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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.
MolecularCancer
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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.
Vincenz et al.
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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.
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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.
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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
66 Preclinical studies
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
72 Preclinical studies
MKC-3946 IRE1a RNase Inhibition of XBP1splicing
Sensitization tobortezomib and othertherapeuticcompounds
44 Preclinical studies
4m8C IRE1a RNase Inhibition of IRE1-mediated XBP1splicing
Inhibition of multiplemyeloma cell growth
73 Preclinical studies
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
UPR in Multiple Myeloma
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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.
Mol Cancer Ther; 12(6) June 2013 Molecular Cancer
Therapeutics840
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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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