in vitro and in vivo selective antitumor activity of a ... · development of novel proteasome...

Cancer Therapy: Preclinical

In Vitro and In Vivo Selective Antitumor Activity of a NovelOrally Bioavailable Proteasome Inhibitor MLN9708against Multiple Myeloma Cells

Dharminder Chauhan1, Ze Tian1, Bin Zhou2, Deborah Kuhn3, Robert Orlowski3, Noopur Raje1,Paul Richardson1, and Kenneth C. Anderson1

AbstractPurpose: The success of bortezomib therapy for treatment of multiple myeloma (MM) led to the

development of structurally and pharmacologically distinct novel proteasome inhibitors. In the present

study, we evaluated the efficacy of one such novel orally bioactive proteasome inhibitor MLN9708/

MLN2238 in MM using well-established in vitro and in vivo models.

Experimental Design:MMcell lines, primary patient cells, and the humanMM xenograft animal model

were used to study the antitumor activity of MN2238.

Results: Treatment of MM cells withMLN2238 predominantly inhibits chymotrypsin-like activity of the

proteasome and induces accumulation of ubiquitinated proteins. MLN2238 inhibits growth and induces

apoptosis in MM cells resistant to conventional and bortezomib therapies without affecting the viability of

normal cells. In animal tumor model studies, MLN2238 is well tolerated and inhibits tumor growth with

significantly reduced tumor recurrence. A head-to-head analysis of MLN2238 versus bortezomib showed a

significantly longer survival time inmice treated withMLN2238 thanmice receiving bortezomib. Immunu-

nostaining ofMMtumors fromMLN2238-treatedmice showedgrowth inhibition, apoptosis, andadecrease

in associated angiogenesis.Mechanistic studies showed thatMLN2238-triggeredapoptosis is associatedwith

activation of caspase-3, caspase-8, and caspase-9; increase in p53, p21,NOXA, PUMA, and E2F; induction of

endoplasmic reticulum (ER) stress response proteins Bip, phospho-eIF2-a, and CHOP; and inhibition of

nuclear factor kappa B. Finally, combining MLN2238 with lenalidomide, histone deacetylase inhibitor

suberoylanilide hydroxamic acid, or dexamethasone triggers synergistic anti-MM activity.

Conclusion: Our preclinical study supports clinical evaluation of MLN9708, alone or in combination,

as a potential MM therapy. Clin Cancer Res; 17(16); 5311–21. �2011 AACR.

Introduction

Normal cellular processes such as DNA replication, cellcycle, cell growth and survival, inflammation, transcription,and apoptosis are modulated by the ubiquitin-proteasomesignaling pathway (UPS; ref. 1–3), which facilitates proteo-lysis of key regulatoryproteins. Importantly, deregulation in

UPS is linked to the pathogenesis of various humandiseases(3), and targeting components of UPS therefore offersgreat promise in novel therapeutic strategies. Bortezomib(Velcade) is the first-in-class proteasome inhibitor,approved by Food and Drug Administration, for the treat-ment of multiple myeloma (MM) and relapsed mantle celllymphoma (3–7). Although very effective, dose-limitingtoxicities and the development of resistance limit itslong-term utility (8, 9), and there is therefore a need fordevelopment of novel proteasome inhibitors with equipo-tent efficacy and improved safety profile.

Recent preclinical pharmacology studies showed that asecond-generation small-molecule proteasome inhibitor,MLN9708 (Millennium Pharmaceuticals, Inc.), has ashorter proteasome dissociation half-life than bortezomib,as well as improved pharmacokinetics, pharmacody-namics, and antitumor activity in xenograft models (10).In contrast to bortezomib, MLN9708 is an orally bioavail-able proteasome inhibitor and shows efficacy at variousdosing routes and regimens. Upon exposure to aqueoussolutions or plasma, MLN9708 rapidly hydrolyzes to itsbiologically active form MLN2238. Similar to bortezomib,

Authors' Affiliations: 1The LeBow Institute for Myeloma Therapeutics andJerome Lipper Myeloma Center, Department of Medical Oncology, DanaFarber Cancer Institute, Harvard Medical School, Boston, Massachusetts;2Institute for Nutritional Sciences, Shanghai Institute for BiologicalSciences, Chinese Academy of Sciences, Shanghai, China, and Children'sHospital, Boston, Massachusetts; and 3Department of Lymphoma andMyeloma, The University of Texas M.D. Anderson Cancer Center, Hous-ton, Texas

Note: D. Chauhan and Z. Tian contributed equally to this work.

Corresponding Author: Kenneth C. Anderson or Dharminder Chauhan,Dana-Farber Cancer Institute, M561, 450 Brookline Ave, Boston, MA02115. Phone: 617-632-4563; Fax: 1-617-632-2140; E-mail:[email protected] [email protected]

doi: 10.1158/1078-0432.CCR-11-0476

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 5311

on July 26, 2020. © 2011 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 30, 2011; DOI: 10.1158/1078-0432.CCR-11-0476

http://clincancerres.aacrjournals.org/

MLN2238 is a boronic acid analogue that was identified byscreening a large pool of boron-containing proteasomeinhibitors with physiochemical properties distinct frombortezomib (10, 11). In the present study, we examinedthe antitumor activity of MLN2238 using both in vitro andin vivo MM models.

Materials and Methods

Cell cultureMM.1S [dexamethasone (Dex)-sensitive], MM.1R (Dex-

resistant), RPMI-8226, OPM1, OPM2, H929, and INA-6(IL-6–dependent) human MM cell lines were cultured incomplete medium (RPMI-1640 media supplemented with10%FBS,100units/mLpenicillin, 100mg/mLstreptomycin,and 2mmol/L L-glutamine). ANBL-6-bortezomib–sensitive(ANBL-6.WT) and -resistant (ANBL-6.BR) were kindly pro-vided by Dr. Robert Orlowski (M.D. Anderson CancerCenter, Houston, TX). Tumor cells from MM patients werepurified (>95%purity) by CD138þ selection using the AutoMACS magnetic cell sorter (Miltenyi Biotec Inc.). Informedconsent was obtained from all patients in accordance withthe Helsinki protocol. Peripheral blood mononuclear cells(PBMC) from normal healthy donors were maintained inculture medium, as described earlier. Bonemarrow stromalcells (BMSC)werederivedfromCD138�cellsobtainedfromMM patients and cultured in Dulbecco’s modified Eagle’smediummedium containing 20% FBS. Drug sources are asfollows: MLN2238 from Millennium: The Takeda Oncolo-logy Company; lenalidomide, bortezomib, and suberoyla-nilidehydroxamicacid(SAHA)werepurchased fromSelleckChemicals LLC; and Dex was obtained from Calbiochem.

In vitro proteasome activity assaysProteasome activity assay was conducted using the 20S

Proteasome Assay Kit, SDS-Activated (Calbiochem) as pre-

viously described (12, 13), with some modifications.Briefly, MM.1S cells were lysed in radioimmunoprecipita-tion assay (RIPA) buffer and 20 mg (10 mL) of protein wasused in a total volume of 200 mL reaction buffer (20 mmol/L HEPES, pH 7.6, 0.5 mmol/L EDTA) with 0.03% SDSexcept for trypsin-like (T-L) activity assay. The substratesused for measuring chymotrypsin-like (CT-L), T-L, or cas-pase-like (C-L) proteasome activity were Suc-Leu-Leu-Val-Try-AMC (10 mmol/L), Bz-Val-Gly-Arg-AMC (50 mmol/L),and Z-Leu-Leu-Glu-AMC (10 mmol/L), respectively. Thereaction was initiated by adding 10 mL of each substrate,and free 7-amino-4-methylcoumarin (AMC) fluorescencewas quantified using a 380/460-nm filter set in a Spestra-Max M2e fluorometer (Bucher Biotec AG).

Cell viability, proliferation, and apoptosis assaysCell viability was assessed by MTT (Chemicon Interna-

tional Inc.; ref. 14) and CellTiter-Glo (Progema) assays,according to the manufacturer’s instructions. Cell prolif-eration analysis in coculture experiments with patient-derived BMSCs was carried out using thymidine incorpora-tion, as described previously (14). Apoptosis was quanti-fied using Annexin V/propidium iodide (PI) staining kit,as per manufacturer’s instructions (R&D Systems, Inc.),and analyzed on a FACSCalibur flow cytometer (BectonDickinson).

ImmunoblottingWestern blot analysis was carried out as previously

described (15), using antibodies (Ab) against PARP (BDBioscience Pharmingen), caspase-3, caspase-8, caspase-9,p21, E2F, cyclin D1, Cdk6, p-Rb (Cell Signaling), p53,NOXA, PUMA, Bip, CHOP, eIF2-a, or b-actin (Santa CruzBiotechnology). Blots were then developed by enhancedchemiluminescence (ECL; Amersham).

NF-kB and HtrA2/Omi activity assayMM.1S cells were treated with MLN2238 (12 nmol/L) at

various times and harvested; total cellular proteins (1 mg)were subjected to p65 and p52 nuclear factor kappa B (NF-kB) activity analysis using ELISA, as per the manufacturer’sinstructions (TransAM NF-kB Transcription Factor AssayKits; Active Motif). The effect of MLN2238 versus bortezo-mib on HtrA2/Omi serine protease activity was determinedby measuring cleavage of HtrA2/Omi substrate b-casein inan in vitro enzyme–based assay, as per manufacturer’sinstruction (R&D Systems).

Human plasmacytoma xenograft modelAll animal experiments were approved by and conform

to the relevant regulatory standards of the InstitutionalAnimal Care and Use Committee at the Dana-Farber Can-cer Institute. MLN2238 was dissolved in 5% 2-hydroxy-propyl-b-cyclodextrin at 2 mg/mL concentration. Thehuman plasmacytoma xenograft tumor model was usedas previously described (13, 15, 16). CB-17 severe com-bined immunodeficient (SCID) mice (n ¼ 21; Taconic)were subcutaneously inoculated with 5.0 � 106 MM.1S

Translational Relevance

The favorable clinical outcome of bortezomib therapyin multiple myeloma (MM) patients provided impetusfor the development of second-generation small-mole-cule proteasome inhibitors with the goals of improvingefficacy of proteasome inhibition, enhancing antitumoractivity, and reducing toxicity, as well as providingflexible dosing schedules and patient convenience. Inthe present study, we used both in vitro and in vivoMM xenograft models to show antitumor efficacy of anovel orally bioactive proteasome inhibitor, MLN9708.Moreover, combination regimens of MLN9708 withsuberoylanilide hydroxamic acid, lenalidomide, or dex-amethasone induce synergistic anti-MM activity. Ourpreclinical data showing efficacy of MLN9708 in MMdisease models provide the framework for clinical eva-luation of MLN9708, either alone or in combination, toimprove outcome in MM.

Chauhan et al.

Clin Cancer Res; 17(16) August 15, 2011 Clinical Cancer Research5312




cells in 100 mL serum-free RPMI-1640 medium and ran-domized to treatment groups when tumors reached 250to 300 mm3. Mice were treated with vehicle, bortezomib(1 mg/kg; i.v.) or MLN2238 (11 mg/kg; i.v.) twice weeklyfor 3 weeks. Animals were euthanized when their tumorsreached 2 cm3.

In situ detection of apoptosis and assessment ofangiogenesisMice tumor sections were subjected to immunohisto-

chemical (IHC) staining for terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling (TUNEL)and for caspase-3 activation (13). Ki-67 was assessed byIHC staining to quantify proliferation. Tumor angiogenesiswas assessed by IHC staining for VEGF receptor 2(VEGFR2), and PECAM a-SMA expression (13). Immunos-tained tissues were imaged using confocal microscopy(FV1000; Olympus).

Statistical analysisStatistical significance of differences observed in

MLN2238- or bortezomib-treated versus control cultures

was determined using the one-way ANOVA test. The mini-mal level of significance was P < 0.05. Survival of mice wasmeasured using the GraphPad Prism software (version 5).Isobologram analysis (17) was carried out using "Calcu-Syn" software program (Biosoft). Combination index (CI)values of less than 1.0 indicate synergism and values morethan 1.0 antagonism.

Results and Discussion

Effect of MLN2238 on proteasome activity in vitroWe first examined the ability of MLN2238 to inhibit all 3

proteasome activities in MM cells. MM.1S cells were treatedwith various concentrations of MLN2238 for 3 hours andharvested; cell extracts were then analyzed for CT-L, C-L,and T-L proteasome activities, using specific fluorogenicpeptide substrates. MLN2238 significantly inhibited CT-Lproteasome activity with an IC50 at 5 nmol/L (Fig. 1A; P <0.05). Higher concentrations of MLN2238 showed inhibi-tory activity against C-L and T-L proteasome activities(Fig. 1B and C, respectively). We next compared the effectsof MLN2238 versus bortezomib on CT-L proteasome

Figure 1. Proteasome inhibitor MLN2238 inhibits proteasome activity in vitro. A–C, MM.1S cells were treated with MLN2238 at indicated concentrationsfor 3 hours and harvested; cell extracts were then analyzed for CT-L, C-L, and T-L proteasome activities. Results are represented as percent inhibitionin proteasome activities in drug-treated versus vehicle control. D, MM.1S cells were treated with IC50 concentrations of MLN2238 or bortezomib for indicatedtimes and harvested; cell extracts were then analyzed for CT-L proteasome activity. E, left, MM.1S cells were treated with MLN2238 (12 nmol/L) at indicatedtimes and harvested; protein lysates were subjected to immunoblotting using anti-ubiquitin and anti-actin Abs. Blots shown are representative of 3independent experiments. E, right, MM.1S cells were treated with MLN2238 for 24 hours and harvested; protein lysates were subjected to immunoblottingusing anti-ubiquitin and anti-actin Abs. Blots shown are representative of 3 independent experiments. F, recombinant human HtrA2 enzyme was incubatedwith its substrate b-casein in assay buffer (R&D Systems), followed by SDS-PAGE, silver staining, and quantification of cleaved b-casein. The bargraph represents percent inhibition of HtrA2-induced b-casein cleavage in the presence of bortezomib (3 nmol/L) or MLN2238 (12 nmol/L). Datapresented are means � SD (n ¼ 2; P < 0.05).

Proteasome Inhibitor MLN9708 as Myeloma Therapy

www.aacrjournals.org Clin Cancer Res; 17(16) August 15, 2011 5313




activity. MLN2238 triggered a significant and similar degreeof CT-L inhibition as bortezomib (Fig. 1D; P < 0.05). It iswell established that proteasome inhibition causes stabili-zation and accumulation of ubiquitinated proteins, and inagreement with this observation, ML2238 induced amarked increase in ubiquitinated proteins in a time- anddose-dependent manner (Fig. 1E).

A recent study showed that peripheral neuropathy asso-ciated with bortezomib therapy may be, in part, due toblockade of neuronal cell survival protease HtrA2/Omi(18). In the present study, treatment of MM cells withbortezomib inhibited HtrA2/Omi, and importantly, nosignificant inhibition of HtrA2/Omi was noted in responseto MLM2238 treatment (Fig. 1F; P < 0.004). These datahighlight another distinction between bortezomib andMLN2238; however, the effect of MLN2238 on otherproteases remains to be examined. Nonetheless, our find-ings suggest that MLN2238 targets proteasomes and,importantly, retains the potency and selectivity of borte-zomib against CT-L proteasome activity.

Anti-MM activity of MLN2238 in vitroHuman MM cell lines (MM.1S, INA-6, RPMI-8226,

MM.1R, H929, OPM1, and OPM2) were treated withvarious concentrations of MLN2238 for 48 hours, followedby assessment for cell viability by MTT assays. A significantconcentration-dependent decrease in viability of all celllines was observed in response to treatment withMLN2238(Fig. 2A, P < 0.05; n ¼ 3). Moreover, MLN2238-induceddecrease in viability is due to apoptosis, as evidenced by asignificant increase in the Annexin Vþ/PI� apoptotic cellpopulation in MM.1S, H929, OPM1, and OPM2 cells(Fig. 2B; P < 0.005, n ¼ 3). The anti-MM activity ofMLN2238 was observed in MM cell lines sensitive andresistant to conventional therapies, as well as representingdistinct cytogenetic profiles. For example, we examinedisogenic cell lines Dex-sensitive MM.1S and Dex-resistantMM.1R with t(14;16) translocation and c-maf overexpres-sion; RPMI-8226 with TP53, K-Ras, and EGFR mutations;INA-6, an IL-6-dependent cell line with N-Ras activatingmutation; H929 with t(4;14) translocation and mutated

Figure 2. Anti-MM activity of MLN2238. A, MM cell lines were treated with or without MLN2238 at the indicated concentrations for 48 hours, followed byassessment for cell viability by MTT assays. Data presented are means � SD (n ¼ 3; P < 0.05 for all cell lines). B, MM.1S, H929, OPM1, and OPM2cell lines were treated withMLN2238 (IC50 concentrations) and analyzed for apoptosis using Annexin V/PI staining assay. C, purified patientMMcells (CD138þ)were treated with indicated concentrationsMLN2238 for 24 and 48 hours, and cell viability wasmeasured using CellTiter-Glo assay. Data presented aremeans� SD of triplicate samples (P < 0.001 for all patient samples). D, bortezomib-sensitive (ANBL-6.WT) and -resistant (ANBL-6.BR) MM cell lines were treated withincreasing concentrations of bortezomib and MLN2238 for 48 hours, followed by assessment for cell viability by MTT assays. The IC50 value of MLN2238 andbortezomib for ANBL-6.WT or ANBL-6.BR was derived. The bar graph shows the IC50 ratio (ANBL-6.BR/ANBL-6.WT) of ML2238 and bortezomib. Datapresented are means � SD (n ¼ 3). E, PBMCs from healthy donors were treated with indicated concentrations of MLN2238 for 48 hours and then analyzedfor viability by CellTiter-Glo assay. Data presented are means � SD of quadruplicate samples (P < 0.001 for all PBMC samples).

Chauhan et al.





Ras; and OPM2 with t(4;14)(p16;q32) translocation, andabnormal TP53 (19–24). Thus, the variable IC50 ofMLN2238 observed against MM cell lines may be due totheir distinct genetic background and/or drug resistancecharacteristics (19, 21).To determine whether MLN2238 similarly affects pur-

ified patient MM cells, tumor cells from 6 MM patients,including those relapsing after multiple prior therapiessuch as bortezomib, lenalidomide, and Dex, were treatedwith MLN2238. Patients were considered refractory tospecific therapy when disease progressed on therapy orrelapsed within 2months of stopping therapy. A significantdose-dependent decrease in viability of all patient MM cellswas noted after MLN2238 treatment (Fig. 2C; P < 0.001 forall patients). In addition, a parallel treatment of MM cellsobtained from bortezomib-refractory patients showedincreased in vitro cytotoxicity in response to MLN2238versus bortezomib (data not shown). These findings showthe ability of MLN2238 to trigger cytotoxicity even intumor cells obtained from patient resistant to bortezomib,Dex, or lenalidomide therapies. To further determinewhether MLN2238 overcomes bortezomib resistance, weused previously characterized (25) bortezomib-sensitive(ANBL-6.WT) and -resistant (ANBL-6.BR) MM cell lines.As seen in Figure 2D, the IC50 ratio (ANBL-6.BR/ANBL-6.WT) of MLN2238 is significantly less than bortezomib (P <0.01; n ¼ 3), showing the ability of MLN2238 to overcomebortezomib resistance. In the context of mechanism(s)mediating bortezomib resistance, a recent study has linkedit to increased signaling through the insulin-like growthfactor-1–Akt axis (25); however, involvement of othersignaling cascades cannot be excluded. Furthermore, adifferential proteasome content/activity profile, abnormalor mutated proteasome subunits, and/or endoplasmic reti-culum (ER) stress levels may contribute to bortezomibresistance. These issues remain to be examined in a broaderpanel of MM cell lines and patient cells. Finally, in thepresent study, MLN2238 at the IC50 for MM cells does notsignificantly affect the viability of normal PBMCs (Fig. 2F),suggesting specific anti-MM activity and a favorable ther-apeutic index for MLN2238.

MLN2238 inhibits human MM cell growth in vivo andprolongs survival in xenograft mouse modelHaving shown that MLN2238 induces apoptosis in MM

cells in vitro, we examined the in vivo efficacy of MLN2238given intravenously or orally using a human plasmacytomaMM.1S xenograft mouse model (13, 16). Treatment oftumor-bearing mice with intravenous injection ofMLN2238 significantly (P ¼ 0.001) inhibited MM tumorgrowth and prolonged survival (87%; P < 0.001) of thesemice (Fig. 3A and B, respectively). Bortezomib treatmentalso reduced tumor progression (Fig. 3A), albeit to a lesserextent than MLN2238. Moreover, we found thatMLN2238-treated mice survived for a longer time thanmice receiving bortezomib (P < 0.01; CI ¼ 95%). Inaddition, blood chemistry profiles of MLN2238-treatedmice showed normal levels of creatinine, hemoglobin,

and bilirubin (Fig. 3C). Examination of the xenograftedtumor sections showed that MLN2238, but not vehiclealone, dramatically increased the number of cleaved cas-pase-3–positive (red color) cells (Fig. 3D). Similarly,MLN2238 increased the number of TUNEL-positive (greencolor) cells compared with vehicle treatment (Fig. 3D). Inagreement with these data, a significant decrease in pro-liferation marker Ki-67 (red color) was noted in tumorsections from MLN2238-treated mice relative to tumorsfrom control mice (Fig. 3D). Finally, treatment of tumor-bearing mice with oral doses of MLN2238 significantly(P ¼ 0.001) inhibited MM tumor growth and prolongedsurvival (P < 0.01) of these mice (Fig. 3E and F, respec-tively). These in vivo data confirm our in vitro findingsshowing potent apoptotic activity of MLN2238 againstMM cells.

Prior studies have established that MM cell growth isassociated with angiogenesis, which predominantly occursvia VEGF pathway (26, 27). To determine whetherMLN223 triggers antiangiogenic activity, we directly eval-uated tumors harvested from mice by immunostainingusing 2 distinct markers of angiogenesis, VEGFR2 andPECAM. As seen in Figure 3D, MLN2238 decreased thenumbers of VEGFR2- and PECAM-positive cells. These datasuggest that besides inducing apoptosis, MLN2238 alsoinhibit tumor-associated angiogenic activity. Takentogether, our findings show that MLN2238 reduces tumorgrowth, prolongs survival, and is well tolerated in vivo.

Mechanisms mediating anti-MM activity of MLN2238Studies to date provide evidence for activation of pleio-

tropic cell death signaling cascades in response to protea-some inhibition (28, 29). This is likely due to the fact thatthe majority of cellular proteins undergo degradationthrough proteasome, and blockade of proteasome nega-tively affects this normal cellular process resulting in accu-mulation of unwanted proteins and subsequent activationof multiple cell death signaling. In the light of this notion,we examined the effect of MLN2238 on some of the majorapoptotic signaling pathways triggered by similar class ofagents in MM cells.

Effect of MLN2238 on caspases. Treatment of H929 andMM.1S MM cells with MLN2238 triggered a markedincrease in proteolytic cleavage of PARP, a signature eventduring apoptosis (Fig. 4A and B). Furthermore, MLN2238induced cleavage of caspase-3, an upstream activator ofPARP (Fig. 4A and B). Mitochondria mediate apoptoticsignaling via activation of cell death initiator caspase,procaspase-9 (30). Similarly, Fas-associated death-domain(FADD) protein is an essential component of the death-inducing signaling complexes (DISC), resulting in auto-activation of procaspase-8. Our data show that MLN2238induces activation of both caspase-9 (intrinsic) andcasapse-8 (extrinsic) apoptotic pathways in H929 andMM.1S and cells (Fig. 4A and B, respectively). Studies usingbiochemical inhibitors showed that inhibition of eithercaspase-8 (IETD-FMK) or caspase-9 (LEHD-FMK) resultedin marked abrogation of MLN2238-triggered cytotoxicity






(Fig. 4C). In addition, pan-caspase inhibitor (Z-VAD-FMK)also attenuated MLN2238-induced cytotoxicity (Fig. 4C; P< 0.005). Simultaneous blockade of caspase-8 and caspase-9 led to 89 � 4.4% attenuation of MLN2238-triggered celldeath. These findings suggest that (i) MLN2238 triggersboth mitochondria-dependent and -independent signalingpathways and (ii) MLN2238-induced apoptosis occurs in acaspase-dependent manner.

Effect of MLN2238 on p53 pathway. The molecular

pathways leading to caspase induction includes activationof tumor suppressor p53, which coordinates cellularresponse to stress-signaling pathways via cell-cycle arrest,and apoptosis (31, 32). Examination of MLN2238-treatedMM cells showed an increase in both p53 and p21(Fig. 4D). The induction of p21 may account forMLN2238-induced growth arrest (data not shown). Priorstudies have also linked the p53 pathway to activation ofmitochondrial apoptotic signaling via BH3-only proteinsNOXA and PUMA (33, 34); also, we found that MLN2238triggered robust induction of NOXA and PUMA (Fig. 4D).This finding is consistent with the observed MLN2238-induced caspase-9 induction that occurs via mitochondria

(Fig. 4A and B). Furthermore, the p53-signaling cascade isknown to communicate with retinoblastoma (Rb)–E2Faxis (35). Treatment of MM.1S and MM.1R cells withMLN2238 downregulated pRb with an expected upregula-tion of its downstream target protein E2F (Fig. 4E and F).Similarly, cyclin D1 and its target protein Cdk6 weremarkedly decreased upon treatment with MLN2238(Fig. 4E and F).

Effect of MLN2238 on ER stress pathway. Proteasome

inhibition is associated with induction of the ER stresspathway and the unfolded protein response (13, 36, 37).Analysis of proteins mediating the ER stress pathwayshowed that MLN2238 induces eIf2-a kinase activity andprotein levels of Bip and CHOP/GADD153 (Fig. 4G). Ofnote, ER stress signaling is also linked to activation of p53–NOXA–PUMA signaling axis (38), suggesting a potentialcross-talk between these pathways during MLN2238-induced apoptosis in MM cells. It is conceivable thatMLN2238, like bortezomib, triggers pleiotropic signalingpathways; however, because of the shorter dissociation(t1/2) characteristics of MLN2238 than bortezomib, thekinetics of alterations in stress response signaling may vary

Figure 3.MLN2238 inhibits growth of xenografted human MM cells in CB-17 SCID mice. A, average � SD of tumor volume (mm3) is shown from mice (n ¼ 7/group) versus time (days) when tumor was measured. MM.1S cells (5� 106 cells/mouse) were implanted in the rear flank of female mice (6 weeks of age at thetime of tumor challenge). On days 28 to 30, mice were randomized to treatment groups and treated intravenously with vehicle, MLN2238 (11 mg/kg),or with bortezomib (1 mg/kg) on a twice-weekly schedule for 3 weeks. Data are presented as mean� SD tumor volume. A significant delay in tumor growth inMLN2238-treated mice was noted compared with vehicle-treated control mice. Bars indicate means � SD. B, Kaplan–Meier survival plot showssignificant increase (P < 0.05) in survival of mice receiving MLN2238 (11 mg/kg) or bortezomib (1 mg/kg) compared with vehicle-treated control. C, mice weretreated with vehicle, MLN2238, or bortezomib (as in A); blood samples were obtained and subjected to analysis for bilirubin, creatinine, and hemoglobin levelsusing Quantichrom Creatinine, Bilirubin, and Hemoglobin Assay kits (BioAssay Systems). D, micrographs (top 3 panels) show apoptotic cells in tumorssectioned from untreated or MLN2238-treated mice as identified caspase-3 (Casp3) cleavage (red cells), TUNEL (TUNEL-positive cells, green cells), as well asKi-67 staining. Dotted red/green line indicates a line between tumor and host tissue. Micrographs (bottom 2 panels) show expression of angiogenesismarkers in tumors sectioned from untreated or MLN2238-treated mice, identified by VEGFR2 (green) and PECAM (red) staining. Bar scale, 100 mm(caspase-3 and TUNEL); 50 mm (Ki-67); and 10 mm (VEGFR2 and PECAM). E, tumor-bearing mice were treated orally with vehicle or MLN2238 (8 mg/kg) on atwice-weekly schedule for 3 weeks. Data are presented as mean � SD tumor volume. Bars indicate means � SD. F, the Kaplan–Meier survival plotshows increase in survival of mice receiving oral doses of MLN2238 (8 mg/kg) compared with vehicle-treated control.

Chauhan et al.





and this issue remains to be defined. Nonetheless, ourmechanistic data show that MLN2238-induced apoptosisin MM cells is caspase dependent and correlates withactivation of p53–p21, p53–NOXA–PUMA, Rb–E2F, andER stress signaling pathways.

MLN2238 overcomes the cytoprotective effects of theMM bone marrow microenvironment and inhibits invitro capillary-like tube formationInteraction of MM cells with BMSCs triggers cytokine

secretion, which mediates paracrine growth of MM cells, aswell as protects against drug-induced apoptosis (28, 39,40). To determine whether MLN2238 affects BMSC-trig-gered MM cell growth, MM.1S cells were cultured with orwithout BMSCs in the presence or absence of variousconcentrations of MLN2238. A significant inhibition ofBMSCs-induced proliferation of MM.1S was noted inresponse to MLN2238 treatment (Fig. 5A). These data

suggest that MLN2238 not only directly targets MM cellsbut also overcomes the cytoprotective effects of the MMhost bone marrow microenvironment.

Angiogenesis play an important role in the progression ofMM (26, 41). Our in vivo data using tumor sections fromMLN2238-treatedmice showed a decrease in the expressionof angiogenesis markers. To confirm the antiangiogenicactivity of MLN2238, we utilized in vitro capillary-like tubestructure formation assay using human vascular endothelialcells (HUVEC), which when plated onto Matrigel differ-entiate and form capillary-like tube structures similar to invivoneovascularization, a process dependent on cell–matrixinteraction, cellular communication, and cellular motility.This assay therefore provides evidence for antiangiogeniceffects of drugs/agents. HUVECs were pretreated withvehicle [dimethyl sulfoxide (DMSO)] or MLN2238 (10nmol/L) for 8 hours, washed in media and seeded in 96-well culture plates coatedwithMatrigel, and then incubated

Figure 4.Mechanisms mediating anti-MM activity of MLN2238. A and B, H929 andMM.1S and cells were treated with MLN2238 at the indicated doses for 24hours and harvested; whole cell lysates were subjected to immunoblot analysis with anti-PARP, anti-caspase-3, anti-caspase-8, anti-caspase-9, or anti-actin Abs. FL, full length; CF, cleaved fragment. C, MM.1S cells were pretreated with biochemical inhibitors of caspase-8 (IETD-FMK), caspase-9(LEHD-FMK), or pan-caspase (Z-VAD-FMK) for 1 hour. MLN2238 (12 nmol/L) was then added in cultures for additional 24 hours, followed by analysis of cellviability by MTT assay. Data presented are means � SD (n ¼ 3; P ¼ 0.005). Error bars represent SD. D and E, MM.1S cells were treated withMLN2238 (12 nmol/L) for 24 hours and harvested; protein lysates were subjected to immunoblotting with indicated Abs. F, MM.1R cells were treatedwith MLN2238 (12 nmol/L) for 24 hours and harvested; protein lysates were subjected to immunoblotting with indicated Abs. G, MM.1S cells were treated withMLN2238 (12 nmol/L) for indicated times and harvested; protein lysates were subjected to immunoblotting using indicated Abs. Blots shown in thefigure are representative of 3 independent experiments.






for additional 4 hours, followed by analysis of tube forma-tion using an inverted microscope. As seen in Figure 5B,tubule formation was significantly decreased in theMLN2238-treated cells but not after treatment with DMSOalone (P<0.001,n¼3).HUVECviabilitywas assessedusingtrypan blue exclusion assay: less than 5% cell death wasobserved after MLN2238 treatment, excluding the possibi-lity that drug-induced inhibitionof capillary tube formationwas due to cell death. These in vitro data corroborate withour in vivo findings in animal model (Fig. 3C) to showantiangiogenic activity of MLN2238.

MLN2238 targets NF-kBNF-kB plays a key role duringMM cell adhesion–induced

cytokine secretion in BMSCs, which, in turn, triggers MMcell growth in a paracrinemanner (15, 39, 42). Reports havealso linked constitutive activation of noncanonical NF-kBpathway to the genetic abnormalities/mutations (43, 44),allowing for an autocrine growth of MM cells. Importantly,constitutive NF-kB activity in primary tumor cells fromMMpatients renders these cells refractory to inhibition by bor-tezomib (45), and, in fact, bortezomib induces canonicalNF-kB activity (46). Given the findings from these studies,we examined whether MLN2238 affects NF-kB. ML2238, in

a time-dependent manner, inhibits both constitutive andTNF-a–induced NF-kB activation in MM cells (Fig. 5C–F;P < 0.05; n ¼ 3). These data suggest that MLN2238 is apotent inhibitorof both canonical andnoncanonicalNF-kBpathways.

Combined treatment with MLN2238 andlenalidomide, dexamethasone, or histone deacetylaseinhibitor SAHA induces synergistic anti-MM activity

Preclinical studies (47–49) provided the basis for clinicaltrials of proteasome inhibitor bortezomib in combinationwith lenalidomide, Dex, and histone deacetylase (HDAC)inhibitors (50). Given that MLN2238, like bortezomib, is aboronic acid analogue,we examinedwhetherML2238 simi-larly enhances the anti-MM activity of other agents. MM.1Scells were first treated with both MLN2238 and lenalido-mide simultaneously across a range of concentrations for 48hours and then analyzed for viability by MTT assay. Ananalysis of synergistic anti-MM activity using the Chouand Talalay method (17) showed that the combination oflowconcentrationsofMLN2238andlenalidomide triggeredsynergistic anti-MM activity, with a CI < 1.0 (Fig. 6A).

In addition to proteasomal degradation, intracellularprotein catabolism also occurs via an HDAC-dependent

Figure 5. MLN2238 blocks BMSC-induced MM cell proliferation, inhibits in vitro capillary tubule formation, and targets NF-kB. A, MM.1S cells were treatedwith indicated concentrations of MLN2238 in the presence or absence of BMSCs for 48 hours, followed by measurement of proliferation using tritiatedthymidine incorporation assay. Data presented are means � SD (n ¼ 3; P < 0.005). B, HUVECs were cultured in the presence or absence of MLN2238 for 8hours (cell viability >95%); cells were then washed with plain media and placed on the Matrigel for 4 hours to allow tube formation, followed by photographywith an inverted microscope [magnification: 4�/0.10 NA oil; media: endothelial basal medium (EBM)-2]. In vitro angiogenesis is reflected by capillarytube branch formation. Images are representative of 3 experiments with similar results. C and D, MM.1S cells were treated with MLN2238 (12 nmol/L) atindicated times, harvested, and subjected to NF-kB activity analysis by ELISA. Data presented are the means � SD (n ¼ 3; P < 0.005). E and F, MM.1S cells were treated with MLN2238 (12 nmol/L) at indicated times, followed by the addition of TNF-a during the last 20 minutes before cells were harvested.Cells were then subjected to p65 and p52 NF-kB activity analysis by ELISA. Data presented are the means � SD (n ¼ 3; P < 0.004). Error bars represent SD.

Chauhan et al.





aggressome-autophagy signaling pathway (51–53). Ourprior study showed that inhibition of both mechanismsof protein catabolism by combining bortezomib andHDAC inhibitor induced significant cytotoxicity in MMcells (51). Recent clinical trials combining bortezomib andtheHDAC inhibitor vorinostat showed promising outcomein relapsed and refractory MM, including activity amongsome patients with prior exposure to bortezomib (54). Inthe light of these studies, we examined whether the com-bination of MLN2238 with HDAC inhibitor SAHA triggerssynergistic anti-MM activity. MM.1S cells were treated withbothMLN2238 and SAHA simultaneously across a range ofconcentrations for 48 hours and then analyzed for viability.Isobologram analysis showed that the combination of lowconcentrations of MLN2238 and SAHA triggered synergis-

tic anti-MM activity, with a CI < 1.0 (Fig. 6B). These dataconfirm the potential for clinical trials combiningMLN2238 and HDAC inhibitors.

We next examined whether MLN2238 adds to the anti-MM activity of the conventional anti-MM agent Dex. Aswith lenalidomide and SAHA, the combination ofMLN2238 with Dex triggered synergistic anti-MM activity,evidenced by a significant decrease in viability of MM.1Scells (Fig. 6C). Although definitive evidence of decreasedtoxicity of combination therapy awaits results of clinicaltrials, the synergy observed in vitromay allow for the use oflower doses and decreased toxicity.

Collectively, our preclinical studies therefore showpotent in vitro and in vivo anti-MM activity of MLN2238at doses that are well tolerated in human MM xenograft

Figure 6. Combination of low doses of MLN2238 and lenalidomide, SAHA, or Dex triggers synergistic anti-MM activity. A, MM.1S cells were treatedfor 48 hours with indicated concentrations of MLN2238, lenalidomide, or MLN2238 plus lenalidomide and then assessed for viability by MTT assays.Isobologram analysis shows the synergistic cytotoxic effect of MLN2238 and lenalidomide. The graph (left) is derived from the values given in the table (right).Numbers 1 to 9 in graph represent combinations shown in the table. FaCom, fraction of cells showing decrease in viability with MLN2238 plus lenalidomidetreatment. CI < 1 indicates synergy. All experiments were carried out in triplicate, and the mean value is shown. B, MM.1S cells were treated for48 hours with indicated concentrations of MLN2238, SAHA, or MLN2238 plus SAHA and then assessed for viability by MTT assays. Isobologram analysisshows the synergistic cytotoxic effect of MLN2238 and SAHA. The graph (left) is derived from the values given in the table (right). Numbers 1 to 9in graph represent combinations shown in the table. CI < 1 indicates synergy. C, MM.1S cells were treated for 48 hours with indicated concentrations ofMLN2238, Dex, or MLN2238 plus Dex and then assessed for viability by MTT assays. Isobologram analysis shows the synergistic cytotoxic effect ofMLN2238 and Dex. The graph (left) is derived from the values given in the table (right).






mouse models. These findings provide the framework forclinical trials of MLN9708 both as a single agent andtogether with lenalidomide, HDAC inhibitors, or Dex topotentially increase response, overcome drug resistance,reduce side effects, and improve patient outcome in MM.

Disclosure of Potential Conflicts of Interest

K.C. Anderson, N. Raje, and P. Richardson are advisors to MillenniumPharmaceuticals, Inc. Other coauthors have no competing financialinterests.

Grant Support

This investigation was supported by NIH grants SPORE-P50100707, PO1-CA078378, and RO1CA050947 (to D. Chauhan, P. Richardson, and K.C. Anderson,respectively). K.C. Anderson is an American Cancer Society clinical researchprofessor.

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

Received February 20, 2011; revised May 18, 2011; accepted June 23,2011; published OnlineFirst June 30, 2011.

References1. Rock K, GrammC, Rothstein L, Clark K, Stein R, Dick L, et al. Inhibitors

of the proteasome block the degradation of most cell proteins and thegeneration of peptides presented on MHC class I molecules. Cell1994;78:761–71.

2. Goldberg AL. Protein degradation and protection against misfolded ordamaged proteins. Nature 2003;426:895–9.

3. Adams J. The proteasome: a suitable antineoplastic target. Nat RevCancer 2004;4:349–60.

4. Kane RC, Bross PF, Farrell AT, Pazdur R. Velcade: U.S. FDA approvalfor the treatment of multiple myeloma progressing on prior therapy.Oncologist 2003;8:508–13.

5. Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, IrwinD, et al. A phase 2 study of bortezomib in relapsed, refractorymyeloma. N Engl J Med 2003;348:2609–17.

6. Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA,Facon T, et al. Bortezomib or high-dose dexamethasone for relapsedmultiple myeloma. N Engl J Med 2005;352:2487–98.

7. San Miguel JF, Schlag R, Khuageva NK, Dimopoulos MA, ShpilbergO, Kropff M, et al. Bortezomib plus melphalan and prednisone forinitial treatment of multiple myeloma. N Engl J Med 2008;359:906–17.

8. Richardson PG, Sonneveld P, Schuster MW, Stadtmauer EA, Facon T,Harousseau JL, et al. Reversibility of symptomatic peripheral neuro-pathy with bortezomib in the phase III APEX trial in relapsed multiplemyeloma: impact of a dose-modification guideline. Br J Haematol2009;144:895–903.

9. Lonial S, Waller EK, Richardson PG, Jagannath S, Orlowski RZ, GiverCR, et al. Risk factors and kinetics of thrombocytopenia associatedwith bortezomib for relapsed, refractory multiple myeloma. Blood2005;106:3777–84.

10. Kupperman E, Lee EC, Cao Y, Bannerman B, Fitzgerald M, Berger A,et al. Evaluation of the proteasome inhibitor MLN9708 in preclinicalmodels of human cancer. Cancer Res 2010;70:1970–80.

11. Dick LR, Fleming PE. Building on bortezomib: second-generationproteasome inhibitors as anti-cancer therapy. Drug Discov Today2010;15:243–9.

12. Lightcap ES, McCormack TA, Pien CS, Chau V, Adams J, Elliott PJ.Proteasome inhibition measurements: clinical application. Clin Chem2000;46:673–83.

13. ChauhanD, Singh A, BrahmandamM, Podar K, Hideshima T, Richard-son P, et al. Combination of proteasome inhibitors bortezomib andNPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma.Blood 2008;111:1654–64.

14. Hideshima T, Chauhan D, Shima Y, Raje N, Davies FE, Tai YT, et al.Thalidomide and its analogs overcome drug resistance of humanmultiple myeloma cells to conventional therapy. Blood 2000;96:2943–50.

15. Chauhan D, Catley L, Li G, Podar K, Hideshima T, Velankar M, et al. Anovel orally active proteasome inhibitor induces apoptosis in multiplemyeloma cells with mechanisms distinct from Bortezomib. CancerCell 2005;8:407–19.

16. LeBlanc R, Catley LP, Hideshima T, Lentzsch S, Mitsiades CS,Mitsiades N, et al. Proteasome inhibitor PS-341 inhibits humanmyeloma cell growth in vivo and prolongs survival in a murine model.Cancer Res 2002;62:4996–5000.

17. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships:the combined effects of multiple drugs or enzyme inhibitors. AdvEnzyme Regul 1984;22:27–55.

18. Arastu-Kapur S, Anderl JL, Kraus M, Parlati F, Shenk KD, Lee SJ, et al.Nonproteasomal targets of the proteasome inhibitors bortezomib andcarfilzomib: a link to clinical adverse events. Clin Cancer Res2011;17:2734–43.

19. Bergsagel PL, Kuehl WM. Molecular pathogenesis and a consequentclassification of multiple myeloma. J Clin Oncol 2005;23:6333–8.

20. Bergsagel PL, Chesi M, Nardini E, Brents LA, Kirby SL, Kuehl WM.Promiscuous translocations into immunoglobulin heavy chain switchregions in multiple myeloma. Proc Natl Acad Sci U S A 1996;93:13931–6.

21. Davies FE, Dring AM, Li C, Rawstron AC, Shammas MA, O’ConnorSM, et al. Insights into the multistep transformation of MGUS tomyeloma using microarray expression analysis. Blood 2003;102:4504–11.

22. Greenstein S, Krett NL, Kurosawa Y, Ma C, Chauhan D, Hideshima T,et al. Characterization of the MM.1 humanmultiple myeloma (MM) celllines. A model system to elucidate the characteristics, behavior, andsignaling of steroid-sensitive and -resistant MM cells. Exp Hematol2003;31:271–82.

23. Avet-Loiseau H, Attal M, Moreau P, Charbonnel C, Garban F, Hulin C,et al. Genetic abnormalities and survival in multiple myeloma: theexperience of the Intergroupe Francophone du Myelome. Blood2007;109:3489–95.

24. Moreaux J, Klein B, Bataille R, Descamps G, Maiga S, Hose D, et al. Ahigh-risk signature for patients with multiple myeloma establishedfrom the molecular classification of human myeloma cell lines. Hae-matologica 2011;96:574–82.

25. Kuhn D, Bjorklund C, Magarotto V, Mathews J, Wang M, Baladan-dayuthapani V, et al. Bortezomib resistance is mediated by increasedsignaling through the insulin-like growth factor-1/Akt axis. ASH AnnuMeeting Abstr2009;114:2739.

26. Podar K, Tai YT, Davies FE, Lentzsch S, Sattler M, Hideshima T, et al.Vascular endothelial growth factor triggers signaling cascades med-iating multiple myeloma cell growth and migration. Blood 2001;98:428–35.

27. Vacca A, Ria R, Ribatti D, Semeraro F, Djonov V, Di Raimondo F, et al.A paracrine loop in the vascular endothelial growth factor pathwaytriggers tumor angiogenesis and growth in multiple myeloma. Hae-matologica 2003;88:176–85.

28. Chauhan D, Hideshima T, Anderson KC. Proteasome inhibition inmultiple myeloma: therapeutic implication. Annu Rev Pharmacol Tox-icol 2005;45:465–76.

29. Orlowski RZ, Kuhn DJ. Proteasome inhibitors in cancer therapy:lessons from the first decade. Clin Cancer Res 2008;14:1649–57.

30. Bossy-Wetzel E, Green DR. Apoptosis: checkpoint at the mitochon-drial frontier. Mutat Res 1999;434:243–51.

31. Harris SL, Levine AJ. The p53 pathway: positive and negative feed-back loops. Oncogene 2005;24:2899–908.

32. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature2000;408:307–10.

Chauhan et al.





33. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, et al.Noxa, a BH3-only member of the Bcl-2 family and candidate mediatorof p53-induced apoptosis. Science 2000;288:1053–8.

34. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMA inducesthe rapid apoptosis of colorectal cancer cells. Mol Cell 2001;7:673–82.

35. Sherr CJ, McCormick F. The RB and p53 pathways in cancer. CancerCell 2002;2:103–12.

36. Nawrocki ST, Carew JS, Dunner K Jr, Boise LH, Chiao PJ, Huang P,et al. Bortezomib inhibits PKR-like endoplasmic reticulum (ER) kinaseand induces apoptosis via ER stress in human pancreatic cancer cells.Cancer Res 2005;65:11510–9.

37. Obeng EA, Carlson LM, Gutman DM, Harrington WJ Jr, Lee KP, BoiseLH. Proteasome inhibitors induce a terminal unfolded proteinresponse in multiple myeloma cells. Blood 2006;107:4907–16.

38. Li JLee B, Lee AS. Endoplasmic reticulum stress-induced apopto-sis: multiple pathways and activation of p53-up-regulated modu-lator of apoptosis (PUMA) and NOXA by p53. J Biol Chem 2006;281:7260–70.

39. Chauhan D, Uchiyama H, Akbarali Y, Urashima M, Yamamoto K,Libermann TA, et al. Multiple myeloma cell adhesion-induced inter-leukin-6 expression in bone marrow stromal cells involves activationof NF-kappa B. Blood 1996;87:1104–12.

40. Hideshima T, Anderson KC. Molecular mechanisms of novel thera-peutic approaches for multiple myeloma. Nat Rev Cancer 2002;2:927–37.

41. Vacca A, Ribatti D, Presta M, Minischetti M, Iurlaro M, Ria R, et al.Bone marrow neovascularization, plasma cell angiogenic potential,and matrix metalloproteinase-2 secretion parallel progression ofhuman multiple myeloma. Blood 1999;93:3064–73.

42. Feinman R, Koury J, Thames M, Barlogie B, Epstein J. Role of NF-kBin the rescue of multiple myeloma cells from glucocorticoids-inducedapoptosis by Bcl-2. Blood 1999;93:3044–52.

43. Keats JJ, Fonseca R, Chesi M, Schop R, Baker A, Chng WJ, et al.Promiscuous mutations activate the noncanonical NF-kappaB path-way in multiple myeloma. Cancer Cell 2007;12:131–44.

44. Annunziata CM, Davis RE, Demchenko Y, BellamyW, Gabrea A, ZhanF, et al. Frequent engagement of the classical and alternative NF-

kappaB pathways by diverse genetic abnormalities in multiple mye-loma. Cancer Cell 2007;12:115–30.

45. Markovina S, Callander NS, O’Connor SL, Kim J, Werndli JE, RaschkoM, et al. Bortezomib-resistant nuclear factor-kappaB activity in multi-ple myeloma cells. Mol Cancer Res 2008;6:1356–64.

46. Hideshima T, Ikeda H, Chauhan D, Okawa Y, Raje N, Podar K, et al.Bortezomib induces canonical nuclear factor-kappaB activation inmultiple myeloma cells. Blood 2009;114:1046–52.

47. Hideshima T, Richardson P, Chauhan D, Palombella VJ, Elliott PJ,Adams J, et al. The proteasome inhibitor PS-341 inhibits growth,induces apoptosis, and overcomes drug resistance in human multiplemyeloma cells. Cancer Res 2001;61:3071–6.

48. Mitsiades N, Mitsiades CS, Richardson PG, Poulaki V, Tai YT, Chau-han D, et al. The proteasome inhibitor PS-341 potentiates sensitivityof multiple myeloma cells to conventional chemotherapeutic agents:therapeutic applications. Blood 2003;101:2377–80.

49. Davies FE, Raje N, Hideshima T, Lentzsch S, Young G, Tai YT, et al.Thalidomide and immunomodulatory derivatives augment naturalkiller cell cytotoxicity in multiple myeloma. Blood 2001;98:210–6.

50. Richardson PG, Weller E, Lonial S, Jakubowiak AJ, Jagannath S, RajeNS, et al. Lenalidomide, bortezomib, and dexamethasone combina-tion therapy in patients with newly diagnosed multiple myeloma.Blood 2010;116:679–86.

51. Hideshima T, Bradner JE, Wong J, Chauhan D, Richardson P, Schrei-ber SL, et al. Small-molecule inhibition of proteasome and aggresomefunction induces synergistic antitumor activity in multiple myeloma.Proc Natl Acad Sci U S A 2005;102:8567–72.

52. Grant S, Easley C, Kirkpatrick P. Vorinostat. Nat Rev Drug Discov2007;6:21–2.

53. Dai Y, Chen S, Venditti CA, Pei XY, Nguyen TK, Dent P, et al.Vorinostat synergistically potentiates MK-0457 lethality in chronicmyelogenous leukemia cells sensitive and resistant to imatinib mesy-late. Blood 2008;112:793–804.

54. Badros A, Burger AM, Philip S, Niesvizky R, Kolla SS, Goloubeva O,et al. Phase I study of vorinostat in combination with bortezomib forrelapsed and refractory multiple myeloma. Clin Cancer Res 2009;15:5250–7.






2011;17:5311-5321. Published OnlineFirst June 30, 2011.Clin Cancer Res Dharminder Chauhan, Ze Tian, Bin Zhou, et al. Myeloma CellsBioavailable Proteasome Inhibitor MLN9708 against Multiple

Selective Antitumor Activity of a Novel OrallyIn Vivo and In Vitro

Updated version

10.1158/1078-0432.CCR-11-0476doi:

Access the most recent version of this article at:

Cited articles

http://clincancerres.aacrjournals.org/content/17/16/5311.full#ref-list-1

This article cites 54 articles, 32 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/17/16/5311.full#related-urls

This article has been cited by 32 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/17/16/5311To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-11-0476

http://clincancerres.aacrjournals.org/content/17/16/5311.full#ref-list-1

http://clincancerres.aacrjournals.org/content/17/16/5311.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/17/16/5311


in vitro and in vivo selective antitumor activity of a ... · development of novel proteasome...

Documents