation, ER stress and apoptosis signals including BH3-only proteins at clinically achievable drug concentrations. Our study demonstrates that preventing the accumulation of misfolded proteins and avoidance of catastrophic ER stress has a crucial role in bortezomib resistance by suppressing apoptosis-inducing signals in MM cells. We also document that the mechanism for this effect involves a unique point mutation of PSMB5 in bortezomib-resistant MM cells, which contributes to reducing the accumulation of misfolded proteins and alleviates catastrophic ER stress in MM cells.
Materials and methods
Establishment of bortezomib-resistant MM cell lines
KMS-11 was kindly provided by Professor T Otsuki, Kawasaki Medical University (Okayama, Japan). OPM-2 was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Two bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, were established from their parental lines, KMS-11 and OPM-2, under continuous exposure to bortezomib in RPMI-1640 medium supplemented with 10% fetal bovine serum over a half year. During this time, the concentration of bortezomib was increased stepwise weekly after confirmation of the maintained viability of the cells at the previous dose. After their establishment, the bortezomib-resistant cell lines were incubated in bortezomib-free medium for 2 weeks to confirm the stability of resistance trait, and then subjected to all assays used in our study.
Antibodies, reagents and western blot analysis
Bortezomib was purchased from Toronto Research Chemicals (North York, Ontario, Canada). Antisera against caspase-12 and CHOP were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). Antisera against Bcl-Xl/s, Mcl-1, Noxa, ubiquitin and actin were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Antisera against Bid, Bcl-2, β-galactosidase, V5-tag and caspase-4 were purchased from Medical & Biological Laboratories (Nagoya, Japan). Antisera against β5, β1 and β2 subunits of the 26S proteasome were purchased from BIOMOL International, L.P. (Plymouth Meeting, PA, USA).
Western blot analysis was performed as previously described.8 In brief, protein samples were electrophoresed by SDS polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk, membranes were incubated with primary antibody followed by a horseradish peroxidase-conjugated secondary antibody. The protein band was detected using chemiluminescent substrate.
Cell proliferation and apoptosis assays
The cell proliferation assay was previously described.8 Calculation of the IC50 value used XLfit 4.2 curve-fitting software for Excel. Apoptotic cells after exposure to bortezomib were evaluated using Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen, Franklin Lakes, NJ, USA).
Chymotrypsin-like activity assay
A total of 5 × 105 cells were incubated with or without 10 nM bortezomib for the indicated times. After washing twice with cold phosphate-buffered saline, cells were resuspended in 50 mM Tris (pH 7.4) buffer containing 5 mM MgCl2 and 0.2 μg/ml digitonin, which permeabilizes the cell membrane without disrupting it. Cells were transferred into black 96-well flat-bottom plates at a final concentration of 4 × 104 cells in 160 μl of buffer in each well. Thereafter, 40 μl of fluorogenic substrate, Suc-LLVY-amc (BIOMOL International, L.P.), was added to each well. After incubation for 3 h at 37 °C, fluorescence was measured at 380 nm excitation wavelength and 460 nm emission wavelength.
Total RNA extraction from MM cell lines, followed by reverse transcription into cDNA, was performed as previously reported.8 Exon II of the PSMB5 gene was amplified by means of PCR using the following primer set: forward, 5′-IndexTermTTCCGCCATGGAGTCATA-3′; and reverse, 5′-IndexTermGTTGGCAAGCAGTTTGGA-3′. After purification, the PCR product was directly sequenced by the dye terminator method with the aid of an ABI377 (Applied Biosystems, Foster City, CA, USA).
Transfection by lentiviral infection
The cDNA encoding wild-type or mutated PSMB5 was obtained from KMS-11 or KMS-11/BTZ cells, respectively, by PCR amplification using 5′-attB-added PCR primers (Gateway Technology, Invitrogen, Carlsbad, CA, USA) followed by sequencing. The lentivirus-based expression vector was constructed from the combination of each cDNA-containing entry vector, cytomegalovirus (CMV) promoter-containing vector, and plenti6.4/R4R2/V5-DEST multisite gateway vector by attB-attP and attL-attR reaction. The 293FT packaging cell line was transfected with plenti/CMV/wPSMB5/V5 or plenti/CMV/mPSMB5/V5 plasmids for 24 h and each viral supernatant was collected. After equalization of viral titer, KMS-11 cells were infected by each viral supernatant for 24 h and then selected by incubation with 5 μg/ml blasticidin. After selection, KMS-11 cells stably expressing wild-type PSMB5 (KMS-11/wPSMB5-V5) or mutated PSMB5 (KMS-11/mPSMB5-V5) were assayed for apoptosis after exposure to bortezomib, and were also used for immunoblot analysis. As a control for lentiviral expression, plenti/CMV/lacZ/V5 plasmid was similarly constructed and transfected into KMS-11 cells (KMS-11/lacZ-V5).
Two MM cell lines, KMS-11/BTZ and OPM-2/BTZ, demonstrate acquired resistance to bortezomib treatment
Two bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, showed a 24.7-fold (IC50 148.3 nM) and 16.6-fold (IC50 51.6 nM) higher resistance to bortezomib, respectively, compared with their parental cells, KMS-11 (IC50 6 nM) and OPM-2 (IC50 3.1 nM) after a 72-h exposure (Figure 1a). In addition, these cells showed cross-resistance against another proteasome inhibitor, MG132, but not against doxorubicin (Figure1a and Table 1). In experiments measuring apoptosis induced by dose-escalated bortezomib treatment (Figure 1b), KMS-11/BTZ showed remarkable tolerance to between 3.3 and 100 nM of the drug, and OPM-2/BTZ between 3.3 and 33.3 nM, whereas the parental cells underwent apoptosis even at lower concentrations, 10 nM in KMS-11 and 3.3 nM in OPM-2. We next investigated the biological differences between resistant and parental cells at 10 nM bortezomib in further analyses.
Bortezomib-resistant cells do not activate apoptosis-executing signals induced by bortezomib treatment
To compare the apoptosis-regulating signals following bortezomib treatment, we investigated alterations in the expression of different caspases, Bcl-2 family members, BH3-only proteins, NF-κB activation, and ER stress signaling in the bortezomib-resistant cells and their parental cells in the presence of 10 nM bortezomib for 48 h. As shown in Supplementary Figure S1 (also refer Supplementary Materials and methods), immunoblot analysis indicated that KMS-11/BTZ and OPM-2/BTZ cells failed to activate caspases, that is, cleavage of caspase-3, -8 and -9, which did occur in the parental cells after bortezomib exposure. In Figure 2a, two bcl-2 family proteins, Bcl-2 and Bcl-xL, are shown to be overexpressed in OPM-2/BTZ cells compared with their parental cells, and this was maintained during exposure to bortezomib. However, this was not the case for KMS-11/BTZ cells. We also noted that another Bcl-2 family protein, Mcl-1, was not altered in the bortezomib-resistant cells after exposure to bortezomib, whereas parental cells showed accumulation of long, short and cleaved forms of Mcl-1 after treatment. A BH3-only protein, Noxa, which was rapidly upregulated by bortezomib treatment in parental cells, was significantly suppressed in bortezomib-resistant cells. Similarly, the basal level of Bid, a caspase-8-dependent BH3-only protein, which declined in parental cells after bortezomib exposure, was not changed in bortezomib-resistant cells. Most recently, Wang et al.19 proposed that Noxa was transcriptionally activated by the complex consisting of ATF3 and ATF4, which were upregulated by an inhibitor of ER-associated protein degradation or by bortezomib treatment in tumor cells. As shown in Supplementary Figure S2 (also refer Supplementary Materials and methods), there were no obvious differences in the induced levels of ATF3 and ATF4 between KMS-11/BTZ and the parental KMS-11 line. Moreover, both OPM-2/BTZ and OPM-2 showed low levels of ATF3 expression before and after bortezomib treatment.
Gel shift assays demonstrated that even in the presence of bortezomib, resistant cells maintained NF-κB activation as represented by three (KMS-11/BTZ) or four (OPM-2/BTZ) different DNA–protein complexes, and one of them became abundant at 48 h after bortezomib exposure (depicted by an asterisk in Supplementary Figure S3 and refer Supplementary Materials and methods). On the other hand, in the parental cells, NF-κB activity was suppressed after bortezomib treatment, whereas only one of the complexes (indicated by three asterisks) showed a transient increase at 6 h after bortezomib exposure, followed by a decrease at 12 h and later. Most recently, Hideshima et al.20 have proposed that bortezomib activates the canonical NF-κB pathway through activation of IKKβ resulting in IκBα phosphorylation and degradation, a process mediated by the proteasome. Following this report, the overexpressed band (depicted by triple asterisks in Supplementary Figure S3a) after bortezomib exposure may correspond to the one resulting from activation of the canonical NF-κB pathway. In fact, we have also demonstrated that bortezomib treatment resulted in phosphorylation of p65 and IκBα followed by degradation of IκBα in bortezomib-sensitive MM cells even when they are committed to cell death (Supplementary Figure S3b). On the other hand, two bortezomib-resistant MM cells showed neither phosphorylation of IκBα and p65 nor degradation of IκBα, indicating that the canonical NF-κB pathway in these two resistant lines was not altered by bortezomib exposure. Our study demonstrated that even in the presence of bortezomib, resistant cells maintained constitutively active NF-κB.
We next examined the expression of ER stress-related markers including cytosolic and ER chaperones, which might prevent the aggregation of misfolded proteins and promote their refolding.14 We also assessed the activation of CHOP and caspase. As shown in Supplementary Figure S4 (also refer Supplementary Materials and methods), compared with the parental cells, bortezomib-resistant cells had neither upregulated cytosolic chaperones nor ER chaperones such as Bip and Grp94, thioredoxin family (PDI) or lectin family proteins (calreticulin) with the exception of calnexin. On the other hand, CHOP expression was upregulated immediately after bortezomib treatment in the two parental MM cells. The already-low levels of CHOP completely disappeared in bortezomib-resistant cells (Figure 2b). In addition, the resistant cells showed no activation of caspase-4 and -12, which were induced in susceptible cells.
The ubiquitin–proteasome pathway is altered in bortezomib-resistant cells
To determine whether unfolded proteins accumulated after exposure to bortezomib, intracellular misfolded proteins recognized as polyubiquitinated were assessed by western blot analysis in bortezomib-resistant MM and parental cells before and after bortezomib treatment. As shown in Figure 2c, both KMS-11 and OPM-2 cells gradually accumulated polyubiquitinated proteins after bortezomib treatment. However, only transient accumulation of polyubiquitinated proteins was observed both in KMS-11/BTZ and OPM-2/BTZ cells, which had returned to basal level by 24 and 48 h after exposure, respectively. We next investigated the status of protein biosynthesis in resistant and parental cells. Newly synthesized proteins were labeled and measured before and after bortezomib treatment (Supplementary Materials and methods and Supplementary Figure S5). After bortezomib treatment, the two parental lines continued to synthesize protein 6 h after treatment but this was reduced by 24 h because of progressive apoptosis. In contrast, the two resistant lines both maintained continuous protein synthesis throughout the treatment. These results suggest that bortezomib-resistant MM cells maintain the same level of protein synthesis, unlike the bortezomib-adapted myeloid leukemia HL60a cell that was previously reported.18 We also determined the expression levels of each of the 20S proteasome subunits, β1, β2 and β5. The total amount of all three subunits was comparable between resistant and parental cells before bortezomib exposure (Figure 2c). Only the amount of β2 subunit was slightly increased after bortezomib treatment in the resistant cells, whereas it was gradually decreased in the parental cells.
We next investigated the dose-dependent alteration of accumulated polyubiquitinated proteins and expression levels of CHOP and Noxa, and activation of caspase-3, in bortezomib-resistant and parental MM cells treated with the drug. At a higher concentration of bortezomib than the IC50 value, the two resistant lines showed activation of caspase-3 and Noxa expression, indicating progression to apoptosis. However, they showed moderate accumulation of polyubiquitinated proteins, which was not followed by activation of CHOP. This finding may indicate that intracellular stresses different from proteasome inhibition have occurred, which trigger apoptosis independently of ER stress before excessive unfolded protein accumulation can take place at high concentrations of bortezomib.
To compare bortezomib-induced proteasome inhibition, chymotrypsin-like activity was measured. This was found to decrease in KMS-11 cells to 30–37% of the control level after a 6-h exposure, whereas KMS-11/BTZ cells retained 47–51% of the activity even after a 48-h exposure. In OPM-2 cells, chymotrypsin-like activity on 6-h exposure was reduced to 10–13%, whereas OPM-2/BTZ cells retained 21–23% of the level in unexposed cells and maintained that until 48 h after exposure. These results indicate that the degree of proteasome inhibition is slightly weaker in bortezomib-resistant cells than their parental cells after bortezomib exposure. This subtle difference may contribute to the avoidance of fatal unfolded protein accumulation.
Expression levels of ubiquitin specific proteases, a lysosomal protease,18 before and after bortezomib exposure were also examined. As shown in Supplementary Figure S5 (also refer Supplementary Materials and methods), neither bortezomib-resistant line overexpressed USPs before or after exposure to bortezomib compared with their parental cells. This indicates that USPs have little effect in compensating for impaired proteasome activity in bortezomib-resistant cells.
Alteration of the proteasome β5 subunit in bortezomib-resistant MM cells
Most recently, mutation of the PSMB5 gene (G322A) has been proposed as a possible mechanism responsible for bortezomib resistance in T-cell lymphoblastic/leukemia and myeloid leukemia cells adapted to bortezomib treatment.16, 17 To investigate the presence or absence of genetic alterations in the PSMB5 gene in our MM cells, exon 2 encoding the conserved bortezomib-binding pocket regions in the β5 subunit was sequenced. As shown in Figures 2d and a, substitution at nucleotide position 322 (G/A), which corresponds to the amino-acid change (Ala49Thr) same as previously reported,16, 17 was identified in both the bortezomib-resistant MM cell lines, but not in the parental cells. This reflects the appearance of a G322A-mutated allele in addition to the remaining wild-type allele in bortezomib-resistant MM cells.
The G322A-mutated PSMB5 reduces bortezomib-induced apoptosis through the prevention of ubiquitinated protein accumulation and fatal ER stress in MM cell
To investigate the role of the PSMB5 mutation (G322A) in bortezomib resistance of the MM cell lines, we transfected a G322A-mutated PSMB5 expression construct into KMS-11 cells using a lentiviral vector. As controls, wild-type PSMB5 or lacZ constructs were similarly transfected into KMS-11 cells. In Figure 3a, these transfected cells can be seen to have similar expression levels of V5-tag, which indicates that the transfection efficiency was essentially the same for the mutated PSMB5 and the other genes. After 72-h bortezomib treatment, KMS-11 mPSMB5-V5 cells showed significant reduction of apoptosis compared with KMS-11- wPSMB5-V5-, KMS-11 lacZ-V5-transfected and nontransfected KMS-11 cells (Figure 3b). Similar result was shown in growth inhibition assay when treated with various concentrations of bortezomib for 72 h. The IC50 values were 8.88, 26.38, 8.83 and 113.63 nM in KMS-11 wPSMB5-V5, KMS-11 mPSMB5-V5, parental KMS-11 and KMS-11/BTZ cells, respectively (Figure 3c). There was much less accumulation of polyubiquitinated proteins in mPSMB5-transfected cells compared with wild-type control cells (Figure 3d). Similarly, expression levels of CHOP and Noxa were lower in mutated PSMB5-transfected KMS-11 cells after bortezomib exposure (Figure 3d). These results indicate that G322A-mutated PSMB5 contributed to a reduction in bortezomib-induced apoptosis by preventing ubiquitinated protein accumulation and fatal ER stress in these MM cells.
We have established two novel bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, both of which tolerated the drug even at high concentrations and were also resistant to a different proteasome inhibitor, MG132. These two bortezomib-resistant MM cell lines did not overexpress the β5 proteasome subunit compared with the parental lines, unlike what has been reported in bortezomib-resistant cells other than MM cells.16, 17 However, a unique point mutation, G322A, was identified in the PSMB5 gene as has also been found in previous studies of bortezomib-resistant cells of other hematopoietic lineages.16, 17 In addition, we demonstrated that the G322A point mutation in PSMB5 actually contributes to resistance against bortezomib-induced apoptosis, which is mediated by prevention of polyubiquitinated protein accumulation and fatal ER stress signaling followed by the downregulation of BH3-only protein, that is, Noxa expression. However, the degree of bortezomib resistance in mutated PSMB5-transfected KMS-11 cells was not as great as that of KMS-11/BTZ cells, indicating that as-yet unidentified mechanisms other than PSMB5 mutation may partly contribute to the bortezomib resistance in KMS-11/BTZ cells.
A point mutation of the PSMB5 gene (G322A) identified in both bortezomib-resistant MM cell lines results in replacement of the codon 49 Thr for Ala at the amino-acid level. This would give rise to a conformational change of the bortezomib-binding pocket in the β5 subunit, resulting in the partial disruption of contact between bortezomib and the chymotrypsin-like active site.16, 21 Our study first proved that in MM cells, the G322A point mutation in PSMB5 contributes to resistance against bortezomib-induced apoptosis. Moreover, our study does not support the notion that overexpression of wild-type PSMB5 alone led to bortezomib resistance, unlike previous reports using a bortezomib-adapted monocytic/macrophage cell line.17 Among various hematological malignant cell types, MM cells have the greatest sensitivity to bortezomib even at low doses, probably due to preexisting ER overload. Thus, moderately upregulated PSMB5 may not be able to impart the resistance against bortezomib. In fact, as the mutated PSMB5 is not overexpressed in either of the bortezomib-resistant MM cell lines, the presence of this mutated form may have a major role in acquired resistance against bortezomib. Although bortezomib is a highly selective inhibitor of the chymotrypsin-like activity of PSMB5, another proteasome catalytic site, PSMB1, is also inhibited to some extent by bortezomib. Another subunit, PSMB6, is known to act as a scaffold to stabilize the interaction between bortezomib and the active site of PSMB5.21 However, we could not find any mutations in PSMB1 and PSMB6 in our bortezomib-resistant MM cells (data not shown).
We have not found any PSMB5 gene mutations in a small number (n=4) of clinically available specimens derived from MM patients who showed resistance against bortezomib so far. However, further investigations using a larger number of patients who have acquired bortezomib resistance during or after treatment are required.
In conclusion, we have established two different stable bortezomib-resistant MM cell lines, both of which were found to have acquired exactly the same point mutation (G322A) in the bortezomib-binding pocket of PSMB5. These cells, unlike their parental cells, did not accumulate misfolded proteins and thus avoided the catastrophic ER stress, which triggers CHOP expression and caspase-4 and -12 activations. Apoptosis triggered by Noxa induction was also suppressed. These cell lines will provide tools for the better understanding of the underlying mechanisms of bortezomib resistance, and may lead to the development of novel treatment strategies for overcoming bortezomib resistance in patients with MM.
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We thank Ms Chiori Fukuyama for her skillful technical assistance. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (No. 17016065 & 16062101 for RU) from the Ministry of Education, Culture, Science, Sports and Technology, Japan; and Grants-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare, Japan (No. 17S-1, 17-16 anf 21-8-5 for SI). This research was also funded in part by Kyowa Hakko Kirin Co., Ltd, Tokyo, Japan.
TN, HM and YS are employees of Kyowa Hakko Kirin Co., Ltd., Japan. SI received research funding from Kyowa Hakko Kirin. SI declares honoraria from Janssen Pharmaceutical K.K., Dainippon Sumitomo Pharmaceutical Co., Ltd, Chugai Pharmaceutical Co., Ltd and Novartis Pharma K.K.
Supplementary Information accompanies the paper on the Leukemia website
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Ri, M., Iida, S., Nakashima, T. et al. 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 (2010). https://doi.org/10.1038/leu.2010.137
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