Synergistic Myeloma Cell Death via Novel … · proteasome inhibitor (PI) ... (Stem Cell...

Small Molecule Therapeutics

Synergistic Myeloma Cell Death via NovelIntracellular Activation of Caspase-10–DependentApoptosis by Carfilzomib and SelinexorShaun Rosebeck1, Mattina M. Alonge1, Malathi Kandarpa2, Anoop Mayampurath3,Samuel L. Volchenboum3, Jagoda Jasielec1, Dominik Dytfeld4, Sean P. Maxwell1,Stephanie J. Kraftson2, Dilara McCauley5, Sharon Shacham5, Michael Kauffman5, andAndrzej J. Jakubowiak1

Abstract

Exportin1 (XPO1; also known as chromosome maintenanceregion 1, or CRM1) controls nucleo-cytoplasmic transport ofmost tumor suppressors and is overexpressed in many cancers,including multiple myeloma, functionally impairing tumorsuppressive function via target mislocalization. Selective inhib-itor of nuclear export (SINE) compounds block XPO1-mediatednuclear escape by disrupting cargo protein binding, leading toretention of tumor suppressors, induction of cancer cell death,and sensitization to other drugs. Combined treatment with theclinical stage SINE compound selinexor and the irreversibleproteasome inhibitor (PI) carfilzomib induced synergistic celldeath of myeloma cell lines and primary plasma cells derivedfrom relapsing/refractory myeloma patients and completely

impaired the growth of myeloma cell line–derived tumors inmice. Investigating the details of SINE/PI-induced cell deathrevealed (i) reduced Bcl-2 expression and cleavage and inactiva-tion of Akt, two prosurvival regulators of apoptosis and autop-hagy; (ii) intracellularmembrane-associated aggregation of activecaspases, which depended on caspase-10 protease activity; and(iii) novel association of caspase-10 and autophagy-associatedproteins p62 and LC3 II, which may prime activation of thecaspase cascade. Overall, our findings provide novel mechanisticrationale behind the potent cell death induced by combiningselinexor with carfilzomib and support their use in the treatmentof relapsed/refractorymyeloma and potentially other cancers.MolCancer Ther; 15(1); 60–71. �2015 AACR.

IntroductionCancer cells acquire several stochastic characteristics that pro-

vide growth and survival advantages (1). Multiple cellular factorsaffecting these processes, including tumor suppressor and onco-genic proteins (2–4), are deregulated in cancer, perturbing theirnatural function. One manner in which these proteins may bedysregulated is via mislocalization within the cell. Exportin 1(XPO1; or chromosome maintenance region 1, CRM1) is theprominent nuclear export receptor that controls nucleo-cyto-plasmic localization of many proteins (2) and is overexpressedin many cancer types both solid and hematologic in origin,including multiple myeloma as shown by us and others (4–6),promoting aberrant cytoplasmic accumulation of its cargo. Smallmolecule, selective inhibitors of nuclear export (SINE) force

nuclear retention of deregulated targets by preventing XPO1/cargo interaction, thereby reestablishing normal tumor suppres-sive functions and rendering cancer cells responsive to otherchemotherapeutics. We and others have demonstrated the cyto-toxic effects of SINE in multiple myeloma (4–7). Currently,multiple phase I/II clinical trials are enrolling to test the efficacyof the orally available SINE, selinexor, in patients with advancedstage pancreatic, prostate, salivary gland, and rectal cancers,glioblastoma,melanoma, non-Hodgkin's lymphoma, acutemye-loid leukemia, and relapsed/refractory multiple myeloma (R/Rmultiple myeloma).

Despite significant advances in treatment options, multiplemyeloma remains incurable and the majority of patients willrelapse and become refractory to most agents. Proteasome inhi-bitors (PI), including the second-generation irreversible PI carfil-zomib, and immunomodulatory agents are among several classesof drugs that have improved the rate and duration of patientresponses (8). Both classes of drugs target not only the myelomatumor cell but also the bonemarrowmicroenvironment, the latterof which supports tumor cell growth and drug resistance, likelycontributing to their efficacy when used together. Despite thesuccess of combination treatment regimens using these drugs,many patients fail to achieve a durable response, necessitatingfurther investigation into novel therapeutic options.

The induction of cell death and autophagy are underlyingcommonalities highlighted by the cellular effects of PI and SINE.Recently, autophagy emerged as a myeloma cell death pathwayand alternate to the ubiquitin–proteasome pathway by which

1Department of Medicine, University of Chicago, Chicago, Illinois.2University of Michigan Comprehensive Cancer Center, Ann Arbor,Michigan. 3Computation Institute, University of Chicago, Chicago,Illinois. 4Poznan University of Medical Sciences, Poznan, Poland. 5Kar-yopharm Therapeutics, Newton, Massachusetts.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Andrzej J. Jakubowiak, University of Chicago, 5841 S.Maryland Avenue, MC2115, Chicago, IL 60637. Phone: 773-702-4400; Fax: 773-702-0963; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0488

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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Published OnlineFirst December 4, 2015; DOI: 10.1158/1535-7163.MCT-15-0488

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excess and/or dysfunctional proteins and organelles are degradedand recycled (9–13). RNA knockdown of XPO1 is sufficient toinduce autophagy (14) and is cytotoxic tomyeloma cells (4), and,similarly, SINE compounds induce the degradation of XPO1protein. PI promote accumulation and aggregation of proteinsdestined to be degraded by the proteasome, and these excessproteins are instead degraded via the autophagic machinery.Myeloma cells require basal autophagy for survival and perturb-ing the balance between limited and hyperactive autophagypromotes apoptosis-independent cell death (12). Given theknown effects of both SINE and PI on apoptosis, the establishedeffects of PI on autophagy, and the putative effects of SINE onautophagy, we hypothesized that the combination of selinexorand carfilzomib could augment the antimyeloma activity of eitheragent alone and sought to determine cellular mechanisms bywhich these drugs exert their effects.

Materials and MethodsCell lines and patient samples

Human myeloma cell lines (HMCL) were obtained from theAmerican Type Culture Collection (ATCC) in the last 3 yearsand authentication was not performed. HMCL and patientplasma cells (PC) were cultured in RPMI 1640, 10% FBS, andantibiotics in a 5% CO2 humidified incubator. Carfilzomib-resistant RPMI 8226/Dox40 cells were previously described(15, 16). PC were purified from multiple myeloma patientbone marrow aspirates using immunomagnetic positive selec-tion of CD138þ cells (Stem Cell Technology) and determinedto be >80% enriched by examining Wright-Giemsa stainedcytospins. After Institutional Review Board approval, informedconsent to treatment and sample procurement was obtainedfrom patients.

Detection of autophagy and aggresome formationAutophagy was monitored in live cells using the Cyto-ID

Autophagy Detection Kit (Enzo Life Sciences) according to man-ufacturer recommendations by fluorescence microscopy.

Aggresome formation was detected in fixed cells using theProteoStat Aggresome Detection Kit (Enzo Life Sciences) accord-ing to the manufacturer's recommendations. Images were cap-tured using a Panoramic Scan whole slide scanner (CambridgeResearch and Instrumentation) and analyzed using 3DHistechsoftware. Fold changes in the mean fluorescence intensity (FI;determined using ImageJ software) from the Hoescht and aggre-some dye signals were calculated from ten random fields fromeach slide/treatment and determined by [(FI aggresome dye)/(FIHoescht)].

Immunofluorescence microscopyHMCL were cytospun onto slides, fixed in formaldehyde (4%

in 1� PBS), permeabilized with methanol, and quenchedovernight with sodium borohydride (2 mg/mL in PBS). Slideswere blocked with 2% BSA in PBS, incubated with primaryantibody for 2 hours at room temperature, washed, and incu-bated in secondary antibody for 1 hour at room temperaturewhile protected from light. After washing, slides were mountedwith coverslips and images were captured using a Zeiss Axio-plan upright fluorescence microscope equipped with a CCDcamera (Sensys) and pseudocolored using IPLab imaging soft-ware (Becton Dickinson).

MTT assayHMCL were plated in 96-well plates at 4 � 105 cells/mL after

drugdilutionsweremade and treated for 72hours.MTT (thiazolylblue tetrazoliumbromide; Amresco)was added and incubated forup to 6 hours at 37�C.Cells were lysed and absorbancewas read at570 nm on a Tecan Safire microplate reader.

GST pull-downGST pull-downs were performed as previously described (17)

using MM1.S cells (2 � 107) left untreated or treated withselinexor (50 nmol/L) and carfilzomib (50 nmol/L) for 18 hours.

Murine xenograft studyNOD-SCIDmice were inoculated subcutaneously with 1� 107

NCI H929 cells. When tumors reached a mean volume of100mm3,mice were randomized into groups of eight and treatedwith vehicle (0.6%Pluronic F-68, 0.6%PVP inwater), selinexor (5or 10mg/kg) thrice weekly per os, or twice weekly carfilzomib (1.5or 3mg/kg) intravenously, alone or in combination.Weightswererecorded daily and tumor volume was measured thrice weekly.Animals with greater than 13% weight loss were rested fromdosing until weight loss recovered to no more than 5%. Thisstudy was carried out by Pharmamodels LLC in accordance withthe guidelines for animal welfare and with the approval of theIACUC.

Statistical analysisGraphPad Prism software was used for calculation of all sta-

tistics of significance and IC50 values. Combination indices weredetermined using Calcusyn software (Biosoft).

ResultsSINE and PI treatment both induce hallmarks of apoptosis andautophagy

First, we tested the effects of XPO1 inhibition alone and incombination with carfilzomib on induction of autophagy mar-kers. Characteristically, autophagy involves sequestration of cyto-plasmic contents into double-membrane vesicles, known asautophagosomes, for delivery to lysosomes and degradation.Formation of autophagy-associated membranes can be moni-tored by the processing and accumulation of microtubule-asso-ciated light chain 3 (LC3), a protein that undergoes lipidation andcleavage upon insertion into nascent membranes (18). RPMI8226 and MM1.S cells were treated up to 96 hours with eitherselinexor or carfilzomib alone and in combination. In response toselinexor alone,Western blot analysis revealed prompt processingand accumulation of LC3 II (Fig. 1A) and sequestosome 1 (p62),an LC3-interacting protein and scavenger of ubiquitinated pro-teins fated for degradation via autophagy that is also consumedbyautophagy (19). Caspase-10 levels decreasedmodestly during thecourse of treatment, which is associated with the induction ofautophagy in myeloma cells (12). Treatment with carfilzomibalso promoted LC3 II processing, accumulation and degradationof p62 and LC3 II, and loss of caspase-10, albeit in a faster timeframe than selinexor. IRF4, a transcriptional regulator of caspase-10, BCLAF1, and Bcl-2 expression were impaired, which mayenhance autophagy induction (20). Combination treatment withSINE and PI accelerated these effects, which were similarly dem-onstrated in response to prolonged pan-caspase inhibition, amyeloma cell autophagy inducer (Supplementary Fig. S1; ref. 12).

Targeting Myeloma by Proteasome and XPO1 Inhibitor

www.aacrjournals.org Mol Cancer Ther; 15(1) January 2016 61




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Figure 1.Effects of selinexor and carfilzomib on autophagy. A, Western blot analysis of RPMI 8226 (left) and MM1.S (right) cells treated for the indicated times with selinexor (50nmol/L) and carfilzomib (50 nmol/L, RPMI 8226; 10 nmol/L, MM1.S) alone and in combination. B, Wright-Giemsa stained cytospins of HMCL treated with selinexor(50 nmol/L) or carfilzomib (50 nmol/L) alone or in combination. 100�; scale bar, 10 mm. C, MM1.S cells treated as in B were loaded with an autophagy-specific fluorescentdye. 63�; scale bar, 10 mm. D, changes in aggresome formation in RPMI 8226 cells treated as indicated for 18 hours (� , P ¼ 0.0489; �� , P ¼ 0.0064; ��, P ¼ 0.0065).

Rosebeck et al.

Mol Cancer Ther; 15(1) January 2016 Molecular Cancer Therapeutics62




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Figure 2.Combined selinexor and carfilzomib treatment results in synergistic myeloma cell death and suppresses tumor growth. A, dose responses of HMCLtreated with selinexor and carfilzomib for 72 hours determined by MTT assay. Combination index (CI) values at ED50 are indicated; values less than 1 indicatesynergy. B, Western blot analysis of MM1.S cells treated as indicated for 24 hours detecting markers of apoptosis and autophagy. C, Western blotanalysis of PC from a R/R multiple myeloma patient treated as indicated for 24 hours. D, as in B, combination dose responses in carfilzomib-resistantDox40 cells. E, effects of single agent and combination treatment in NOD-SCID mice implanted with NCI H929 cells subcutaneously (s.c.). Treatmentschedule is depicted below (� , P < 0.0001).






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Figure 3.Caspase-10 activity is necessary for selinexor/carfilzomib-induced cell death. A, caspase-10 protease activity and cleavage were determined from thesame lysates in response to the indicated treatments of MM1.S cells for 24 hours (� , P < 0.0001). B, relative caspase activity was determined afterselinexor (50 nmol/L) and carfilzomib (50 nmol/L) treatment �/þ Q-AEVD-OPH (25 mmol/L) for 24 hours. (Inset) Western blot confirms inhibition ofcaspase-10 and PARP cleavage. C, MM1.S cells were treated with selinexor and carfilzomib (KPT/CFZ; 50 nmol/L each) alone or with increasing amounts ofLEHD.fmk caspase-9 inhibitor (10–100 mmol/L) for 24 hours and analyzed by Western blot. (Continued on the following page.)

Rosebeck et al.





Next, we examined the morphology of myeloma cell linestreated with each inhibitor alone and in combination afterWright-Giemsa staining. Features of both autophagy and apopto-sis were prominent in all three myeloma cell lines. Both selinexorand carfilzomib alone promoted accumulation of cytoplasmicvacuoles, marginalization and degradation of nuclear material,and breakdown of cell membranes and blebbing of cellularcontents (Fig. 1B), which occurred faster in the presence of both.MM1.S cells loaded with a dye that recognizes autophagosomeformation demonstrated a moderate increase in dye accumula-tion with either drug alone and this effect occurred faster with thecombination (Fig. 1C).We alsomonitored aggresome formation,which is enhanced in the setting of diminished proteasomeactivity (21), and detected a significant increase in response toeither inhibitor alone and with the combination (Fig. 1D andSupplementary Fig. S2). These data support our hypothesis thatSINE and PI have common cellular effects, warranting furtherinvestigation into their interaction.

Synergistic antimyeloma activity of selinexor and carfilzomibNext,we tested the effects of combined treatmentwith selinexor

and carfilzomib on myeloma cell viability. Statistical analysis ofMTT assays determined drug combination indices (CI) to be lessthan 1 in response to treatment with both selinexor and carfilzo-mib in three myeloma cell lines, indicating a synergistic relation-ship (Fig. 2A). Western blot analysis of MM1.S cells treated withthe combination demonstrated augmented activation of caspase-8, -9, and -3 and increasedPARP cleavage as comparedwith single-agent treatment (Fig. 2B).Wedetected concomitant accumulationof LC3 II and loss of caspase-10, further supporting that inductionof autophagy correlates with activation of apoptotic machinery.Importantly, we investigated the effects of selinexor and carfilzo-mib in PC derived from R/R multiple myeloma patients whofailed on combination treatment regimens that included carfil-zomib.Western blot analyses demonstrated that PC from three ofthe four R/R multiple myeloma patients responded well to eitherselinexor or carfilzomib alone, and clearly had enhanced markersfor autophagy (patient 7) and apoptosis (patients 7, 8, and 9)when the combination was used. Interestingly, PC from patient10, who progressed on a carfilzomib-containing regimen, did nothave a robust response to carfilzomib as a single agent, butresponded to selinexor (Fig. 2C and Supplementary Fig. S3). Totest the effects of these drugs in a more defined setting of drugresistance, we also assessed the cytotoxicity of SINE and PI in asubclone of RPMI 8226 cells (16), Dox40, which were created bycontinuous exposure to doxorubicin and subsequently found tooverexpress themultidrug resistance p-glycoprotein and are cross-resistant to PI (15, 16, 22). Although selinexor alone was able toreduce the viability of the Dox40 cells, there were no additive orsynergistic effects of the combination (Fig. 2D).

We further investigated the effects of selinexor and carfilzomibin a murine myeloma xenograft model. Although treatment witheither carfilzomib or selinexor alone reduced growth of xeno-grafts, the combination of both drugs completely abrogated

tumor growth (Fig. 2E). Single-agent treatment was well-tolerat-ed; however, two mice from the highest dose combination grouphad to be rested from treatment due to excessive weight loss(Supplementary Fig. S4). Antitumor responses of all nine treat-ment groups are shown in Supplementary Fig. S5. Altogether,these data underscore the potent synergistic antimyelomaeffects of selinexor and carfilzomib and provide preclinical ratio-nale for deploying this drug combination for multiple myelomatreatment.

Role of caspase-10 activation in SINE/PI-induced cell deathNext, we investigated the mechanism of cell death induced by

combined XPO1 and proteasome inhibition. We focused oncaspase-10because itwas recently highlighted as a key cell survivalfactor for all genotypes of myeloma (12) and is less well charac-terized due to its absence from the rodent lineage.Our results thusfar suggest that full-length caspase-10 levels decrease in responseto selinexor or carfilzomib treatment, which possibly accounts forincreased autophagy because caspase-10 limits autophagy inHMCL (12). Examination of other caspases shows cleavage andactivation of the caspase-10 ortholog caspase-8, and caspase-9and -3. To determine if caspase-10 is also activated in response toselinexor and carfilzomib, we assayed its protease activity and thepresence of cleaved/activated fragments and found both increasewith either selinexor or carfilzomib alone, and significantly sowith the combination (Fig. 3A). As an initiator caspase in thecontext of certain death receptor apoptotic pathways (23), wesought to determine whether caspase-10 enzymatic activity wasnecessary for activation of other caspases. Wemonitored proteaseactivity of caspase-1 (negative control), -3, -8, -9, and -10 inresponse to combined selinexor/carfilzomib treatment and alsocotreated cells with a peptide inhibitor of caspase-10, Q-AEVD-OPH (Fig. 3B). Inhibition of caspase-10 catalytic activity abol-ished SINE/PI-induced caspase activity as well as PARP cleavage(Fig. 3B and inset).

In response to certain stimuli, caspase-9 can promote caspase-10 activation (24). Therefore, we alsomonitored the effects of thecaspase-9 inhibitor LEHD.fmk on SINE/PI-induced caspase acti-vation. Western blot analysis showed that, although LEHD.fmkaffected caspase-9 activation at the lowest dose (10 mmol/L),activation of caspase-3, -8, and -10 was only modestly impaired(Fig. 3C). Furthermore, unlike caspase-10 inhibition, PARP cleav-age could not be abrogated even at the highest dose of LEHD.fmk(100 mmol/L). We also tested the effects of siRNA-mediatedinhibition of caspase-9 expression and found that loss of cas-pase-9 had no effect on activation of caspase-3, -8, and -10 andPARP cleavage (Fig. 3D). We monitored overall cell viability inresponse to cotreatment with either LEHD.fmk,Q-AEVD-OPH, orpan-caspase inhibitor Q-VD-OPH and found that while all threeinhibitors were able to significantly protect against selinexor/carfilzomib-induced cell death, caspase-10 inhibition more sig-nificantly reversed cell death than either pan- or caspase-9 inhi-bition (Fig. 3E). Treatment with each inhibitor alone had nosignificant effect on cell viability (Supplementary Fig. S6).

(Continued.) D, 293T cells were transfected on two consecutive days with pools of either control or caspase-9–specific siRNAs. After 48 hours, cells weretreated as indicated with selinexor and carfilzomib (KPT/CFZ; 100 nmol/L each) for 18 hours and analyzed by Western blot. E, effects of pan-caspase inhibitorQ-VD-OPH (VD; 25 mmol/L), caspase-9 inhibitor (LEHD; 50 mmol/L), or caspase-10 inhibitor Q-AEVD-OPH (AEVD; 25 mmol/L) on overall cell viability inMM1.S and RPMI 8226 cells treated with selinexor and carfilzomib (KPT/CFZ) at 50 nmol/L each for 24 hours (�, P < 0.0001; �� , P < 0.001). F, 293T cells werestably transfected with a pool of caspase-10 (C10)-targeting or control (Ctl) CRISPR/Cas 9 plasmids. Multiple clones were tested for sensitivity toselinexor and carfilzomib alone and in combination (K, C, K/C; 100 nmol/L each).






Importantly, CRISPR-mediated stable knockdown of caspase-10prevented apoptosis in response to single agent and combinedSINE/PI treatment (Fig. 3F). Together, these results underscore thenovel role of caspase-10 as a key upstream caspase in the contextof SINE/PI-induced apoptosis.

Novel mechanism of intracellular caspase-10 activationDuring proteasome inhibition, and by extension autophagy,

ubiquitinated caspase-8 forms an apoptosis-inducing complex onintracellular membranes that depends on LC3 and p62 (25, 26).p62 has a newly-appreciated role in modulating cell fate byinteracting with key signaling proteins marked by ubiquitinationand can promote apoptosis or survival depending on the stimulus(27). Given the high degree of homology between caspase-8and -10, we wanted to determine whether there was any relation-ship between caspase-10 and p62 or LC3. MM1.S cells weretreated with SINE/PI and CHAPS-soluble and insoluble fractions,the latter containing intracellularmembranes derived fromautop-hagosomes, were analyzed byWestern blot.Wewere struck by thepresence of "preactivated" cleaved caspase-10, -8, and -9 exclu-sively in the insoluble fractions of untreated cells, yet only aminorpresence of the corresponding full-length caspase (Fig. 4A). Levelsof p62 were also detectable, albeit by long exposure (not shown),and LC3 I was cytoplasmic, whereas LC3 II, which becomesembedded in nascent autophagosomes, was predominantly inthe insoluble fraction. Upon treatment with selinexor/carfilzo-mib, we detected dramatically increased levels of cleaved/activeforms of caspase-10, -8, and -9 almost exclusively in the CHAPS-insoluble fraction. Caspase-3 did not have membrane-associated"preactivated" amounts in untreated cells and its distribution wasmarkedly less exclusively separated upon selinexor/carfilzomibtreatment. Total levels of p62 dramatically increased in responseto SINE/PI treatment, and its distribution was comparably spreadbetween the CHAPS-soluble and insoluble fractions. Two frag-ments of p62 appeared in the insoluble fraction and themolecularweights correspond to previously-identified caspase-cleaved pro-ducts (28). Q-AEVD-OPH cotreatment suppressed activation ofall caspases, as shown in Fig. 3B, and impaired p62 cleavage, buthad no effect on LC3 processing or accumulation. Q-AEVD-OPHalone was not able to reduce the levels of "preactivated" caspase-10, -9, and -8 below that of mock-treated cells, suggesting thatrelocalization and recruitment to intracellular membranes mayinfluence caspase activation.

To further explore this intriguing and novel relationshipbetween p62 and caspase-10, we assessed their subcellular dis-tribution and ability to interact. Using immunofluorescencemicroscopy, untreated cells showed diffuse, occasional punctatestaining of caspase-8 and -10 and p62. However, selinexor/car-filzomib-treated cells demonstrated overt intracellular punctatestaining and colocalization of caspase-10 and -8 with p62 (Fig.4B). Detection of cell surface expression of Fas and its lack ofcolocalization with p62-containing puncta further supports theintracellular nature of these aggregates (Supplementary Fig. S7).GST-tagged p62 associated with full-length endogenous caspase-10 and presumably full-length caspase-8modified by ubiquitina-tion from untreated cell lysates, as well as LC3 II and poly-ubiquitinated proteins, but not LC3I or caspase-9 or -3 (Fig.4C). Interestingly, despite their colocalization, the associationbetween caspase-8 and -10 and GST-p62 was lost in lysates fromselinexor/carfilzomib-treated cells; however, the presence of LC3II and poly-ubiquitinated proteins increased.

Next, we tested the effects of LY294002 (LY), a PI3K inhibitorthat blocks autophagosome membrane nucleation, on the dis-tribution of caspases and autophagy players. LY treatment alonereduced levels of "preactivated" caspase-8 and -10 and accumu-lation of LC3 II in the insoluble fraction and pretreatment with LYprior to SINE/PI modestly reduced cleavage and redistribution ofcaspase-8 and -10 and p62 to the insoluble fraction (Fig. 5A).siRNA-mediated knockdown of Beclin 1, a key upstream autop-hagy initiator (29), impaired redistribution and activation ofcaspase-10 and -8 (Fig. 5B). Interestingly, p62 levels were dra-matically reduced, which could account for diminished caspase-10 and -8 recruitment and activation, although more work isneeded to determine specifically how impaired Beclin 1 expres-sion influences caspase-10 and -8 activation beyond the potentialinhibition of autophagosome formation.

Together, these results suggested that perhaps p62 plays a rolein promoting assembly of caspase-activating complexes alongintracellular/autophagic membranes and that caspase-10 is anovel upstream caspase activator in the setting of combinedXPO1and proteasome inhibition.

Combined selinexor/carfilzomib treatment overcomescytoprotective autophagy

Because autophagy can be cytoprotective in the setting oflimited nutrients or other cell-stressing conditions (11) yet alsoserve as a myeloma cell death pathway, we wanted to determinethe contribution of autophagy to the overall lethal effects of SINE/PI. We tested the effects of bafilomycin, which blocks the finalsteps of autophagy by preventing lysosomal acidification, onapoptosis induction and cell viability. High doses of bafilomycinhad minimal effects on apoptosis either alone or with combina-tion SINE/PI treatment, but promoted LC3 II accumulation asexpected (Fig. 6A and left inset Fig. 6B) and reduced overall cellviability (right inset Fig. 6B). We assessed differences in cellviability at sublethal yet autophagy-inhibiting doses of bafilomy-cin in combination with increasing doses of selinexor/carfilzo-mib, but did not detect significant changes, suggesting thatautophagy may not provide a cytoprotective effect in the settingof strong commitment to apoptosis promoted by the combina-tion of SINE and PI (Fig. 6B). These data show that, in this context,autophagy is neither protective against nor additive to selinexor/carfilzomib-induced cell death.

To further explore the relationship between the induction ofapoptosis and autophagy, we performed a time course with SINE/PI and assessed caspase activation, cleavage of apoptotic sub-strates, and LC3 processing. We also assessed levels of total Aktand its activation state given its pivotal role in myeloma cellsurvival and drug resistance (30), and its ability to limit autop-hagy (31). Interestingly, we found simultaneous activation ofcaspase-10, -8, -9, and -3 and cleavage of both PARP and Aktbeginning 6 hours after treatment (Fig. 6C), which may accountfor the rapid phenotypic changes associated with apoptosis andautophagy seen in Fig. 1B and C. Akt is cleaved during apoptosis,resulting in its inactivation and subsequent induction of autop-hagy (31, 32). Levels of active Ser473 phosphorylated Aktincreased until 8 hours, when Akt cleavage peaked, then declinedto baseline levels. p62 levels accumulated until 16 hours whenwe saw a dramatic increase and its cleavage. LC3 II accumulatedearly at 2 hours, suggesting autophagy-associated membraneformationmay precede apoptosis induction, until 16 hours whenthe presence of LC3 I was no longer detectable and LC3 II levels

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Figure 4.Novel mechanism of intracellular caspase-10 activation. A, MM1.S cells were treatedwith selinexor/carfilzomib (KPT/CFZ; 50 nmol/L each)�/þ caspase-10 inhibitor(AEVD; 25 mmol/L) for 18 hours. CHAPS detergent-soluble (sol.) or insoluble (insol.) fractions were analyzed by Western blot. GAPDH and calnexin serve asindicators for the soluble and insoluble fractions, respectively. B, indirect immunofluorescent detection of caspase-10, caspase-8, and p62 inMM1.S cells untreated orSelinexor/CFZ-treated (50 nmol/L each) for 18 hours. 100�; scale bar, 10 mm. C, GST pull-down using GST only or GST-p62 as bait and lysates from untreated orSelinexor/CFZ-treated (18 hours; 50 nmol/L each) cells as input. A portion of the 18 hour-treated lysate serves as positive control for detecting copurifyingproteins. Ponceau staining of the blot confirms presence of GST and GST-p62.






began to decrease, likely due to autophagic flux. These resultssuggested that Akt activity may increase to offset induction ofapoptosis and to slow the rate of autophagy. However, concom-itant XPO1/proteasome inhibition promotes accumulation ofp62 and LC3II, which can act as a caspase-activating platform,resulting in cleavage and inactivation of Akt and other survival-associated factors, potently and promptly committing to apopto-sis (Fig. 6D). Altogether, our data suggest that the induction ofautophagymayplay a priming role to allowmaximal activation ofcaspases and fulminant apoptosis.

DiscussionWe report mechanistic characterization of selinexor and its

functional interaction with the PI carfilzomib. We found thatboth drugs induce autophagy and combination treatment accel-erates the process. Autophagy has emerged as an alternative celldeath pathway whose balance governs myeloma cell survival andcan occur in the absence of apoptotic characteristics (12). Ourresults, however, suggest that selinexor and carfilzomib-inducedcell death was associated with concurrent hallmarks of bothapoptosis and autophagy. Combination treatment synergisticallypromoted myeloma cell death and suppressed xenograft tumorgrowth, suggesting the possibility that apoptosis and autophagymechanisms may work in concert in the context of combinedXPO1 and proteasome inhibition.

To explore the molecular basis for synergistic cell death byselinexor and carfilzomib, we focused on caspase-10, a relativelyuncharacterized caspase family member that was recentlyhighlighted as an arbiter of thebalance between limited,myelomacell survival-associated autophagy and hyperactive autophagy

(12). Our results suggested that both selinexor and carfilzomibcan promote activation of caspase-10 and combination treatmentsignificantly increases its activity as well as that of other caspases,and inhibition of caspase-10 activity blocks apoptosis and sig-nificantly reverses cytotoxic effects of SINE/PI. We uncoveredthree novel characteristics of caspase-10. First, we determinedthat caspase-10 as well as caspase-8 and -9 were distributedbetween the cytosol and a membrane-associated localization.Full-length pro-caspases remain predominantly cytoplasmic,whereas active fragments aggregate to an insoluble fraction.Intriguingly, we found preactivated levels of caspase-10, -8, and-9 in the insoluble fractionof untreated cell lysates, suggesting thatlocalization to this compartment could be related to caspaseactivation state. Combined treatment with selinexor and carfilzo-mib increased the levels of active caspases in the insoluble fractionwithout changing levels of the pro-caspase form in the same. Wefound caspase-cleaved p62 solely in the insoluble fraction, sug-gesting this subcellular location harbors increased caspase activ-ity. Second, we colocalized caspase-10 and p62. Caspase-8 redis-tributes to intracellular membranes in the setting of proteasomeinhibition,whichdepends onp62and its ability to bindLC3 (26).We found that, in the context of SINE/PI treatment, caspase-8 and-10 colocalize with p62 to intracellular aggregates, again suggest-ing an aggregation to an intracellular membrane-associated loca-tion. Third, we found full-length pro-caspase-10, but not its activefragments, was able to associate with p62 in untreated cells andthis association was almost completely lost in treated lysates,possibly due to its cleavage. Although caspase-8 is known to beubiquitinated, and we found presumably ubiquitinated forms ofcaspase-8 in association with p62, we did not detect evidence ofmodification by ubiquitination for caspase-10. As expected, we


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Figure 5.Inhibition of autophagy nucleation reducesintracellular redistribution and activation ofcaspase-10 and -8. A, MM1.S cells were treatedas indicated with LY294002 (LY; 10 mmol/L)for 2 hours prior to treatment with selinexorand carfilzomib (KPT/CFZ; 50 nmol/L)overnight. CHAPS soluble (sol.) and insoluble(insol.) fractions were analyzed as in Fig. 4A.Detection of Akt phospho-Serine 473 showsLY impairs PI3K activity. B, 293T cells weretransfected on two consecutive days withpools of either control or Beclin 1-specificsiRNAs. After 48 hours, cells were treated asindicated with selinexor and carfilzomib(KPT/CFZ; 100 nmol/L each) for 18 hours andsoluble and insoluble fractions were analyzedby Western blot.


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detected binding of LC3 II and poly-ubiquitinated proteins to p62in untreated cells, and these associations increased dramaticallywith selinexor and carfilzomib treatment. Proteasome inhibition

may cause p62 binding preferences to shift toward the increasingnumbers of aggregated ubiquitinated proteins, which could out-compete pro-caspase-8 and -10. This suggests that perhaps the

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Figure 6.Autophagy cannot compensate for the potent apoptotic insult of selinexor and carfilzomib treatment. A, Western blots analysis of MM1.S cells treated for 18 hourswith the combination of selinexor and carfilzomib (50 nmol/L each) with either increasing amounts of bafilomycin or Q-AEVD-OPH. B, effects of cotreatment withincreasing doses of bafilomycin on selinexor/carfilzomib-induced cell death in MM1.S cells. Left inset, Western blot analysis of bafilomycin dose responsedemonstrates accumulation of LC3 II. Right inset, MTT assay shows the effects of increasing amounts of only bafilomycin on cell viability. C, Western blot analysisof MM1.S cells treated for the indicated times with selinexor and carfilzomib. D, putative model of caspase activation in response to combined XPO1 andproteasome inhibition.






association with p62, and by extension LC3, and membranelocalization is a priming event for activation of caspase-8 and-10 in the setting of combined proteasome and XPO1 inhibition.The nature of the novel association between caspase-10 and p62requires further clarification, such as which domains of p62 arerequired and whether binding is direct. Overall, these data sup-port a novel mechanism of caspase-10 activation that uses intra-cellular membranes and autophagy-associated machinery toorchestrate fulminant caspase activation. These results also drivefurther investigation of the newly-appreciated role of p62 and itsability to regulate cell survival.

In summary, the combination of SINE and PI is a potentantimyeloma treatment with amechanism of action that involvesstrong induction of apoptosis. Selinexor/carfilzomib treatmentsynergistically kills myeloma cells and has significant antitumoractivity in vivo. Our results also suggest that selinexor may con-tribute to overcoming carfilzomib resistance, which can be moredirectly established in the clinical setting. The FDA has recentlyapproved a phase I trial of combination treatment with selinexorand carfilzomib in R/R multiple myeloma, including patientsrefractory to carfilzomib, which is currently enrolling at theUniversity of Chicago and other sites within the Multiple Mye-lomaResearch Consortium (NCT02199665). Preliminary patientresponses are very promising. The first 3 enrolled patients, whoprogressed on very active carfilzomib combinations, respondedwith >50% disease reduction after one cycle of treatment withselinexor and carfilzomib. Although still early, these results sup-port our preclinical rationale for using this combination. Seli-nexor is also being explored as a treatment option for patientswith different types of advanced stage hematologic and solidcancers, underscoring the broad efficacy of this agent. It will beof great interest to determine whether our novel mechanisticobservations are found analogously in other malignancies, which

may inform design of future clinical trials and treatment regimensacross a broad range of cancers.

Disclosure of Potential Conflicts of InterestD.Dytfeld reports receiving a commercial research grant from Janssen and is a

consultant/advisory board member for Janssen, Amgen, Celgene, and Novartis.M. Kauffman has ownership interest (including patents) in a KaryopharmTherapeutics. A.J. Jakubowiak is a consultant/advisory board member forKaryopharm Pharmaceuticals and Onyx/Amgen. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: S. Rosebeck, M. Kandarpa, D. McCauley, S. Shacham,M. Kauffman, A.J. JakubowiakDevelopment of methodology: S. Rosebeck, M.M. Alonge, M. Kandarpa,S.L. Volchenboum, D. Dytfeld, D. McCauley, A.J. JakubowiakAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Rosebeck, M.M. Alonge, M. Kandarpa, J. Jasielec,S.P. Maxwell, D. McCauley, A.J. JakubowiakAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Rosebeck, M.M. Alonge, A. Mayampurath,S.L. Volchenboum, S.P. Maxwell, A.J. JakubowiakWriting, review, and/or revision of the manuscript: S. Rosebeck, S.L. Volchen-boum, J. Jasielec, S. Shacham, M. Kauffman, A.J. JakubowiakAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Rosebeck, M.M. Alonge, D. McCauley,A.J. JakubowiakStudy supervision: S. Rosebeck, A.J. JakubowiakOther (technical laboratory support and experiment optimization):S.J. Kraftson

The costs of publication of this articlewere defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 12, 2015; revised September 24, 2015; accepted October 14,2015; published OnlineFirst December 4, 2015.

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2016;15:60-71. Published OnlineFirst December 4, 2015.Mol Cancer Ther Shaun Rosebeck, Mattina M. Alonge, Malathi Kandarpa, et al.

Dependent Apoptosis by Carfilzomib and Selinexor−Caspase-10Synergistic Myeloma Cell Death via Novel Intracellular Activation of

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