CRM1 inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular mechanisms and therapeutic implications

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1 Lukmi () 8, & Mmilln Pulishrs Limit All rights rsrv 8879/ ORIGINAL ARTICLE CRM inhiition inus tumor ll ytotoxiity n impirs ostolstognsis in multipl mylom: molulr mhnisms n thrputi implitions YT Ti, Y Lnsmn, C Ahry, Y Cll, MY Zhong, M C, D Tnnnum, A Cgntt, M Rgn, AA Munshi, W Snpis, JR SintMrtin, T Kshyp, S Shhm, M Kuffmn,YGu,LWu, I Ghoril, F Zhn, AL Kung, SA Shy, P Rihrson, NC Munshi n KC Anrson Th ky nulr xport protin CRM/XPO my rprsnt promising novl thrputi trgt in humn multipl mylom (MM). Hr w show tht hromosom rgion mintnn (CRM) is highly xprss in ptints with MM, plsm ll lukmi lls n inrs in ptint lls rsistnt to ortzomi trtmnt. CRM xprssion lso orrlts with inrs lyti on n shortr survivl. Importntly, CRM knokown inhiits MM ll viility. Novl, orl, irrvrsil sltiv inhiitors of nulr xport (SINEs) trgting CRM (KPT8, KPT) inu ytotoxiity ginst MM lls (ED o nm), lon n oultur with on mrrow stroml lls (s) or ostolsts (OC). SINEs triggr nulr umultion of multipl CRM rgo tumor supprssor protins follow y growth rrst n poptosis in MM lls. Thy furthr lok my, Ml, n nulr ftor kb (NFkB) tivity. SINEs inu protsompnnt CRM protin grtion; onurrntly, thy uprgult CRM, ptrgt, poptosisrlt, ntiinflmmtory n strssrlt gn trnsripts in MM lls. In SCID mi with iffus humn MM on lsions, SINEs show strong ntimm tivity, inhiit MMinu on lysis n prolong survivl. Morovr, SINEs irtly impir ostolstognsis n on rsorption vi lok of RANKLinu NFkB n NFAT, with miniml impt on ostolsts n s. Ths rsults support linil vlopmnt of SINE CRM ntgonists to improv ptint outom in MM. Lukmi () 8, ; oi:.8/lu.. Kywors: multipl mylom (MM); sltiv inhiitors of nulr xport (SINEs) ginst CRM/XPO; ostolsts (OC); nulr ftorkb (NFkB) tivtion; nulr xport protin; tumor supprssors INTRODUCTION Multipl mylom (MM) is hrtriz y xss on mrrow plsm lls n monolonl protin, ommonly ssoit with ostoporosis n lyti on iss. Although onsirl progrss hs n m in th vlopmnt of novl trtmnts for MM, iss rlps ours in th vst mjority of ptints. Intrtions twn MM lls, ostolsts (OC), n on mrrow stroml lls () hv ntrl rol in iss pthognsis n mit rsistn to onvntionl MM thrpis. Thrfor, novl trgt thrpis with innovtiv mhnisms of tion to inhiit oth MM ll growth n OCmit on strution r urgntly n. Exportin (XPO), th ky nulr xport protin mor ommonly ll CRM (hromosom rgion mintnn ), is rquir for trnsporting rgo protins with luinrih nulr xport squns from th nulus to th ytoplsm. Spifilly, th CRMrgo omplx is tivly trnsport through th nulr por omplx to th ytoplsm, whr th rgo is rls ftr RnGAPtlyz GTP hyrolysis. CRMmit shuttling of ths protins is tightly ontroll, thry rgulting whih protins r tivt or rprss y spifi stimuli. Importntly, this pross is rgult t multipl lvls in nr lls, with CRMpnnt rgo protins inluing tumor supprssor (TSP) n rgultory protins p,, p CIP,p7 KIP, FOXO,, inhiitor of nulr ftor kb (NFkB) (IkB) 7 n BRCA.,8 Ths TSPs n only prvnt nr initition, progrssion n rug rsistn whn proprly loliz insi th nulus, n r oftn mutt, lt or rrntly lot within th ytosol in nr lls, thry isling thm from prforming thir norml funtions within th nulus. Importntly, s CRM is th sol nulr xportr of ths protins, norml llulr loliztion of TSPs in tumors my mit y CRM. In, inrs xprssion of CRM is ssoit with tumor sizs in ostosrom, 9 pnrs nr n ovrin nr. Morovr, ovrxprssion of CRM in lung n rvil nr lls hs ky rol in thir prolifrtion n survivl., Wholgnom squning of hroni lymphoyti lukmi hs rntly intifi rurrnt muttions in highly onsrv rgion of CRM,, suggsting rol of CRM in iss pthognsis of hroni lymphoyti lukmi. At prsnt, it rmins unlr whthr CRM is involv in th pthophysiology of humn MM, n whthr trgt inhiition of CRM my rprsnt novl thrputi molity in MM. LBow Institut for Mylom Thrputis n Jrom Lippr Cntr for Multipl Mylom Cntr, DnFrr Cnr Institut, Hrvr Mil Shool, Boston, MA, USA; Dprtmnt of Biology, Kryophrm Thrputis In, Ntik, MA, USA; Dprtmnt of Hmtologil Miin, King s Collg Lonon, Lonon, UK; Dprtmnt of Molulr Gntis n Miroiology, Shns Cnr Cntr, Univrsity of Flori, Ginsvill, FL, USA; Division of Hmtology, Onology, n Bloo n Mrrow Trnsplnttion, Dprtmnt of Intrnl Miin, Univrsity of Iow, Iow City, IA, USA; Luri Fmily Imging Cntr, Dprtmnt of Pitri Onology, DnFrr Cnr Institut, Hrvr Mil Shool, Boston, MA, USA n 7 Dprtmnt of Hmtology, King s Collg Hospitl Fountion NHS Trust, Lonon, UK. Corrsponn: Dr YT Ti, Dprtmnt of Mil Onology, DnFrr Cnr Institut, M, Brooklin Avnu, Boston, MA, USA. Emil: yutzu_ti@fi.hrvr.u Riv 8 Frury ; rvis 7 Mrh ; pt April ; pt rtil prviw onlin April ; vn onlin pulition, 7 My

2 Trgting CRM in th mylom on mrrow mironvironmnt Inhiition of CRM ws first monstrt using lptomyin B, whih rstors funtion of TSPs y prvnting thir xport to th ytoplsm., CRM lok vi lptomyin B mintin topoisomrs II in th nulus n grtly nhns th ytotoxi fft of th topoisomrs II inhiitors oxoruiin n VP (toposi) in oxoruiinrsistnt MM lls. 7 Howvr, th linil vlopmnt of lptomyin B hs n limit y its toxiity n nrrow thrputi winow in prlinil niml mols. 8 A novl rvrsil CRM inhiitor CBS9 with improv tolrility ws rntly intifi. 9 In ition, novl orlly iovill smllmolul sltiv inhiitors of nulr xport (SINEs) spifilly n irrvrsily in to rsiu Cys8 in th rgoining groov of CRM, thry rogt mny of its trnsport funtions., Signifint ntitumor tivity of ths gnts hs rntly n rport in prlinil stuis of ut myloi lukmi n hroni lymphoyti lukmi. Importntly, SINE KPT n KPT8 hv potnt ntilukmi tivity, with ngligil toxiity to norml hmtopoiti lls., Whthr SINEs r fftiv ginst humn MM is unknown. In this stuy, w hrtriz CRM funtion in MM, n furthr invstigt th ffiy n molulr mhnisms of novl orl SINE CRM ntgonists in MM. Our stuis fin th mhnisms whry ths gnts inhiit MM ll growth n inu poptosis in th on mrrow (BM) mironvironmnt. Morovr, w furthr fin novl rol of CRM in OC iffrntition, suggsting itionl nfits of trgting CRM in MM. MATERIALS AND METHODS Cll lins, ptint smpl prossing n ultur of s n OCs All CD8 þ MM ll lins wr grown s sri prviously., Ptint MM smpls wr otin ftr inform onsnt, in orn with th Dlrtion of Hlsinki n unr th uspis of DnFrr Cnr Institut Institutionl Rviw Borpprov protool. CD8 þ plsm lls from MM ptints wr purifi y CD8miros (Miltnyi Bioth, Auurn, CA, USA). Rsiul CD8ngtiv lls wr ultur in RPMI /% FCS to gnrt s or stimult with MCSF/RANKL for OC iffrntition., Lntivirl CRM smll hirpin RNA (shrna) trnsution CRM shrna vs ontrol plko. (shcnt) or GFP (shgfp) lntivirus (RNAi Consortium t DnFrr Cnr Institut) wr trnsu into MM ll lins. Thr ys post trnsution, CspsGlo /7 (Promg, Mison, WI, USA) n CllTitrGlo Luminsnt Cll Viility Assys (Promg) wr prform. Cll yl profiling n poptosis ssys MM lls wr trt with SINEs, follow y stining with romo oxyuriin/propiium ioi (PI) n flow ytomtri nlysis. Apoptosis ws msur y nnxin V/PI stining n flow ytomtri nlysis, s wll s y CspsGlo Assy. NFkB p DNAining tivity MM lls n CD þ OC prursor (OCP) lls wr prtrt with KPT8 or KPT for h n stimult with prolifrtioninuing lign (APRIL, ng/ml, R&D Systms, Minnpolis, MN, USA) n RANKL ( ng/ml, R&D Systms), rsptivly. Nulr protin ws thn xtrt for NFkB tivity using TrnsAM NFkB p ELISA Kit (Ativ Motif, Crls, CA, USA). Cytokin msurmnts Multiplx ytokin msurmnts wr on using th Luminx systm (EMD Millipor, Billri, MA, USA). Rltim quntittiv rvrs trnsriptionpcr RNA ws xtrt from lls n mrna for init gns ws quntifi using th ViiA7 RlTim PCR Systm n nlyz y th V. softwr (Lif Thnologis). Dissmint MM mol All xprimntl prours n protools wr pprov y th Institutionl Animl Cr n Us Committ (DnFrr Cnr Institut). SCIDig mi wr injt intrvnously with MMSLuNo (MMSlu) lls n img wks ltr to trmin slin ioluminsn. Mi wr ivi into thr groups with similr mn ioluminsn (n ¼ 8 pr group): mi riv KPT or KPT7 (h t mg/kg), or vhil (.% (w/v) Pluroni F8,.% (w/v) PVP K9/ in stril wtr) vi orl gvg thr tims pr wk on nononsutiv ys. On y, KPT n KPT7 wr oth os intnsifi to 7 mg/kg. Wight n ioluminsn t wr ollt vry 8 ys for on month, t whih point mi wr kill u to hin lim prlysis. Som mi wr kill ftr th son os. BM lls wr ollt for poptosis ssys for MMSlu lls. Extrmitis wr ollt from ths mi n tissu stions wr prpr for histologi n immunohistohmil nlysis for CD8, p n lv sps. Sttistil nlysis In vitro xprimnts wr prform in triplit n rpt t lst two tims; rprsnttiv xprimnt is shown (mn±s..). Sttistil signifin of iffrns (st t Po.) ws trmin using onwy nlysis of vrin with Bonfrroni s post ho omprison (for mor thn thr groups) or twotil unpir Stunt s ttst (for two groups) in GrphP Prism vrsion. (GrphP Softwr, Sn Digo, CA, USA). SINE KPT8 ws hrtriz most xtnsivly in in vitro ssys, ut hs poor PK proprtis unsuitl for us in vivo. KPT n 7 r lss potnt ginst CRM thn KPT8, ut hv similr spifiity n goo orl iovilility, thry suitl for in vivo stuis. KPT, nrly s potnt s KPT8, hs optiml orl iovilility n systmi xposur, n is thrfor urrntly unrgoing Phs tsting in ptints with vn hmtologi n soli tumor mlignnis. Immunolotting Antiois wr otin from Cll Signling (Dnvrs, MA, USA), xpt nticrm n mf As (Snt Cruz Biothnology, Snt Cruz, CA, USA). Immunofluorsn nlysis MMS lls wr trt with KPT8, fix with % prformlhy, prmiliz with.% Triton n stin with init ntioy to tt iniviul TSP (grn) n with imiinophnylinol (Lif Thnologis, Crls, CA, USA) to tt nuli (lu). RESULTS CRM is highly xprss in MM lls n ssoit with survivl n on lysis High CRM protin xprssion ws first shown y immunolotting in CD8 þ ptint MM lls (n ¼ 8) n MM ll lins (n ¼ ; Figur ). Gn xprssion nlyss monstrt inrs CRM xprssion in nwly ignos MM lls (n ¼ ) vs norml plsm lls (n ¼ ; Po. unr ssion numr GSE9 (rf. ) n GSE8 (rfs. 7,8) in Figur ; P ¼. in GSE9 (rf. 9) in Supplmntry Figur A) n in plsm ll lukmi vs MM (Po. in GSE, Supplmntry Figur A). Intrstingly, CRM trnsript is lso highr in ptints unrsponsiv to ortzomi trtmnt (Po. in GSE987, Figur ). Th orrltion of CRM xprssion n survivl ws furthr trmin y th Pvlu n hzr rtio t th st xprssion signl utoff using Rpkg unr GSE8. 7,8 Highr CRM is signifintly ssoit with poor outom (P ¼. for vntfr survivl n P ¼. for ovrll survivl, Figur ) n th xtnt of on lyti lsion in th Totl Thrpy ohort (P ¼.8, Lukmi () & Mmilln Pulishrs Limit

3 Trgting CRM in th mylom on mrrow mironvironmnt CRM βtin CRM βtin Norml MMS MMR INA INAGFP ANBL KMS8 KMS MM ptints RPMI8 MOLP8 8BM PE XG U CRM xprssion NPC (n=) p =.7 MGUS (n=) p <. MM (n=) CRM xprssion 9 NR (n=78) p<. R (n=8) Rspons to Bortzomi 7 Proportion of Css Evnt Fr Survivl Evnts/n Low CRM 9/9 High CRM 9/ p =. Ovrll Survivl OS Dths/n Low CRM /9 High CRM / p =. Bon lyti lsion with CRM xprssion MRI MRI Totl 7 > 7 (n) Low CRM 7 8 High CRM 7 7 p (Chisqur).8 Months from Strt of Thrpy Months from Strt of Thrpy CRM PARP CL PARP CL sps GAPDH MMR shcnt shcrm# shcrm# shcnt MMS shcrm# shcrm# shcnt U shcrm# shcrm# f Viility (fol hng) Csps /7 tivity (AU, x ).8... ** ** * ** * * ** * * * * ** MMR MMS U shcnt shcrm# shcrm# Figur. CRM is highly xprss in ptint with MM lls n CRM ownrgultion inhiits MM ll viility. () CRM lvl ws xmin y immunolotting in CD8 þ plsm lls from norml onors (n ¼ ) n MM ptints (n ¼ 8; uppr pnl), s wll s MM ll lins (lowr pnl). Atin srv loing ontrol. () CRM gn xprssion ws nlyz unr ssion numr GSE9 n GSE8, whih inlus CD8 þ lls from norml onors (norml plsm ll (NPC), n ¼ ), monolonl gmmopthy of untrmin signifin (MGUS, n ¼ ) n nwly ignos MM (MM, n ¼ ). Po.: MM vs MGUS. () CRM gn xprssion ws nlyz in GSE978 t, inluing ptints not rsponsiv (NR) n rsponsiv (R) to ortzomi trtmnt. Po.. () Kpln Mir plots on prognosti rlvn of CRM xprssion on vntfr n ovrll survivl for MM ptints (GSE8). Th lu n r lins rprsnt ptint groups with lowr n highr CRM xprssion, rsptivly (lft). Assoition of CRM xprssion n lyti lsion (p7 vs7) on mgnti rsonn imging (MRI) ws lso ssss (right). () Thr MM ll lins wr inft with two iffrnt lntivirl smll hirpin (sh)crm (#, #) or ontrol plko. (shcnt), follow y immunolotting for CRM. GAPDH srv s loing ontrol. CL PARP, lv PARP; CL sps, lv sps. (f) Thr ys following lntivirl trnsution, lls in wr sujt to luminsnt ll viility (*Po.; **Po.) n sps/7 ssys (*Po.; **P ¼.). GAPDH, glyrlhy phospht hyrogns. Figur ). Ths rsults suggst tht lvt CRM xprssion is importnt for MM pthophysiology. CRM ownrgultion rss MM ll growth n survivl W irtly ownrgult CRM in xmthsonsnsitiv MMS n rsistnt MMR lls with wiltyp p (pwt), s wll s in U lls hroring mutt p (pmt). CRM ownrgultion in MM lls using four sprt shcrm lntiviruss trgting iffrnt CRM squns onsistntly lok ll growth n survivl (Po., Figurs, f n Supplmntry Figurs B E). Trnsution with shcrm lntiviruss, ut not ontrol shcnt or shgfp lntiviruss, signifintly ru ll viility n nhn oth sps/7 tivity n lvg of sps/poly ADP rios polymrs (PARP). Intrstingly, SINE CRM ntgonist KPT8 signifintly ru CRM protin n strongly inu lvg of PARP n sps in xmthsonrsistnt MMR lls (Supplmntry Figur D). Thus, CRM funtion is ritil for mintnn of mlignnt growth n survivl of MM lls. SINEs trgting CRM inu growth rrst n poptosis in MM lls ssoit with nulr umultion of multipl TSP gns As th ffts of KPT8 mimik thos of CRM shrna, w trmin whthr smllmolul, ruglik, sltiv CRM & Mmilln Pulishrs Limit Lukmi ()

4 8 Trgting CRM in th mylom on mrrow mironvironmnt SINEs (Figur ) inu omprl ytotoxiity ginst MM lls. KPT8 hs th grtst potny for CRM, ut is unsuitl for in vivo us. KPT n KPT7 hv intrmit PK proprtis n mort potny,, whrs KPT, urrntly in phs linil trils, is nrly s potnt s KPT8 n hs optiml PK proprtis. Multipl MM ll lins wr inut with KPT8 or KPT for ys, follow y luminsn ll viility ssy (Figurs, ). Both SINEs ru ll viility, with ED s for KPT8 n KPT of o no nm, rsptivly. Aitionl ssys furthr onfirm tht KPT8 n KPT potntly inhiit growth n survivl of MM lls, rgrlss of thir rug snsitivity or p sttus (Supplmntry Figur ). Nxt, ll yl nlyss show tht thr SINEs similrly rs Sphs prolifrting MMS lls, follow y n inrs in sug poptoti lls (Figur ). Using immunofluorsn stining, prominnt nulr umultion of p n IkB ourr s rly s h ftr KPT8 trtmnt (Figur ). Nulr loliztion of p7, ownstrm trgt of p n FOXOA signling importnt for growth rrst n poptosis, s wll s FOXOA n PPA ws vint t ltr tim points ( h). Immunolotting of nulr vs ytoplsmi xtrts prpr from MMS lls following KPT trtmnt furthr onfirm nulr umultion of p, IkB, p n p7 (Figur ). Brg vlit purity of nulr xtrt prprtion s Brg is unttl in ytoplsmi xtrt. Similr rsults wr otin in MMR lls trt with KPT8 or KPT (t not shown). Ovrnight trtmnt with KPT8 inu 9 to fol inrs in poptoti MMS lls, s omin % of nnxin V þ n nnxin V þ /PI þ lls is.% (ontrol) vs 8.9% t. mm KPT8 n 8.% t. mm KPT8 (Figur f). Bus ths four SINEs spifilly lok CRM n thir ffts r similr to thos in th shrna xprimnts sri ov, it is likly tht CRM inhiition itslf, rthr thn n offtrgt fft, mits ntimm proprtis. SINEs spifilly inu ytotoxiity ginst MM lls oultur with s or OCs, n r tiv ginst MM ptint lls Th ntimm ffts of SINEs wr nxt stui in MM lls in th ontxt of th BM mironvironmnt. SINEs lok prolifrtion of MM lls oultur with ithr or OCs (Figurs, ). In ontrst, SINEs inu miniml ytotoxiity ginst ptintriv s lon (Figurs, n Supplmntry Figurs A C). Trtmnt with SINEs ovrnight potntly tivt sps/7 n 9 in MMSlu (pwt) n RPMI8 (pmt) lls, ultur lon n with (Figur ). SINEs lso signifintly tivt sps8 in MMSlu lls, lon or oultur with (Figur ). In ontrst, spss wr not tivt in ptnt s (n) unr th sm trtmnt onitions (Figurs, n Supplmntry Figurs A C). Immunolotting lso onfirm sltiv ytotoxiity of KPT8 n KPT ginst MMS lls vs s. As signifint inrss in p, s wll s lvg of PARP n sps, wr tt in KPT8 or KPTtrt MMS lls ultur lon n with s, ut not in s lon (Figur ). Thus, SINEs signifintly ovrm th growth n survivl vntg in MM lls onfrr y s n OCs, with miniml impt on ths BM ssory lls. Furthrmor, omin trtmnt with KPT8 n ortzomi nhn lvg of PARP n sps thn ithr rug lon in MMR (Figur f) or MMS (Supplmntry Figur D) lls. KPT8ru CRM protin ws rogt in th prsn of ortzomi, initing tht protsoms r involv in SINEru CRM protin xprssion n suggsting tht ths rugs in omintion might nhn poptosis in MM lls. Importntly, KPT8 is tiv ginst th mjority of MM ptint lls (n ¼ ; Supplmntry Figur E), with poptosis Figur. KPT8 KPT7 KPT KPT Chmil Formul: C 7 H F N 7 O Molulr Wight:. Chmil struturs of SINEs trgting CRM. mit vi sps/7 tivtion triggr y ovrnight trtmnt with KPT8 (n ¼ ; Figur g). Mhnisms of tion of CRM inhiition ginst MM lls in th BM mironvironmnt Immunolotting nxt monstrt tht KPT8, in os n timpnnt mnnr, triggr inution of p n Bx, long with lvg of PARP n sps, in MMS lls (Figur n Supplmntry Figur A). In ition, moluls ssoit with MM ll prolifrtion, tht is, my n pikb, s wll s th ntipoptoti protin Ml, wr signifintly rs ftr KPT8 trtmnt. Rution in BlxL ws lso sn, lthough not s signifint s for Ml. Ths molulr hngs wr orrlt with os n timpnnt rss in CRM protin in MMS lls. Intriguingly, lthough KPT8 n KPT ru CRM protin, it simultnously inu CRM mrna xprssion in MM lls (MMS, INA n ANBL, Figurs, n Supplmntry Figurs B, C) ultur lon n with s, whrs xmthson i not (ANBL in Supplmntry Figur C). This inution in CRM mrna following trtmnt with SINEs is likly in rspons to loss of CRM funtionl tivity, n my thrfor provi usful iomrkr for SINE tion. In ition to inrs CRM mrna triggr y ovrnight trtmnt with KPT8 ( nm), p, p, PUMA, BAX, CHOP, Corf, MIC n IkB wr lso inrs in MMS lls, ultur lon n in th prsn of s (Figurs, ). Thrfor, SINEs trgting CRMmoult xprssion lvls of CRM rgos in MM lls, thry rgulting vrious funtions inluing ll yl n poptosis, s wll s strss rsponss. NFkB is tivt in MM n ontriuts to tumor growth, survivl n rug rsistn, s wll s OC tivtion n ostolysis. As KPT8 inu nulr umultion of IkB, w nxt xmin whthr KPT8 loks NFkB tivity in MM lls. KPT ( h trtmnt) rs slin NFkB p tivity in nulr protin xtrt from MMS n MMR lls (Figur, uppr pnl). KPT8 lso onsistntly lok p DNAining tivity in MMS lls with or without APRIL stimultion (Figur, Lukmi () & Mmilln Pulishrs Limit

5 Trgting CRM in th mylom on mrrow mironvironmnt % viility (RLU) 8 KPT8 (nm) MMS MMR H99 8BM ANBL ANBLVR OPM OPM DOX LR U p IκB DMSO FITC KPT8 Mrg: FITC+DAPI DMSO KPT8 9 % viility (RLU) 8 KPT (nm) MMS MMR H99 8BM ANBL ANBLVR MMSGFP INAGFP MOLP8 XG FOXOA FOXOA p7 PPA KPT8 CE NE f % of lls in ll yl KPT KPT p p IκB CRM p7 Brg DMSO KPT lnk DMSO KPT PI.%.% 7.%.%.8%.%.μM.μM Annxin V y y y y Figur. SINEs, spifi CRM inhiitors, inu potnt ytotoxiity ginst MM lls vi nulr umultion of tumor supprssors n growth rrst, follow y poptosis. MM ll lins snsitiv or rsistnt to onvntionl n novl gnts wr inut with KPT8 ()or KPT () ( nm) for ys, follow y ll viility ssys. () MMS lls wr trt with SINEs n stin ily with romo oxyuriin/pi to trmin ll yl profils using flow ytomtri nlysis. () MMS lls wr trt with KPT8 ( mm) for h (p n IkB) or 8 h (FOXOA, FOXOA, p7, PPA), follow y immunofluorsn stining. () MMS lls wr trt with KPT ( nm) for 8 h, follow y ytoplsmi (CE) n nulr protin xtrtion (NE) for immunolotting. Brg srv s NE ontrol. (f) MMS lls trt with KPT8 ovrnight wr stin with nnxin V/PI for flow ytomtri nlysis. DAPI, imiinophnylinol; DMSO, imthyl sulfoxi; FITC, fluorsin isothioynt; RLG, rltiv light units. lowr pnl n Supplmntry Figur D). As APRIL, BM ytokin, tivts NFkB to support MM ll growth n survivl, ths rsults suggst tht SINEsinu inhiition of NFkB is ruil for th rs in MM viility triggr y ths gnts in th BM mironvironmnt. Bus SINEinu MM ytotoxiity is not signifintly ttnut y n SINEs r not irtly ytotoxi to, w lso ssss th ffts of CRM inhiition on ytokin proution y ths ssory lls. Ovrnight trtmnt with KPT8 (p nm) rs th srtion of multipl ytokins, inluing IL, VEGF, MIP, IL, from s, ultur lon or with MM lls (Figur n Supplmntry Figur E). KPT, KPT7 n KPT signifintly inhiit growth of MM lls in vivo Th in vivo ntimm tivity of SINEs ws nxt xmin in SCIDig orthotopi MM mol, whih llows for th stlishmnt of iffus MM on lsions similr to humn iss. Shorttrm orl trtmnt of mi with ithr KPT or KPT7 & Mmilln Pulishrs Limit Lukmi ()

6 Trgting CRM in th mylom on mrrow mironvironmnt R.L.U. x ANBLVR MMR no MM KPT (,,,,,, nm) MM no MM + Viility ( R.L.U. x ) MMS MMS+OC OC KPT8 (nm) R.L.U. x Csps /7 Csps 9 oultur MMSlu MMSlu + R.L.U. x 8 KPT (nm) KPT (nm) oultur RPMI8 KPT KPT7 KPT KPT7 ginst CRM p PARP CL PARP Csps CL Csps βtin MMS+ KPT8 KPT MMS KPT8 KPT KPT8 KPT Dx f Bor (nm) KPT8 (nm) hr CRM Csps CL Csps GAPDH g Csps /7 R.L.U. x MM MM MM KPT8 (nm) Figur. SINEs spifilly inu toxiity vi sps tivtion in MM lls oultur with or ostolsts. () Bortzomirsistnt ANBLVR, xmthson (x)rsistnt MMR lls n MM ptint lls (MM), ultur lon ( ) or with ( þ ) s, wr trt with KPT ( nm) for ys, follow y ll viility ssys. () MMS lls, lon or with ostolsts (OC), wr trt with KPT8. () MMSlu (uppr pnl, MMS ll xprssing luifrs gn) n RPMI8 (lowr pnl) lls, ultur lon or with s, wr trt with KPT ovrnight, follow y sps/7 n 9 tivity ssys. () MMSlu lls wr oultur with s ovrnight with KPT,7, n wr ssy for sps8 tivity. () MMS lls wr ultur ovrnight lon or with s, in th prsn or sn of KPT8 or KPT ( nm). Cll lysts wr thn prpr n sujt to immunolotting. (f) MMR lls wr trt with KPT8 ( nm) lon or with Bor ( nm) t init tim points. Cll lysts wr prpr for immunolotting. (g) MM ptint lls (n ¼ ) wr trt ovrnight with KPT8 ( nm) n ssy for sps/7 tivity; RLG, rltiv light units. (two oss t 7 mg/kg) inu MMSlu tumor ll th n lvg of sps in vivo (Figur ). This ws onfirm y fol inrs in % of nnxin V þ n nnxin V þ /PI þ lls, s wll s.8 to fol highr sps/7 tivity in MMSlu tumors hrvst from SINE vs vhil ontroltrt mi (Supplmntry Figurs A, B). Immunohistohmil stuis furthr show inrs p n lv sps in tumors xis from SINE vs ontrol vhiltrt mi (Figur ). In longrtrm stuis in this mol, KPT or KPT7 trtmnt ru tumor urn (Figur ) strting from y 9 (Supplmntry Figur C, Po.), without signifint fft on oy wight in trt mi (Supplmntry Figur D). A signifint survivl nfit ws sn in KPT n KPT7 trt vs vhiltrt mi: ys for SINEtrt vs ys for vhiltrt mi (logrnk tst, Po.; Figur ). Thr ws no iffrn in tumor urn or survivl twn KPT n KPT7 groups. In ition, KPT trtmnt lso ompltly lok MMS tumor growth in murin suutnous xnogrft mol (Supplmntry Figur E, n ¼ 8 for h group). Ths rsults init tht ths SINEs CRM ntgonists show omprl ntimm ffts in vivo. Highrsolution CT imging of th mi ring issmint MMSlu tumors furthr show mrk MMinu ostolysis n rution in on minrl nsity in vhiltrt nimls (Figur ). In ontrst, improvmnt of on lsions rsmling ontrol mi with no MMSlu tumor ll injtion ws osrv in SINEtrt vs vhiltrt mi. SINEs furthr prvnt OC iffrntition n on rsorption vi inhiition of NFkB n NFAT W furthr invstigt whthr th ffts of SINEs on on wr mit only through ntimm tivity, or y itionl irt ffts on ostolstognsis. As NFkB tivtion y ssntil OC Lukmi () & Mmilln Pulishrs Limit

7 Trgting CRM in th mylom on mrrow mironvironmnt MMS MMS+ MMS MMS+ KPT8 (nm) Dospnny.. CRM 9 CHOP.. slin CRM p PARP CL PARP Csps CL sps Ml BlxL My HSP9 HSP7 Fol hng 8 p p PUMA..... KPT8 (nm) Corf BID IκBα NFκ B p tivity (OD) APRIL (min) KPT8 (μm). Inution y APRIL βtin hr CRM p PARP CL PARP CL sps mf Ml BAX Hsp9 piκbα GAPDH Timpnny 8 8 CRM BAX Corf p PUMA MIC 8 p CHOP.. KPT (μm).. Fol hng. MMS MMS+ IL (ng/ml) MMS+ * * KPT8 KPT KPT8 (nm) Figur. Funtionl squl following SINE trtmnt in MM lls, ultur lon n with s. () MMS lls wr trt ovrnight with. nm (uppr pnl) n nm KPT8 t init tim intrvls (lowr pnl), rsptivly. Cll lysts wr prpr follow y immunolotting. Atin n glyrlhy phospht hyrogns (GAPDH) srv s loing ontrols. (, ) Totl RNA ws xtrt from MMS lls, ultur lon or with in th prsn of KPT8 ( nm), follow y quntittiv rvrs trnsriptionpcr for CRM n its potntil trgts. 8S n GAPDH srv s intrnl ontrols for normliztion. () Nulr xtrts wr prpr from MMS (&) n MMR ( ) lls trt with KPT for h (uppr pnl), nulr xtrts wr furthr m from MMS lls trt with KPT8 with APRIL ( ng/ml; lowr pnl). NFkB tivity ws thn trmin s on p DNAining tivity. () IL ws msur in suprntnts of oulturs of MMS with s, with or without SINEs ( nm). *Po.. Error rs in rprsnt mn þ s.. from thr inpnnt xprimnts. CL PARP, lv PARP. iffrntition ytokin RANKL long with NFAT (rf. ) ontrol ostolstognsis, w xmin whthr SINEs inhiit ths ky OC rgultors to lok OC formtion. KPT lok RANKLinu NFkB p tivity in CD þ OC prursor lls (Figur 7). KPT lso lok RANKLinu phosphoryltion of p (Figur 7), furthr initing n inhiition of NFkB signling s. As xpt, RANKL/MCSF profounly inu NFAT ftr ys; importntly, KPT lok NFAT inution t oth protin n trnsriptionl lvls (Figurs 7, ). Using rltim quntittiv rvrs trnsriptionpcr, ownstrm trgts of NFkB n NFAT tht r ruil for hsion, fusion n funtion of prostolsts, inluing ACP/TRAP, intgrin (ITG), ITGv, DCSTAMP (DCST), 7,8 wr lso ownrgult (Figur 7). KPT8 lso prou similr ffts on ths OCrlt moluls (Supplmnt Figurs A C) In wk MM ptint OC ulturs (n ¼ ), KPT8 signifintly ru th numr of TRAP þ multinult mtur OC lls (Figur 7), onsistnt with rs srtion of TRAP, spifi mtur OC mrkr (Supplmntry Figurs D, E). Immunofluorsn stining of th tin ytosklton n nulus furthr show tht SINEs inhiit tin lt formtion ruil for mtur OC funtion; n prvnt fusion of prostolsts, thry rsing th numr of multinult mtur OC (Figur 7, Supplmntry Figur F). Finlly, pit formtion ssys on ntin slis onfirm tht ithr KPT8 or KPT signifintly impir % rosion r (Figur 7f, Supplmntry Figur G). W simultnously ssss th fft of SINEs on ostognsis riv from msnhyml stm lls of norml hlthy onors (n ¼ ). KPT lok lium position (Figur 7g). KPT8 i not furthr xrt INA MM llmit inhiition of & Mmilln Pulishrs Limit Lukmi ()

8 % survivl Trgting CRM in th mylom on mrrow mironvironmnt BLI (ph/s/m /sr) 8 CRM sps Clv sps GAPDH.E+.E+ 8.E+9.E+9 Vhil KPT KPT7.E+9.E+9.E+ Trtmnt (ys) Vhil vhil KPT7 Trtmnt (ys) mrkr vhil p<. KPT KPT7 KPT No MM p Clv sps L7 L L L CD8 H&E KPT vhil KPT KPT7 KPT7 Vhil BV/ BMD TV (mg/) No MM 8% 7 KPT % KPT7 % Vhil 7% Figur. SINEs inu signifint ntimm tivity n inhiit MMinu on lysis in issmint murin mol of humn MM. SCIDig mi wr inoult intrvnously with MMSlu lls. At th first ttion of MM ll growth, trtmnt ws strt with KPT (7 mg/kg), KPT7 (7 mg/kg) or vhil vi orl gvg thr tims pr wk on nononsutiv ys. For shorttrm trtmnts (two oss in totl), (, ) BM mononulr lls wr ollt from h mous n sujt to immunolotting (). Glyrlhy phospht hyrogns (GAPDH) srv s loing ontrol. () Th xtrmitis from ths mi wr stin with hmtoxylin n osin (H&E) n immunohistohmilly nlyz for th init protins. Originl mgnifition,. Longtrm trtmnt (n ¼ 8 pr group) signifintly ru tumor growth s ssss y ioluminsn (BLI) (), n prolong survivl of mi using logrnk (Mntl Cox) nlysis () ( ys in ontrol group vs n ys in KPT n KPT7 groups, rsptivly, Po.). () On trtmnt y, mi unrwnt highrsolution quntittiv omput tomogrphy imging of lumr vrtr to ssss ostolysis n on minrl nsity. Top: omprisons of thrimnsionl volum rnring of LL7 from iffrnt mi s on CT imgs (isply winow: HU); ottom: rprsnttiv sgittl CT slis monstrting LL7 n volumofintrst us for quntifition (isply winow: HU). BV/TV: rtio of on volum (HU) vs totl volum (HU) of LL7; BMD: vrg on minrl nsity in LL7 (ollgn rfrn: mg/). lium position from ostolsts (Supplmntry Figur H). Thus, SINEs spifilly lok OC formtion n thir on rsorption tivity, without vrs ffts on on formtion tivity. DISCUSSION W first show tht high CRM xprssion in CD8 þ MM ptint lls is ssoit with lyti on iss n shortr survivl, n tht ortzomirsistnt ptint MM lls xprss highr CRM lvls, suggsting pthogni rol of CRM in MM. Using four sprt shcrm lntiviruss trgting four iffrnt rgions in CRM gn in svrl MM ll lins, w osrv tht th gr of CRM ownrgultion orrlt with lvls of poptosis. Thus, CRM funtion is ritil for th mintnn of mlignnt growth n survivl of MM lls, onsistnt with rnt rport of CRM/XPO s MM survivl gn. 9 W thn show tht loking CRM using SINEs (KPT8, KPT, KPT7, n KPT ) inu growth rrst n poptosis in MM lls, lon n ultur with or OC. KPT8 n KPT furthr inhiit NFkB tivity in MM n OC ling lls, thry ovroming tumor ll growth n rug rsistn in th BM miliu. Importntly, this is th first rport to fin novl rol of CRM in OC formtion, s CRM knokown signifintly loks th ky OC iffrntition rgultor NFAT inu y RANKL. KPT8 n KPT onsistntly inhiit oth NFkB tivity n OC iffrntition gns ownstrm of NFAT (ACP/TRAP, ITGv, ITG n DCST), thry prvnting hsion n fusion of prostolsts to form funtionl OC. Ths rsults thrfor provi omplling vin to trgt CRM to oth inu poptosis in MM lls n improv on iss. Lukmi () & Mmilln Pulishrs Limit

9 Trgting CRM in th mylom on mrrow mironvironmnt. NFκB p. tivity. RANKL(min) KPT (nm) Fol hng (TRAP+ mtur OC).8... * * KPT8 (nm) ** ** MM MM MM MM RANKL/MCSF NFAT pp GAPDH KPT & + + KPT nm nm nm NFAT ACP ITGαv ITGβ x DCST.x. RANKL/MCSF KPT f KPT g Normliz Asorn KPT (nm)..8. nm nm... ** ** ** nm 8 % ro r nm... KPT (μm) KPT (μm) Figur 7. KPT n KPT8 irtly lok ostolstognsis n on rsorption without vrs ffts on ostolstognsis. () CD þ ostolst prursor (OCP) lls wr prtrt with KPT n thn stimult with or without RANKL ( ng/ml, min), follow y nulr lyst xtrtion to trmin NFkB p tivity. () OCPs wr stimult with RANKL/MCSF for ys, with or without KPT. Protin lysts wr prpr n immunolott with init As. () Totl RNA ws prpr from KPTtrt OCP stimult with RANKL/MCSF for ys n sujt to rltim quntittiv rvrs trnsriptionpcr for OC iffrntition gns. Exprssion ws normliz with 8S s n intrnl ontrol. () Thrwk MM ptint OC ulturs (n ¼ ) with or without KPT8 ( nm) wr nlyz for multinult trtrtrsistnt i phosphts (TRAP) þ mtur OC lls ( nuli, mm pr ll). () Aitionl MM ptint OC ulturs with KPT wr prform on glss ovrslips, follow y immunofluorsn stining using Alx 8onjugt phlloiin for tin (r) n imiinophnylinol for nuli (lu). Extn spring r pr multinult OC ( nuli) n numr of nuli pr OC ws ru in ospnnt mnnr. (f) OC ultur ws prform on ntin slis with n without KPT for wks. Brightfil imgs of ri ntin iss wr thn quir n pross with Photoshop 7. to otin inry img. Bon pits wr shown y lk pixls (uppr pnls). Th prntg of th r oupi y lk pixls orrspons to th % on rosion r (lowr pnl). *Po.; **Po.. (g) Humn ostoprognitor lls riv from norml onors (n ¼ ) wr ultur in ostogni mi, with or without KPT. Clium position y Alizrin r ws quntifi y ELISA t th n of y ultur. Rprsnttiv imgs of Alizrin r stining r shown; sl rs rprsnt mm. Th mhnisms of CRM inhiitionmit ytotoxiity of ths SINEs in MM lls wr lint. SINEs rpily ru Sphs prolifrting MMS lls, follow y inrs sug poptoti lls. As shown y immunofluorsn stining n immunolotting of nulr n ytoplsmi lysts, yling MMS lls xprss low ovrll, with low nulr n high ytoplsmi lvl, of ky TSP protins inluing p, IkB, FOXOA, FOXOA, p7 n PPA. Importntly, KPT8 n KPT potntly rstor nulr loliztion n lvt lvls of ths TSPs in MMS lls. Trnsripts of p n its ownstrm trgts p, BAX, PUMA wr lso inu y KPT8, initing tht nulr loliztion of p ls to tivtion of its TSP funtion. PUMA, whih is rmtilly ( to fol) uprgult y KPT8 in MMS lls, is lso rgult y pinpnnt mhnism to inu poptosis in vivo. For xmpl, FOXO uprgults PUMA in rspons to pinpnnt poptoti stimuli. & Mmilln Pulishrs Limit Lukmi ()

10 Trgting CRM in th mylom on mrrow mironvironmnt As KPT8 lso inus nulr umultion of FOXO, it furthr nhns PUMA xprssion. Inrs PUMA vi FOXO my thrfor ontriut to KPT8inu ll th in MM lls hroring mutt p. Along with inrs propoptoti PUMA n BAX, KPT8 lso rs ntipoptoti protins Ml n BlxL in MM lls, onsistnt with ytotoxiity of SINEs ginst tumor lls rsistnt to onvntionl thrpy. Ml is rgult y NFkB n mits survivl n rug rsistn in MM, 9 furthr highlighting th potntil linil utility of its inhiition y SINEs. Importntly, KPT n KPT7 signifintly inrs p n lv sps in MMS tumors growing within th ons of SCID mi, onfirming rugrlt inution of tumor ll poptosis in vivo. Surprisingly, SINEs ru lvls of MM growth rivrs n onogns inluing my. Th nulr xport of my is not mit y CRM, n it is not lr why CRM inhiition l to my rution. A rnt stuy rport tht my n CRM r oorintly rgult, n tht p tivtion rus lvls of oth gns. This osrvtion is onsistnt with th prsn of p rprssivining sit in th CRM promotr. Intrstingly, trtmnt of ut myloi lukmi with KPT8 ws rntly rport to ownrgult th onogni rivrs Flt n Kit, whih is not u to gnrl inrs in protin grtion s p, p, IkB n othr protins r inrs. Morovr, SINE trtmnt lvts lvls of th propoptoti gns BAX n PUMA, whrs ruing th ntipoptoti protins Ml. Thus, it is likly tht nulr ntrpmnt of TSPs protts thm from ytoplsmi protsommit grtion; n onvrsly, tht CRM inhiition n riv th strution of onogni protins. SINEs inu ro ntitumor tivity in MM. Thy r tiv ginst MM lls hroring mutt p, whih ours in 7% in MM ll lins n inrsingly in th stting of rlps n rfrtory MM. Ths rsults init pinpnnt mhnisms whry SINEs mit thir ntinr tivity. Blok of NFkB tivtion y SINE oul mit pinpnnt SINEinu MM ytotoxiity, in ition to PUMA inution sri ov. Importntly, SINEs inhiit NFkB p DNAining tivity, whih my ount, t lst in prt, for th ility of SINEs to ovrom MM ll growth n onvntionl rug rsistn onfrr y th BM miliu. Spifilly, NFkB is implit in miting intrinsi rug rsistn in MM n othr tumors, s wll s rgulting hsion molul xprssion on MM n BM ssory lls miting thir intrtion n ining, whih onfrs rsistn to onvntionl hmothrpy in our stuy. KPT8 n KPT lok NFkB p tivity, t lst in prt, y inrsing nulr IkB lvls n inhiiting pikb in oth MM lls n OC prursors. Inhiition of RANKLinu NFkB tivity in OC prursors furthr lok xprssion of rly OC iffrntition gn ACP n NFAT. Intrstingly, SINEs us miniml poptosis of s n ostolsts t th onntrtions whih triggr 8% MM ll poptosis (log iffrns), onsistnt with th miniml ffts of SINEs on othr nonnoplsti ll typs., Signifintly, SINEs ru srtion of ytokins supporting MM ll growth, survivl, rug rsistn n ngiognsis, inluing IL, VEGF, MIP n IL, from s, lon or in oulturs with MM lls. As proution of mny of ths MM growth ftors/ytokins ours vi NFkBpnnt prrin mhnisms in s, SINEinu inhiition of NFkB tivity in s n othr ssory lls my ontriut to thir ytotoxiity ginst MM lls in th BM mironvironmnt. In ition to s, ostoprognitor lls r lso lss snsitiv to SINEs thn ithr MM lls or OCs. Although inhiiting ostolstognsis, SINEs h no impt on ostognsis in vitro n i not furthr rs MM llsupprss lium position from ostolsts, suggsting tht SINEs i not fft nw on formtion. Ths osrvtions, long with th miniml toxiity of SINEs on norml hmtopoiti lls, tht is, CD þ prognitor, NK, T lls,, suggst tht SINEs my hv mor fvorl thrputi winow thn prviously intifi CRM inhiitors. Furthrmor, KPT, urrntly in phs trils in vn mlignnis, show no signifint fft on norml immunogloulin lvls in stuis in monkys n rts trt ovr 8 ys with y rovry (Shhm t l., unpulish osrvtions). Ths osrvtions init tht SINE CRM ntgonists show improv sltivity for noplsti vs norml ll typs, n my thrfor hv fvorl sifft profil. In summry, this stuy fins rol of CRM lvtion in MM pthophysiology, oth tumor ll growth n rug rsistn s wll s OC iffrntition. Importntly, SINE trtmnt inu potnt n rpi poptosis of MM lls in vitro n in vivo, n furthr irtly rs on rsorption. Ths t thrfor provi th prlinil rtionl for ongoing linil vlopmnt of th orl KPT to improv ptint outom n rs on iss in MM (Clinil Tril: NCT789). CONFLICT OF INTEREST YL, WS, JRSM, TK, SS, MK r mploys of Kryophrm Thrputis In., whos prout ws us in this rsrh PR srvs on visory ors to Millnnium, Clgn, Novrtis, Johnson & Johnson n BristolMyrs Squi NCM srvs on visory ors to Millnnium, Clgn n Novrtis KCA srvs on visory ors to Onyx, Clgn, Gil n SnofiAvntis. Th rmining uthors lr no onflit of intrst. ACKNOWLEDGEMENTS W r grtful for Ho Wng, Mtt M, Mihll Chn, Yiguo Hu n Sunyoung Kong for xllnt thnil support to th projt. W thnk th input from Hoqing Ying n Dilr MCuly from Kryophrm Phrmutis. W lso thnk th nursing stff n linil rsrh oorintors of th LBow Institut for Mylom Thrputis n th Jrom Lippr Multipl Mylom Cntr of th DnFrr Cnr Institut for support n hlp in proviing primry tumor spimns for this stuy. This stuy ws support y Ntionl Instituts of Hlth Grnts RO 97, PO 7878 n DF/HCC SPORE in Multipl Mylom PCA77; n th Low Fun to Cur Mylom (KCA); KCA is n Amrin Cnr Soity Clinil Rsrh Profssor. AUTHOR CONTRIBUTIONS YTT, YL, SS, MK, KCA onptuliz rsrh n form th hypothsis of this ppr; YTT, YL, YC, SS, ALK sign, prform xprimnts, nlyz t; FZ, AC, AAM nlyz xprssion t; CA, MC, YC, MYZ, AC, MR, DT, JRS M, TK, YG, WS, IG, LW, prform th in vitro rsrh n ollt t; SS, ALK, CA, MC, MYZ sign, prform n nlyz niml work; YL, YC, SS, MK, LW, SAS provi rgnts, nlyti tools n input to stuis; NCM, PR, KCA provi MM ptint smpls; YTT wrot th mnusript; YTT, SS, MK, KCA ritilly vlut n it th mnusript. REFERENCES Xu D, Grishin NV, Chook YM. NES: ts of NESontining CRM rgos. Mol Biol Cll ; : 7 7. 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Nt Rv Cnr 7; 7: Mutk SC, Yng WQ, Dong SD, Wr SL, Crig DA, Timmrmns PB t l. Intifition of nulr xport inhiitors with potnt ntinr tivity in vivo. Cnr Rs 9; 9: 7. Supplmntry Informtion ompnis this ppr on th Lukmi wsit ( & Mmilln Pulishrs Limit Lukmi ()