Endoplasmic Reticulum Stress and the Unfolded Protein Response: Targeting the Achilles ... ·...

Review

Endoplasmic Reticulum Stress and the Unfolded ProteinResponse: Targeting the Achilles Heel of Multiple Myeloma

Lisa Vincenz1, Richard J€ager1, Michael O'Dwyer2, and Afshin Samali1

AbstractMultiple myeloma is characterized by the malignant proliferating antibody-producing plasma cells in the

bone marrow. Despite recent advances in therapy that improve the survival of patients, multiple myeloma

remains incurable and therapy resistance is the major factor causing lethality. Clearly, more effective

treatments are necessary. In recent years it has become apparent that, as highly secretory antibody-producing

cells, multiplemyeloma cells require an increased capacity to copewith unfolded proteins and are particularly

sensitive to compounds targeting proteostasis such as proteasome inhibitors, which represent one of the most

prominent new therapeutic strategies. Because of the increased requirement for dealingwith secretory proteins

within the endoplasmic reticulum, multiple myeloma cells are heavily reliant for survival on a set of signaling

pathways, known as the unfolded protein response (UPR). Thus, directly targeting the UPR emerges as a new

promising therapeutic strategy. Here, we provide an overview of the current understanding of the UPR

signaling in cancer, and outline its important role in myeloma pathogenesis and treatment. We discuss new

therapeutic approaches based on targeting the protein quality control machinery and particularly the IRE1a/XBP1 axis of the UPR. Mol Cancer Ther; 12(6); 831–43. �2013 AACR.

The Unfolded Protein ResponseThe endoplasmic reticulum (ER) is the primary cellular

Ca2þ store and the site of biosynthesis of secreted andtransmembrane proteins, both of which enter the ERlumen cotranslationally. Inside the ER proteins are foldedand undergo glycosylation or lipidation. The correct fold-ing and trafficking of these proteins are dependent onchaperones within the ER, which require Ca2þ and ATP,and on an oxidizing environment to facilitate the forma-tion of disulfide bonds between protein chains. Therefore,these processes are sensitive to nutrient deprivation, tochanges in Ca2þ homeostasis and in the cellular redoxstate. Such conditions, as well as a high load of secretedproteins, the presence of folding-deficient mutant pro-teins, impairment of glycosylation, of vesicular traffickingor of protein degradation will lead to an accumulation ofmisfolded or unfolded proteins in the ER. These condi-tions are collectively referred to as "ER stress". As aconsequence, the cell triggers a set of signaling pathways

termed the unfolded protein response (UPR) that initiallyaims to restore homeostasis, but can also induce apoptosisif the stress cannot be resolved (1).

The initial phase of the UPR aims at resolving the stressby expanding the secretory apparatus, increasing ERvolume, decreasing the load of newly synthesized pro-teins, enhancing the removal of unfolded proteins fromthe ER by a process termed ER-associated degradation(ERAD; ref. 2), and by inducing autophagy (3). Thus, theUPR serves as an important physiologic adaptationmech-anism of particular importance in secretory cell types.However, when these attempts to overcome the stress fail,cell death ensues. ER stress-induced apoptosis proceedsprimarily via the mitochondrial pathway, which is con-trolled by the BCL-2 family of proteins (4).

UPR signaling pathwaysIn mammals, the 3 major ER stress sensors are the ER

transmembrane proteins inositol-requiring enzyme 1(IRE1, ERN1), PKR-like ER kinase (PERK, EIFA2K3), andactivating transcription factor 6 (ATF6). The ER luminaldomains of these proteins interact with the ER chaperone78 kDa glucose-regulated protein [GRP78, or immuno-globulin binding protein (BiP)]. As unfolded proteinscompete for binding with GRP78, their accumulationleads to dissociation of GRP78 from the luminal domainsof the ER stress sensors, allowing their activation (Fig. 1).

PERK is a serine/threonine protein kinase that phos-phorylates eukaryotic initiation factor 2a (eIF2a) to inhibitthe initiation step of mRNA translation, thus loweringoverall protein load of the ER. However, eIF2a phos-phorylation promotes increased translation of activation

Authors' Affiliations: 1Apoptosis Research Centre, National University ofIreland Galway; and 2Department of Haematology, University HospitalGalway, Galway, Ireland

Current address for L. Vincenz: Department of Cellular Biochemistry, Max-Planck-Institute of Biochemistry, Martinsried, Germany; and currentaddress for R. J€ager: University of Applied Sciences Bonn-Rhein-Sieg,Rheinbach, Germany.

Corresponding Author: Afshin Samali, Apoptosis Research Centre,National University of Ireland Galway, Galway, Ireland. Phone: 353-91-492440; Fax: 353-91-495504

doi: 10.1158/1535-7163.MCT-12-0782

�2013 American Association for Cancer Research.

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transcription factor 4 (ATF4), which induces a set of genesinvolved in apoptotic and in adaptive responses duringER stress (5, 6). A second target of PERK is the transcrip-tion factor, nuclear factor-erythroid 2–related factor 2(NRF2), whose phosphorylation liberates it from its inhib-itor Kelch-like ECH-associated protein 1 (KEAP1), allow-ing the expression of genes involved in oxidative stress/redox signaling (7).

ATF6 is synthesized as a transmembrane protein and isoccluded from the nucleus by tethering to the ER mem-brane. Dissociation of GRP78 allows for transport of ATF6

to the Golgi where it is cleaved from its transmembranedomain, allowing for nuclear translocation (8). ATF6regulates the expression of a set of genes involved inprotein quality control and ERAD (9) and stimulatesexpression of the X-box binding protein 1 (XBP1) genewhose transcript is a target of IRE1a (10).

IRE1 is a type I ER transmembrane protein. It has both akinase activity and an endoribonuclease activity. Thereare two IRE1 isoforms; IRE1a is ubiquitously expressed,whereas IRE1b expression seems to be restricted to gas-trointestinal epithelial cells. Dissociation ofGRP78 during

Figure 1. Signaling pathways of theunfolded protein response. Whenunfolded proteins accumulate inthe ER lumen, they are bound byGRP78, leading to activation of theER stress sensors ATF6, PERK,and IRE1, which induce a signalingcascade termed theUPR.The UPRinvolves the downregulation oftranslation and the activation oftranscription factors that regulategenes promoting ER homeostasisand cell survival. During prolongedor severe ER stress, however,genes that induce apoptosis areupregulated. See main text fordetails.

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ER stress leads to the activation and autophosphoryla-tion of the cytoplasmic kinase domain of IRE1a, followedby oligomerization that activates the RNase activity. Thekinase domain of IRE1a recruits the E3 ubiquitin ligase,TNF receptor-associated factor 2 (TRAF2) that mediatesactivation of c-jun-NH2-kinase (JNK; ref. 11), and of NF-kB (12) signaling pathways, which may be involved incell death induction or expression of prosurvival genesand/or cytokines, respectively. The RNase activity inconjunction with a RNA ligase removes an intron fromthe XBP1 mRNA (13). The unspliced mRNA encodes anunstable protein, XBP1u, which expresses a DNA bind-ing domain, but is mainly cytoplasmic. XBP1 splicingresults in a shift in the open reading frame of XBP1 andallows for translation of an alternative C-terminus thatharbors a nuclear translocation signal as well as a trans-activation domain. The spliced XBP1 protein (XBP1s),therefore, is a potent transcription factor that controlsgenes involved in ER membrane biosynthesis, proteinimport, chaperoning, ERAD, and cell type-specific gene-tic programs (14).The RNase activity of IRE1a has also been implicated in

the degradation of specific mRNAs, mostly encoding ER-synthesized proteins, a process termed regulated IRE1-dependent decay (RIDD; ref. 15). The roles of RIDD in ERstress and cell death are not fully understood but maycomprise adaptive functions arising from lessening pro-tein load at the ER or proapoptotic functions due to thedegradation of transcripts encoding proteins importantfor cell survival.How these specific activities of IRE1a are coordinated

to launch adaptive, cytotoxic, and inflammatoryresponses is currently not well understood. As a compo-nent of a complex protein platform, referred to as theUPRosome (Fig. 2), IRE1a activity ismodulated by severalinteracting proteins (e.g., BAX, BAK, BI-1, HSP90, HSP70,and RACK; ref. 16). The interaction with the BCL-2 familyproteins BAX and BAK is reported to be crucial for IRE1aactivation during ER stress (16). This interaction is coun-teracted by the ER-resident transmembrane protein BAXInhibitor-1 (BI-1) whose overexpression inhibits IRE1aactivity, and its deficiency increases XBP1 splicing andincreases secretory activity of B cells (16). Interestingly, BI-1 abundance is regulated at the level of protein stability bythe ER-associated RING type E3 ligase bifunctional apo-ptosis regulator, BAR, which mediates ubiquitination ofBI-1, thus initiating proteasomal degradation and remov-ing the block of IRE1a activation imposed by BI-1 (17).

Regulation of proliferation, autophagy, andapoptosisBeyond restoring ER homeostasis, the UPR impacts on

the proliferation and apoptosis equilibrium, thus helpingcells or tissues to copewith the consequences of ER stress.The main mechanism of PERK-induced apoptosis is

thought to be through increased expression of the tran-scription factor C/EBP-homologous protein (CHOP;refs. 1, 18), which is mediated by ATF4/ATF3 (5). The

exact mechanism by which CHOP can induce apoptosishas not yet been delineated.

Apart from increasing the folding capacity of the ER,little is known about how IRE1a/XBP1s signaling exertsits prosurvival function during ER stress at the level ofthe apoptotic machinery. Recently, it was reported thatXBP1s overexpression leads to increased BCL-2 expres-sion in a breast cancer cell line (19). In a hematopoietic cellline that undergoes apoptosis upon interleukin (IL)-3withdrawal, overexpression of XBP1s was cytoprotectiveand attenuated induction of the proapoptotic BCL-2 fam-ily member BIM (20). However, there is no evidence thatBCL-2 familymembers aredirect transcriptional targets ofXBP1s. Since IRE1a interacts with BCL-2 family membersat the ER membrane (21), it is possible that the activationstatus of IRE1amight influence their pro- or antiapoptoticactivities.

ER stress can also activate autophagy as a mechanismfor removing unfolded proteins or damaged ER. Autop-hagy can be induced by both the PERK and the IRE1a armof the UPR (22).

Role of the UPR in CancerUPRpathways are frequently activated andplay crucial

roles in tumorigenesis and therapy response (23). Evi-dence suggests that the UPR is of particular importancefor adaptation of cancer cells to hypoxic conditions. Forexample, PERK was shown to be involved in growth andhypoxia resistance of tumors derived from transformedmouse embryonic fibroblasts inoculated into mice (6).XBP1 splicing by IRE1a has also been implicated inadaptation to hypoxia (24). As oxygen is the preferredterminal electron acceptor in the redox relay required fordisulphide bond formation, hypoxia leads to an increasein misfolded proteins triggering IRE1a activation andXBP1 splicing (25). In fact, a XBP1 splicing reporter trans-gene revealed activation of IRE1a at sites of hypoxiawithin tumors in a transgenic breast cancer model (26).Furthermore, all 3 arms of the UPR have been shown to atleast partially control VEGF levels in hepatoma cell linesand fibroblasts, respectively (27). Thus, UPR signaling intumors seems to be important for switching on angiogen-esis in response to local hypoxia.

Remarkably, IRE1a is one of the most frequentlymutated kinases in cancer (28). Because at least someof the mutations result in loss of kinase and RNaseactivity (29), this would suggest that in certain cancers,IRE1a signaling counteracts tumorigenesis, possibly viaJNK activation or via degradation of essential mRNAsby RIDD. Mutations in XBP1 have been found in anumber of cancers including multiple myeloma (30);however, neither their relevance nor functional conse-quences have been shown thus far.

Other studies, in contrast, point towards a protumori-genic role of IRE1a, in particular of XBP1 splicing activity.Intriguingly, XBP1 splicing seems to play a driving role inthe pathogenesis of multiple myeloma (31).

UPR in Multiple Myeloma

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How Myeloma Cells Deal with Protein Load andER Stress

Multiple myeloma is a malignancy of post-germinalcenter B lymphocytes in the bone marrow that are clas-sified by a number of chromosomal abnormalities andgenetic mutations. Multiple myeloma cells share pheno-typical characteristics with long-lived plasma cells andexpress extensively hypermutated immunoglobulingenes. As multiple myeloma cells actively produce andsecrete immunoglobulin, they are prone to ER stress andtherefore require strict regulation of ER stress for survival.

Cellular strategies to maintain ER homeostasis includeactivation of the UPR, induction of chaperones, andautophagy, all of which have been shown to play impor-tant roles in myeloma pathogenesis (32). A significantnumber of genes involved in protein synthesis as well asthe UPR are frequently mutated in patients with multiplemyeloma (30).

TheUPR is highly active inmultiplemyeloma cells, andthis activity increases in advanced disease stages (32). Theexpression of UPR genes such as XBP1may be a selectionfactor during the progression of multiple myeloma by

Figure 2. IRE1 signaling during ERstress. Upon accumulation ofunfolded proteins in the ER lumen,the ER chaperone GRP78dissociates from the ERtransmembrane protein IRE1,allowing it to oligomerize andautophosphorylate causingactivation of its endoribonucleasedomain. This domain then splicesthe mRNA of XBP1 leading totranslation of the activetranscription factor XBP1s, whichregulates the transcription ofgenes involved in re-establishingER homeostasis. Theendoribonuclease domain alsocleaves other transcripts, leadingto their degradation (RIDD). Inaddition, activated IRE1 binds tothe adaptor protein TRAF2, leadingto the activation of ERK, JNK, andNF-kB. IRE1 interacts with otherproteins, such as Bax/Bak, HSP90,or HSP70, that modulate itsactivity.

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promoting cell survival during ER stress (32). At the sametime,multiplemyelomacells are exceptionally sensitive toER stress-induced apoptosis caused by the proteasomeinhibitor, bortezomib, compared with other cancer cells(33). This could be explained by the high immunoglobulinproduction leading to high basal ER stress before treat-ment (Fig. 3). Indeed, a correlation between the levels ofimmunoglobulin production and sensitivity of multiplemyeloma cells to ER stress was observed (34). AlthoughtheUPRpromotes survival ofmultiplemyeloma cells anddisease progression, the balance between prosurvival andproapoptotic signalingof theUPR is easily tipped towardsdeath when multiple myeloma cells are exposed to exog-enous ER stress. Thus, drug-induced ER stress by target-ing the protein quality control machinery as well asinhibition of prosurvival signaling pathways of the UPRemerge as promising strategies for treatment of multiplemyeloma.

Central role of XBP1s in multiple myelomaIn response to antigenic stimuli, mature B cells in the

germinal center differentiate into antibody producing

plasma cells, a process mediated by a complex transcrip-tional program involving the coordinated expression of anumber of transcription factors. This involves upregula-tion of the transcriptional repressor B-lymphocyte–induced maturation protein 1 (BLIMP-1), which sup-presses expression of genes maintaining earlier develop-mental stages and proliferation, and of XBP1, which is acritical transcription factor for plasma cell differentiationand immunoglobulin production (35). In line with thisimportant role in normal plasma cell biology, XBP1 isfrequently overexpressed inmultiplemyeloma (36). Inter-estingly, mutations in XBP1 have been found in a smallpercentageof patientswithmyeloma, further suggesting apotential causative role in disease pathogenesis at least insome patients (30).

The prognostic role for XBP1s overexpression hasrecently been recognized in the clinic. High levels ofspliced XBP1 mRNA were consistently detected in allsamples from a group of 253 newly diagnosed patients,and high ratios of spliced versus unspliced XBP1 mRNAdirectly correlated with lower median overall survival,which was independent of other previously known

Figure 3. Model depicting howinhibition of proteasome and/or IRE1may work in synergy to induce celldeath. A, myeloma cells have to dealwith a large load of unfolded proteinsdue to extensive immunoglobulinsynthesis. Unfolded proteins areremoved by the ERAD pathway.Accumulation of unfolded proteins inthe ER also induces IRE1 activation.IRE1 splices XBP1 mRNA to yieldXBP1s, an active transcription factorthatmediates adaptation of the ER tohigh secretory demand. XBP1u,translated from unspliced XBP1mRNA, is unstable and removed bythe proteasome. Also, the IRE1inhibitory protein BI-1 is degraded bythe proteasome. B, proteasomeinhibitors block the ERAD pathway,leading to accumulation of unfoldedproteins and consequently activationof IRE1, PERK, and ATF6. AlthoughXBP1splicingmayoccur, XBP1uandpossibly BI-1 also accumulate, thelatter interfering with IRE1 activation.Blocking XBP1 splicing with small-molecule IRE1 inhibitors actssynergistically by removing theXBP1s-induced adaptation andsurvival pathways.






prognostic factors (37). Patients treatedwith thalidomide-based regimes on the MRC Myeloma IX trial had inferioroutcome in the presence of increased XBP1s transcripts(37). Accordingly, XBP1s was proposed as an indepen-dent prognostic marker and a predictor of thalidomideresponse (37). In a separate study, analysis of samplesfrom 22 patients showed increased levels of spliced XBP1mRNA in stage III patients compared with stage I and IIpatients (32). Thus, high levels of XBP1s are correlatedwith advanced disease stages and poor prognosis fortreatment response and disease outcome. In contrast tothe findings in thalidomide-treated patients, a recentstudy found that low XBP1 mRNA levels predicted poorresponse to bortezomib, both in vitro and in patients (38).In this study, the ratio of spliced versus unspliced XBP1transcripts did not correlate with bortezomib sensitivity.These findings suggest that XBP1 levels are a correlate ofUPR induction in cells and, as shown by others (33), theUPR renders myeloma cells sensitive to proteasome inhi-bition. Therefore, the prognostic value of XBP1 levelsmaydepend on the type of therapy and how this influences theUPR.

A clear indication that XBP1 might play a causal role inmyeloma pathogenesis is a transgenic mouse, in whichXBP1s is overexpressed under the control of the Em-pro-moter in the B-cell lineage (31). At only 40 weeks of age,Em-XBP1s mice developed monoclonal gammopathy ofundetermined significance (MGUS), which resemblesmultiple myeloma in the secretion of paraprotein and canresult in the development of multiple myeloma, and 26%of mice spontaneously developed multiple myelomawithin 2 years (31). This study shows that XBP1 over-expression alone can drive transformation of plasma cellsand promote multiple myeloma pathogenesis. B cellsfrom these mice showed an enhanced proliferation rateand increased secretion of immunoglobulin comparedwith control mice. Microarray analysis identified morethan 1,000 genes that were differentially expressed in Em-XBP1s myeloma cells compared with B cells from youngneoplasm-free mice, including genes involved in the reg-ulation of cell-cycle progression and proliferation. Thisstrongly suggests a role of XBP1s in the expression of thesegenes. In particular, XBP1s may play an important role inthe regulation of IL-6, a cytokine essential for the survivalof plasma and myeloma cells (39).

Thus, XBP1s has been identified as a driving survivalfactor of multiple myeloma cells and may, in fact, deter-mine selective survival of more resilient tumor cells dur-ing the progression of multiple myeloma (31, 32, 37).

Although there is growing evidence implicating XBP1sin the pathogenesis of multiple myeloma, very little isknown about how the IRE1a/XBP1 pathway is regulatedin this disease. We recently reported that the 70 kDa HSP(HSP70, HSP72, or HSPA1) protects cells from ER stress-induced apoptosis by prolonging XBP1 splicing (40).HSP70 is an important survival factor in myeloma andhas been implicated in drug resistance (41). Adherence ofmyeloma cells to bonemarrow stromal cells or fibronectin

results in integrin-dependent upregulation of HSP70,inducing resistance to treatment with melphalan (41).HSP70 is also associated with bortezomib resistance(42). HSP70-mediated drug resistance may be, at least inpart, due to the enhanced XBP1 splicing.

Recent studies suggest that the bone marrow microen-vironment in multiple myeloma is hypoxic, with a major-ity of multiple myeloma cells residing in a hypoxic niche(43). Moreover, multiple myeloma cell lines grown in ahypoxic environment show activation of IRE1a withincreased XBP1 splicing (44). IRE1a activation secondaryto hypoxia may play an important role in the survival ofmultiple myeloma cells in the hypoxic bone marrowniche. As such sites may also be the location of tumor-initiating cells, IRE1a activation may play a role in theirsurvival during therapy.

Targeting Protein Turnover and Quality ControlProteasome inhibition

Currently, the single most important class of antimye-loma therapeutics is the proteasome inhibitors. Thedipeptide boronic acid analogue bortezomib (Velcade,PS-341; Fig. 4) is a potent, highly selective, and reversibleinhibitor of the 26S proteasome complex and inducesapoptosis in multiple myeloma cells (45). Many differentmechanisms of action may account for this activity ofbortezomib in multiple myeloma. One mechanism mayinvolve blockade of ERAD, resulting in accumulation ofunfolded proteins and induction of ER stress. In fact,bortezomib treatment rapidly activates PERK and eIF2aphosphorylation in multiple myeloma cells, followed byinduction of ATF4 and of CHOP (Table 1; ref. 33).

The induction of the proapoptotic BH3-only proteinNOXA may be critical for the proapoptotic effect ofbortezomib following induction of ER stress in myelomacells (46) and may be mediated by the activation of ATF3andATF4 (47). JNK is activated downstreamof IRE1a andfacilitates apoptosis by its ability to regulate BCL-2 familyproteins. Phosphorylation of BCL-2 by JNK suppresses itsantiapoptotic activity, whereas phosphorylation of theproapoptotic BH3-only member BIM enhances its proa-poptotic potential. Bortezomib has previously beenshown to induce the phosphorylation of JNK and itsdownstream targets c-JUN and ATF2 in myeloma cells.Inhibition of JNK activity reduced bortezomib-inducedapoptosis (48). These findings suggest that treatmentwithbortezomib induces ER stress, leading to activation of theproapoptotic arm of the IRE1a signaling pathway whilesimultaneously suppressingprosurvivalXBP1s signaling,leading to cell death.

Several mechanisms of bortezomib resistance havebeen described. These include mutation or overexpres-sion of proteasome subunits (49), induction of HSPs (42),the formation of aggresomes, induction of autophagy andfinally, a reduction in protein biosynthesis (in particularimmunoglobulins). In the case of acquired resistanceat the level of the proteasome, this may be overcome

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with alternative proteasome inhibitors, such as carfilzo-mib (PR-171), marizomib (NPI-0052), or ixazomib(MLN9708; Fig. 4), which, unlike bortezomib, irreversiblyblock the proteasome (50). However, resistance despiteadequate proteasome inhibition may require differentapproaches, including the use of HSP inhibitors andhistone deacetylase (HDAC) inhibition to overcomeaggresome-mediated resistance and inhibition of autop-hagy. Aggresomes are inclusion bodies formed by the

accumulation of misfolded proteins when the capacity ofthe intracellular protein folding and degradationmachin-ery is exceeded, such as with proteasome inhibition. Theformation of aggresomes leading to clearance of aggre-gated proteins is cytoprotective, enabling myeloma cellsto overcome anotherwise toxic load of protein aggregates.The formation of aggresomes involves transport by themicrotubule network, a process that is dependent onHDAC6 (51). There is evidence of a potent in vitro synergy

Figure 4. Chemical structures ofsmall molecular compoundstargeting protein quality control andthe UPR in multiple myeloma.






between HDAC6 inhibitors and bortezomib (52). In onestudy, the HDAC6 inhibitor Tubacin markedly augment-ed both JNK phosphorylation and caspase activity inmultiple myeloma cells leading to synergistic cell death(53). The Myc oncogene, which is frequently deregulatedin multiple myeloma, has been shown to regulate aggre-some formation (54).Myc activation leads to an increase in

protein translation inmultiplemyeloma cells, while at thesame time, upregulates HDAC6, favoring aggresomeformation (54). Bortezomib and HDAC inhibitor combi-nation studies are currently the subjects of clinicaltrials and patients with multiple myeloma with elevatedlevels of Myc activity may be particularly sensitive to thisapproach.

Table 1. Therapeutic agents that affect UPR signaling in multiple myeloma

Targeting protein turnover and quality control

Therapeuticagent

Moleculartarget Effect on UPR

In vitro antimyelomaactivity References

Phase of trial formultiple myeloma

Bortezomib(PS-341)

26S proteasome Induction of PERK,ATF4, CHOP

Induction of apoptosis 33, 60 U.S. Food andDrugAdministration–approved

17-AAG(Tanespimycin)

HSP90 Induction of CHOP andATF6, less inductionof XBP1 splicing

Induction of apoptosis 60 Phase III clinicaltrials

Radicicol HSP90 Induction of CHOP andATF6, less inductionof XBP1 splicing

Induction of apoptosis 60 Preclinical studies

MAL3-101 HSP70 Induction of XBP1splicing

Inhibition of proliferation,induction of apoptosis,enhanced effects ofproteasome andHSP90 inhibitors

61 Preclinical studies

CHR-2797(Tosedostat)

M1 amino-peptidases

Induction of CHOP,ATF4, and ATF6; noeffect on XBP1

Inhibtion of proliferationand survival in bonemarrowmicroenvironment,induction of apoptosis

69 Phase II clinical trial

Reolysin(Reovirus)

Whole cell Induction of XBP1splicing

Induction of apoptosis,sensitization tobortezomib


Targeting UPR signaling pathways

Therapeuticagent

Moleculartarget

Effect on UPR inmultiple myelomacells Antimyeloma activity References

Phase of trial formultiple myeloma

Sunitinib Kinases Inhibition of IRE1activity

Inhibition of proliferation 71 Phase II clinical trial

STF-083010 IRE1a RNase Inhibition of XBP1splicing

Cytotoxic in vitro and intransgenic mousemodels


MKC-3946 IRE1a RNase Inhibition of XBP1splicing

Sensitization tobortezomib and othertherapeuticcompounds


4m8C IRE1a RNase Inhibition of IRE1-mediated XBP1splicing

Inhibition of multiplemyeloma cell growth


NOTE: A range of therapeutic agents with antimyeloma activity affect UPR signaling. The table summarizes the stage of trials formultiplemyelomaonly.Someof theagentshavealreadybeenapprovedor tested inclinical studies for other typesof cancerbut havesofar been used for the treatment of multiple myeloma in preclinical studies only.

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Proteasome inhibitionmay increase the requirement formolecular chaperones to maintain accumulating proteinsin a soluble state and vice versa inhibition of molecularchaperones may lead to an increase in misfolded proteinsthat require degradation by the proteasome. In line withthis, HSP90 inhibition showed synergistic antimyelomaactivity in combination with bortezomib in multiple mye-loma cell lines as well as in mouse models (55–58). Acombination of bortezomib with the HSP90 inhibitortanespimycin (Fig. 4) has recently been tested in a phaseI/II study in patients with relapsed multiple myelomawith promising result as it showed activity even inpatients that hadpreviously been treatedwith bortezomib(59). Furthermore, the combinationwaswell tolerated andseemed to be safer than other combinational treatmentswith bortezomib (59).Acquired bortezomib resistance in multiple myeloma

has recently been associated with low levels of XBP1mRNA and of ATF6 protein (38). At the same time,acquired bortezomib resistance was accompanied byincreased levels of phosphorylated eIF2a and reducedimmunoglobulin production. This may suggest that theacquired bortezomib resistance is due to a dampenedimmunoglobulin synthesis leading to a reduction in ERprotein load, basal ER stress, and UPR activity. Intrigu-ingly, the acquired bortezomib resistancemade cellsmoresensitive to the melphalan and the ER stress inducertunicamycin (38). However, as experimentally manipu-lating XBP1 levels in multiple myeloma cell lines onlyslightly affected bortezomib sensitivity (38). These studiestherefore lend support to a therapeutic approach involv-ing the combination with an ER stress-inducing agent toenhance the effectiveness of proteasome inhibition and toovercome bortezomib resistance.

HSP inhibitorsInhibition of HSPs also leads to a disruption in protein

processing and inducesERstress. Indeed, bothHSP70 andHSP90 inhibitors, which show significant antimyelomaactivity, have been shown to induce the UPR in multiplemyeloma cells (Table 1; refs. 60, 61). HSPs are molecularchaperones that can bind client proteins and mediateprotein folding, refolding, stability, degradation, activa-tion, and trafficking. Increased expression of prosurvivalHSP90 and HSP70 has been observed in many types ofcancers and their inhibition has emerged as a promisinganticancer strategy (62). The HSP90 inhibitors 17-AAG(tanespimycin) and radicicol (Fig. 4) induce apoptosis inmultiple myeloma cell lines (60). Cell death induced bythese HSP90 inhibitors is associated with the induction ofthe UPR in multiple myeloma cells. Intriguingly, it wasobserved that ATF6 cleavage and CHOP expressiondownstream of PERK signaling were stimulated by bothcompounds to a higher extent than XBP1 splicing (Table1), as compared with the effects of common pharmaco-logic ER stress inducers (60). The early effects (within 2hours) on XBP1 splicing were similar but the HSP90inhibitors failed to induce further splicing on prolonged

incubation (up to 24 hours; ref. 60). Since HSP90 isinvolved in the regulation and stability of IRE1a, it isconceivable that inhibition may limit the degree of XBP1splicing (63). The net effect of HSP90 inhibition is adominantly proapoptotic UPR response. Stress-inducibleHSP70, a cochaperone ofHSP90was stronglyupregulatedas a survival response followingHSP90 inhibition in vitro,enabling myeloma cells to fold greater quantities of dam-aged proteins (60). In recent clinical trials of HSP90 inhi-bitors in multiple myeloma, consistent induction ofHSP70 has been observed and HSP70 induction is nowconsidered a biomarker of in vivo HSP90 inhibition (64).

Inhibition of HSP70 sensitizes cells to induction ofapoptosis byHSP90 inhibition (61, 65). The HSP70 inhib-itor MAL3-101 (Fig. 4) causes growth arrest and apopto-sis not only in human multiple myeloma cell lines, butalso in primary multiple myeloma cells, without toxicityin healthy control cell populations (61). MAL3-101induces XBP1 splicing at concentrations around 4-foldhigher than its IC50 value and showed synergistic effectsin combinationswith proteasome inhibitorsMG-132 andbortezomib (61).

Oncolytic virotherapyOncolytic viral therapy may represent another novel

therapeutic approach that induces ER stress in multiplemyeloma. The reovirus-based therapeutic Reolysin iswell tolerated in clinical trials for several cancers andshowed potent anticancer activity (66). It has beenproposed that Reolysin has a potential in targetingmultiple myeloma cells as reovirus replication maypromote ER stress-induced apoptosis via the accumu-lation of viral proteins (67). Indeed, Reolysin wasshown to have antimyeloma activity in cell lines, inex vivo patient tumor specimens, and in in vivo mousemodels of multiple myeloma (67, 68). Furthermore,Reolysin induced NOXA-mediated apoptosis in multi-ple myeloma cells and significantly increased the anti-myeloma activity of bortezomib (67). Interestingly,Reolysin induced ER stress in multiple myeloma cellsas determined by increased XBP1 splicing, ER swelling,and increased intracellular calcium levels (Table 1;ref. 67). This effect was significantly increased bycotreatment with bortezomib. Reolysin represents anattractive therapeutic strategy as reovirus was shownto selectively target multiple myeloma cells but nothematopoietic stem cells in a mouse model of multiplemyeloma (68).

Aminopeptidase inhibitorsAminopeptidases have been identified as a target

for cancer therapy, as their inhibition leads to a disruptionof protein turnover. The small-molecule inhibitor CHR-2797 (tosedostat; Fig. 4) targets the M1 family of amino-peptidases and is currently in phase II clinical trials (Table1). CHR-2797 has been shown to inhibit the proliferationof multiple myeloma cells, induce apoptosis, and over-come the protective effect of the bone marrow stroma






microenvironment on multiple myeloma cells (69). CHR-2797 increased the levels of ATF4 and CHOP and stimu-lates the activation of ATF6 in themyeloma cell lineH929,without affecting XBP1 splicing (69), thus primarilyinducing proapoptotic UPR signaling. Similarly to theeffect of proteasome inhibition, inhibition of aminopepti-dases by CHR-2797 leads to the upregulation of HSP70(69).

Targeting IRE1a as a Therapeutic Strategy inMultiple Myeloma

Given the importance of the IRE1a/XBP1 axis in mye-loma, it is not surprising that this has become an attractivetarget for drug development.

The kinase inhibitor sunitinib has antimyeloma activity(70) and is currently in phase II clinical trials. Sunitinibwas shown to reduce XBP1 splicing in multiple myelomacells, which shows the potential of developing specifickinase inhibitors of IRE1a as a means of modulating theUPR in human cells (71).

However, if IRE1a kinase inhibitors also prevent JNKactivation, this could be counterproductive. Severalgroups have recently shown the potential of compoundsthat specifically target the RNase activity of IRE1a andselectively reduce XBP1 splicing. STF-083010 (Fig. 4) wasrecently reported as a novel small-molecule inhibitor ofIRE1a RNase activity in multiple myeloma (72). STF-083010 inhibits XBP1 splicing in multiple myeloma celllines treated with a range of ER stress-inducing agents(Table 1), while not affecting the kinase activity, and alsoinhibited XBP1 splicing in a transgenic mouse expressingan XBP1-luciferase reporter gene. STF-083010 showedcytotoxic activity in multiple myeloma cell lines and wasselectively toxic to transformed cells isolated frompatients with multiple myeloma as compared withhealthy plasma cells, showing the therapeutic potentialof targeting XBP1 splicing (72). Another recent studyreported the aldehyde 8-formyl-7-hydroxy-4-methylcou-marin (4m8C; Fig. 4) as an inhibitor of IRE1a RNaseactivity, inhibiting both XBP1 splicing and RIDD, but notaffecting IRE1a autophosphorylation (73). This studyshowed that 4m8C acts by covalently and selectivelybinding to a lysine residue (K907) within the RNasedomain inhibiting its activity in a noncompetitive fashion,and elucidated the mechanism of action of other reportedIRE1a inhibitors. STF-083010 selectively binds to K907,whereas the compounds described by Volkmann andcolleagues (74) were shown to bind to K907 and K599within IRE1a kinase domain. These findings are consis-tent with the observation that STF-083010 selectivelyinhibits IRE1a endoribonuclease activity without affect-ing its kinase activity, whereas the compounds describedby Volkmann and colleagues (74) inhibited RNaseactivity but could, at higher concentrations, also inhibitautophosphorylation.

The activity of another compound specifically target-ing the endoribonuclease domain of IRE1a has recentlybeen reported (44). MKC-3946 (Fig. 4) was shown to

inhibit growth of multiple myeloma cell lines but notof normal mononuclear cells. Furthermore, MKC-3946inhibited XBP1 splicing after treatment with bortezo-mib or 17-AAG. This sensitized multiple myeloma cellsto cytotoxicity of these compounds and overcame theprotection provided by bone marrow stromal cellsand IL-6 treatment (Table 1). MKC-3946 also inhibitedXBP1 splicing in vivo and significantly inhibited growthof RPMI8226 plasmacytoma in a xenograft murinemodel (44). Treatment of multiple myeloma cells withMKC-3946 did not affect the phosphorylation state ofIRE1a (44). In fact, binding of IRE1a to TRAF2 and phos-phorylation of JNK were both enhanced by MKC-3946treatment.

However, given that IRE1a is involved in the UPR inother cell types, particularly highly secretory cells, sys-temic targeting of IRE1amay have undesired side effectsand caution will be required when IRE1a inhibitors enterclinical development (75).

ConclusionA hallmark of multiple myeloma is the high level of

production and secretion of immunoglobulin putting aheavy load on the secretory machinery and perturbingproteostasis (protein homeostasis) within the ER. Theproteostasis network includes all pathways involved inprotein synthesis, folding, trafficking, and degradation.Disturbances of proteostasis lead to accumulation of mis-folded proteins and induction of cellular stress responsessuch as the UPR in case of proteostatic stress within theER. Because of their high secretory activity, multiplemyeloma cells experience persistent high levels of ERstress and are dependent on the UPR for maintenance ofproteostasis. Consequently, multiple myeloma cells arecharacterized by a high basal UPR activity, and multiplemyeloma cells may be addicted to the UPR for survival.Thus, multiple myeloma cells are highly sensitive tocompounds that target proteostasis, such as proteasomeinhibitors, and in particular, such that directly target ERproteostasis such as IRE1a inhibitors. These compoundsact by shifting the balance between prosurvival and proa-poptotic signaling of the UPR, pushing the cells beyondthe point of no return. Considering these, multiple mye-loma cells are particularly sensitive to agents that furtherdisturbproteostasis. Furthermore, synergistic effects havebeen observed when targeting several pathways of theproteostasis network such as the proteasome and HSPs.Interestingly, bortezomib resistance has been linked toreduced UPR signaling, which indicates that reducedbasal ER stress, possibly due to a downregulation ofimmunoglobulin production,mayplay a role in resistanceto proteasome inhibition. Thus, treatment with ER stress-inducing agents might be a promising strategy to re-sensitize multiple myeloma cells to proteasome inhibi-tion. The UPR-induced transcription factor XBP1s hasbeen identified as a driving survival factor of multiplemyeloma and inhibitors specifically targeting XBP1mRNA splicing by IRE1a show antimyeloma activity.

Vincenz et al.





Furthermore, NF-kB, an important survival factor inmul-tiple myeloma, may be induced by the UPR. Severalantimyeloma compounds target NF-kB and directlytargeting NF-kB is a promising antimyeloma therapy.Targeting prosurvival signaling of the UPR is a novelpromising strategy for myeloma therapy. Taken together,we describe two therapeutic strategies targeting thisAchilles heel of multiple myeloma cells; first, compoundsthat disturb ER proteostasis by targeting components ofthe protein quality control machinery, such as the protea-some or HSPs, leading to ER stress; and second, com-pounds directly targeting the prosurvival signaling armsof the UPR, in particular IRE1a.Targeting proteostasis is themost powerful strategy for

the treatment of multiple myeloma. In particular, theIRE1a/XBP1 pathway seems to play an important rolein multiple myeloma pathogenesis and therapeuticresponses. Targeting ER stress responses emerges as apromising strategy to overcome resistance of multiplemyeloma cells to current treatment modalities.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Vincenz, R. J€ager, M. O’Dwyer, A. Samali.Development of methodology: L. Vincenz, M. O’DwyerAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M. O’DwyerWriting, review, and/or revision of the manuscript: L. Vincenz, R. J€ager,M. O’Dwyer, A. SamaliAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. VincenzStudy supervision: A. Samali

Grant SupportA. Samali is recipient of grants from Science Foundation Ireland (09/

RFP/BIC2371), the Health Research Board (HRA/2009/59), and BreastCancer Campaign (2010NovPR13). L. Vincenz was funded by an IrishCancer Society Scholarship (CRS10VIN).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 31, 2012; revised January 21, 2013; accepted February 6,2013; published OnlineFirst May 31, 2013.

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2013;12:831-843. Published OnlineFirst May 31, 2013.Mol Cancer Ther Lisa Vincenz, Richard Jäger, Michael O'Dwyer, et al. Targeting the Achilles Heel of Multiple MyelomaEndoplasmic Reticulum Stress and the Unfolded Protein Response:

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