A Purine Scaffold HSP90 Inhibitor BIIB021 Has Selective ... · Lymphoma and Blocks vFLIP K13-Induced NF-kB Ramakrishnan Gopalakrishnan, Hittu Matta, and Preet M. Chaudhary Abstract

Cancer Therapy: Preclinical

A Purine Scaffold HSP90 Inhibitor BIIB021 Has SelectiveActivity against KSHV-Associated Primary EffusionLymphoma and Blocks vFLIP K13-Induced NF-kB

Ramakrishnan Gopalakrishnan, Hittu Matta, and Preet M. Chaudhary

AbstractPurpose: Kaposi sarcoma–associated herpes virus (KSHV)–associated primary effusion lymphomas

(PEL) have extremely poor prognosis when treated with conventional chemotherapy. KSHV-encoded viral

FLICE-inhibitory protein (vFLIP) K13 binds to the IkappaB kinase (IKK) complex to constitutively activate

theNF-kB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. The

molecular chaperone HSP90 is a component of the IKK complex and is required for its activity.

Experimental Design:Wehave analyzed the effect of HSP90 inhibitors on the survival and proliferation

of PEL cells and on the activity of the NF-kB pathway.

Results: We show that BIIB021, a purine scaffold–based orally administrable HSP90 inhibitor, shows

preferential cytotoxicity toward PEL cells as compared with non-PEL cells. The cytotoxic effect of BIIB021

against PELwas associated with induction of cell-cycle arrest and apoptosis. BIIB021 blocked the expression

of a number of cellular proteins involved in the regulation of cell cycle and apoptosis. BIIB021 also blocked

constitutiveNF-kB activity present in PEL cells, in part, by blocking the interaction of vFLIP K13with the IKK

complex subunits. In a xenograft model of PEL, BIIB021 significantly reduced tumor growth.

Conclusion: BIIB021 blocks constitutive NF-kB activity in PEL and shows preferential antitumor

activity against PEL in vitro and in vivo. BIIB021 may be a promising agent for treatment of PEL. Clin

Cancer Res; 19(18); 5016–26. �2013 AACR.

IntroductionPrimary effusion lymphoma (PEL) is a variant of non–

Hodgkin lymphoma that is characterized by infection withKaposi sarcoma–associated herpes virus (KSHV; alsoknown as human herpes virus 8; ref. 1). Although admin-istration of cytotoxic chemotherapeutic agents representsthe current standard of care, the prognosis of PEL is extreme-ly poor with a short median survival time of 3 to 6 monthsupon diagnosis (2). Thus, there is an urgent need for saferand more effective therapeutic options for PEL.

NF-kB is a critical transcription factor that controls theexpression of several genes involved in the regulation ofcellular survival, proliferation, and inflammatory response(3). TheNF-kB pathway is constitutively active in amajority

of PEL cells and is believed to be essential for their survivaland proliferation (4–7). The constitutive activity of NF-kBpathway in the PEL cells is primarily due to the activity ofKSHV-encoded viral FLICE-inhibitory protein (vFLIP) K13(5–8). K13 activates the NF-kB pathway by activating amulti-subunit IkappaB kinase (IKK) complex that consistsof 2 catalytic subunits, IKK1/IKKa and IKK2/IKKb, and aregulatory subunit,NEMO/IKKg (5, 8). K13, however, is nota kinase that activates the IKK complex by inducing itsphosphorylation. Instead, K13 activates the complex bydirect interaction via a mechanism believed to involve aconformational change of the IKK complex (9).

HSP90 is an abundant molecular chaperone that func-tions in the proper folding, assembly, and transportation ofa wide range of client proteins (10). Inhibition of HSP90function causes many oncogenic client proteins to adoptaberrant conformations followed by their ubiquitylationand proteasomal degradation. HSP90, along with CDC37,was also shown to be an essential component of the IKKcomplex (11). Treatment with geldanamycin, an HSP90inhibitor, was shown to block TNFa-induced activation ofIKK and NF-kB (11). A subsequent study revealed thatHSP90 is also a component of the K13/IKK complex andK13-induced NF-kB activity could be blocked by geldana-mycin (12).

Most of the currently available HSP90 inhibitorsbelong to 1 of the following 3 categories: (i) geldanamycin

Authors' Affiliation: Jane Anne Nohl Division of Hematology and Centerfor the Study of Blood Diseases, University of Southern California KeckSchool of Medicine, Los Angeles, California

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Preet M. Chaudhary, Jane Anne Nohl Division ofHematology and Center for the Study of Blood Diseases, University ofSouthern California Keck School of Medicine, Los Angeles, CA 90033.Phone: 323-865-3916; Fax: 323-865-0060; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-12-3510

�2013 American Association for Cancer Research.

ClinicalCancer

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derivatives (e.g., 17-AAG and 17-DMAG), (ii) purine scaf-fold–based HSP90 inhibitors (e.g., BIIB021 and PU-H71),and (iii) resorcinol derivatives (e.g., NVP-AUY922 and KW-2478). Although geldanamycin-derivative 17-AAG was thefirst to enter clinical trials, its development has been limitedby hepatotoxicity and limited solubility and stability (13,14). Subsequently, HSP90 inhibitors using purine as ascaffold were designed that lacked the above limitations ofgeldanamycin derivatives, particularly hepatotoxicity (15).In this study, we conducted a comparative analysis of the 3main classes ofHSP90 inhibitors against PEL cells.We showthat while all 3 classes of HSP90 inhibitors are effectiveagainst PEL, the purine scaffold inhibitor BIIB021 showspreferential cytotoxicity toward PEL as compared with theother lymphoma cells.We further show that the cytotoxicityof BIIB021 against PEL is associated with inhibition ofclassical and alternative NF-kB pathways induced by KSHVvFLIP K13.

Materials and MethodsLentivirus constructsTo construct a lentiviral vector expressing K13 from a

tetracycline-inducible promoter, K13 cDNA with a C-ter-minal FLAGepitopewas initially cloned in amodifiedpENTentry vector containing a tetracycline-inducible promoter(TO). Recombination-based subcloning was used to trans-fer the TO-K13-FLAG cassettes into pSLIK destination vector(16). A lentivirus construct expressing an NF-kB–drivenluciferase reporter construct was generated in pLENTI6/V5-based vector (Invitrogen). Recombinant lentiviruseswere generated in the HEK293-FT cells. Postinfection, cellswere cultured in normal growth media containing theappropriate drugs to select positive clones.

Cell viability, cell-cycle, and apoptosis analysisCells from exponentially growing cultures were plated

in untreated flat-bottom 96-well plates at a density of10 � 103 cells per well, treated with an increasing con-centration of the drugs and subsequently assessed for cellviability using the MTS reagent following the manufac-turer’s instructions (Promega). Percent cell survival was

calculated on the basis of the reading of cells grown in thepresence of dimethyl sulfoxide (DMSO) control. IC50 forthe individual drugs were calculated using GraphPadPrism 5 software. DNA content analysis was conductedas described previously (17). Apoptosis was analyzedusing BD AnnexinV-FITC apoptosis detection kit I as permanufacturer’s instructions.

Statistical analysisTwo-tailed unpaired Student t test was used to test for

differences between 2 groups using GraphPad Prism 5software. Differences with a P � 0.05 were consideredstatistically significant. All experiments were repeated aminimum of 3 times.

Additional information about materials and methods isprovided in the Supplementary Information.

ResultsBIIB021 specifically targets KSHV-associated PEL

We treated a panel of logarithmically growing PEL andnon-PEL cells lines for 72 hours with increasing concentra-tions of HSP90 inhibitors BIIB021, 17-DMAG, and NVP-AUY922, respectively (Fig. 1A). While 17-DMAG and NVP-AUY922 inhibited the growth of both PEL and non-PEL celllines equivalently, BIIB021 showed preferential cytotoxicitytoward the PEL cell lines (Fig. 1B). Thus, the IC50 values ofBIIB021 for the PEL cell lines ranged from 41.5 to 71.5nmol/L, whereas its IC50 for non-PEL cell lines ranged from187 to 275 nmol/L (Table 1). In contrast, the IC50 of 17-DMAG forPEL andnon-PEL cell lines ranged from55 to217nmol/L and from 24.2 to 1655 nmol/L, respectively. Sim-ilarly, the IC50 of NVP-AUY922 for PEL and non-PEL celllines ranged from 19.3 to 60.5 nmol/L and from 16.1 to40.5 nmol/L, respectively. The preferential toxicity ofBIIB021 against PEL cells was also seen at 24 and 48 hoursof drug treatment (Fig. 1C).

BIIB021 induces cell-cycle arrest in PELWe next examined the effect of BIIB021 on cell-cycle

progression. Treatment of BC-1 with BIIB021 resulted inG1 arrest as observed by amarked increase in the number ofcells in the G1 phase and concomitant decreases in cells inthe S-phase (Fig. 2A). In contrast, treatment of BC-3 cellswith BIIB021 resulted in cell-cycle arrest in G2–M phaseas observed by an increase in the percentage of cells in theG2–M phase (Fig. 2A). BIIB021 also significantly increa-sed the proportion of cells with reduced DNA content (sub-G0/G1) suggestive of apoptosis (Fig. 2A). BIIB021-inducedcell-cycle arrest was accompanied by reduced expression ofseveral HSP90 clients critical for cell-cycle progression,including CDK2 (cyclin-dependent kinase 2), CDK4,CDK6, and CDK9 (Fig. 2A). In addition, BIIB021 depletedthe levels of c-MYC, cyclin A, and cyclin B1 (Fig. 2A). Incontrast, BIIB021 upregulated the expression of CDK inhib-itor p21 (Fig. 2A), which is a negative regulator of cell-cycleprogression. Finally, BIIB021 decreased the expression ofHSP90 clients AKT, GSK3b, and survivin (Fig. 2A), whichhave been implicated in the pathogenesis of PEL (18–20).

Translational RelevancePrimary effusion lymphoma (PEL) is an aggressive

form of non–Hodgkin lymphoma commonly seen inHIV-positive patients that is universally associated withinfection by Kaposi sarcoma–associated herpes virus.PEL has extremely poor prognosis when treated withconventional chemotherapy, and novel therapeuticstrategies are urgently needed for the treatment of thisdisorder. The work presented in this article shows thatHSP90 inhibitor BIIB021 blocks constitutive NF-kBactivity present in PEL cells and also shows in vitro andin vivo activity against this disease. These results providethe basis for clinical testing of BIIB021 in PEL.

BIIB021 Specifically Targets PEL

www.aacrjournals.org Clin Cancer Res; 19(18) September 15, 2013 5017




BIIB021 induces apoptosis in PEL with the cleavage ofPARP, caspases-9, -7, and -3

We next examined the effect of BIIB021 on the inductionof apoptosis upon staining with nuclear dye Hoechst33258. Treatment of BC-1 and BC-3 cells with BIIB021resulted in appearance of cells with condensed and frag-mented nuclei suggestive of apoptosis, which was con-firmed by staining with AnnexinV/propidium iodide (Fig.2B). Induction of apoptosis by BIIB021 involved activation

of caspases-9, -7, and -3 and cleavage of their downstreamsubstrate, PARP (Fig. 2C). However, BIIB021 had no sig-nificant impact on the levels of expression of BCL-2, BCL-XL, and MCL-1 and on the expression or cleavage of BID(Fig. 2C). Similarly, BIIB021 had no significant effect on thelevel of p53 (Fig. 2C).

To provide further evidence of the preferential toxicityof BIIB021 against PEL cells, we analyzed the cleavage ofPARP (as a measure of caspase activation) in a panel of

Figure 1. HSP90 inhibitorsefficiently target KSHV-associatedPEL. A, chemical structures ofBIIB021, 17-DMAG, and NVP-AUY922. B, indicated PEL andnon-PEL cell lines were treatedwith increasing concentrations ofBIIB021, 17-DMAG, and NVP-AUY922 for 72 hours, and cellviability was measured using anMTS assay as described inMaterials and Methods. An ovalshapedmarking shows preferentialtoxicity of BIIB021 on PEL cell linesat 100 nmol/L. C, BC-1, BC-3,BJAB, and Namalwa cells weretreated with increasingconcentrations of BIIB021 for 24and 48 hours, and cell viability wasmeasured by MTS assay. Thevalues shown aremean�SEMof 2independent experiments carriedout in triplicate.

Table 1. List of cell lines, the associated viruses, diseases, and IC50 doses of HSP90 inhibitors for 72 hours

Cell line DiseaseAssociatedvirus

p53status

BIIB021(IC50, nmol/L)

17-DMAG(IC50, nmol/L)

NVP-AUY922(IC50, nmol/L)

BC-1 PEL KSHV and EBV WT 41.5 55 30BC-3 PEL KSHV WT 62.6 74.8 28.1BCBL-1 PEL KSHV Mut 53.6 217 60.5JSC-1 PEL KSHV and EBV WT 71.5 63.8 40.6UMPEL-1(c) PEL KSHV and EBV N/A 59.6 92.1 19.3BJAB Burkitt lymphoma None Mut 187 33.3 16.1Namalwa Burkitt lymphoma EBV Mut 275 1655 31.2Jurkat T-cell acute lymphoblastic

leukemiaNone Mut 214 410.5 40.5

MM1S Multiple myeloma None WT 203 41.8 24U266 Multiple myeloma None Mut 238 24.2 25.8

Abbreviations: EBV, Epstein–Barr virus; Mut, mutated; N/A, not available; WT, wild-type.

Gopalakrishnan et al.

Clin Cancer Res; 19(18) September 15, 2013 Clinical Cancer Research5018




PEL (BC-3. BCBL-1, and JSC-1) and non-PEL (Jurkat,BJAB, Namalwa, MM1S, and U266) cell lines followingtreatment with BIIB021 (100 nmol/L), 17-DMAG (100nmol/L), and NVP-AUY922 (50 nmol/L) for 48 hours.Consistent with the results of cell viability assay, BIIB021resulted in increased PARP cleavage in all the 3 PELcell lines but in none of the non-PEL cell lines tested

(Fig. 2D). In contrast, NVP-AUY922 increased PARPcleavage in all cell lines, whereas 17-DMAG increasedPARP cleavage in all PEL cell lines and 3 non-PEL celllines (BJAB, MM1S, and U266). Thus, while PEL cells aresensitive to all HSP90 inhibitors, they show preferentialsensitivity to BIIB021 at concentration that is not cyto-toxic to non-PEL cell lines.

Figure 2. BIIB021 induces cell-cycle arrest and apoptosis in PEL cells. A, left, cell-cycle analysis of control and BIIB021-treated BC-1 and BC-3 celllines. BIIB021 (200 nmol/L for 48 hours) results in G1 arrest in BC-1 cells and G2–M arrest in BC-3 cells. Cells were stained with propidium iodide (PI) andanalyzed by flow cytometry. Data are a representative of 3 individual experiments. Right, Western blot analysis showing the effect of BIIB021 on theexpression ofHSP90 client proteins involved in cell-cycle regulation. Lack of effect onCOX-2, a protein that is not anHSP90 client, shows the specificity of theobserved effect. B, left, BC-1 and BC-3 cells were treated with 200 nmol/L BIIB021 or DMSO control for 48 hours. Cells were then stained withHoechst 33258 and photographed. Right, BC-1 and BC-3 cells were treated with 200 nmol/L BIIB021 or DMSO control for 48 hours, stained withAnnexinV-FITC/PI, and analyzed for apoptosis by flow cytometry. C, Western blotting showing the effect of BIIB021 on the cleavage of PARP, activation ofcaspases, andexpressionofBCL-2 familymembers andp53.D,Western blotting showing theeffect ofBIIB021 (100nmol/L, 48hours), 17-DMAG (100nmol/L,48 hours), and NVP-AUY922 (50 nmol/L, 48 hours) treatment on the cleavage of PARP in the indicated cell lines. Cl, cleaved; FL, full length.






Inhibition of NF-kB pathway in PEL by BIIB021NF-kB pathway has been shown to be essential for the

survival of PEL cells. To check whether BIIB021 inhibits theNF-kB pathway in PEL cells, we took advantage of BC-1 andBC-3 cells engineered to express a stably integrated copy ofanNF-kB–driven luciferase reporter construct (NF-kB-Luc).BIIB021 decreased the NF-kB-Luc activity in a dose-depen-dent manner (Fig. 3A), which was accompanied by a reduc-tion in the level of nuclearNF-kBasmeasuredby ap65/RelADNA-binding assay (Fig. 3A). Interleukin 6 (IL-6) is an NF-kB target gene and a known growth factor for PEL cells (21,22). Consistent with its inhibition of the NF-kB pathway,BIIB021 resulted in reduced secretionof IL-6 secretion in thesupernatants of BC-1 and BC-3 cells (Fig. 3B). Finally,

BIIB021 blocked the expression of A20 and XIAP, 2 knowntargets of the NF-kB pathway (refs. 23, 24; Fig. 3C).

To study the mechanism of classical NF-kB inhibition byBIIB021, we studied its effect on IkBa phosphorylation.Treatment with BIIB021 reduced the phosphorylation ofIkBa on conserved Ser32/36 residues in both BC-1 and BC-3 cells as measured by Western blot analysis (Fig. 3C).BIIB021 resulted in a dose-dependent reduction in theexpression of IKKa/b but had no significant effect on theexpression of NEMO/IKKg (Fig. 3C). Furthermore, BIIB021blocked the phosphorylation of IKKa and IKKb on Ser176/Ser180 and Ser177/Ser181, respectively (Fig. 3D). Collec-tively, the above results suggest that BIIB021 blocks classicalNF-kB pathway in PEL by blocking the expression and

Figure 3. Inhibition of NF-kBpathwaybyBIIB021 in PEL cells. A, left, BC-1/NF-kB-Luc andBC-3/NF-kB-Luc cells were treatedwith increasing concentrationsof BIIB021or vehicle control for 15 hours and cell lysates used formeasurement of luciferase activity. Values shownare themean�SEM from1 representativeexperiment of 3 carried out in duplicate. Asterisks (��) and (��) indicate significance at levels of P � 0.001 and P � 0.0001, respectively. Right, anELISA-based NF-kB binding assay showing reduced p65/RelA DNA-binding activity in the nuclear extracts of BC-1 and BC-3 cells treated with BIIB021(200 nmol/L for 48 hours). Values shown are the mean � SEM from 1 representative experiment of 3 carried out in duplicate. Asterisks (��) indicatesignificance at levels of P � 0.0001. B, BIIB021 blocks interleukin (IL)-6 secretion. IL-6 levels were measured using ELISA in the supernatants of BC-1 andBC-3 cells treated with 200 nmol/L of BIIB021 or DMSO control for 24, 48, and 72 hours. The values (mean � SEM) shown are from a representativeof 3 independent experiments carried out in triplicate. C, Western blot analyses showing the effect of BIIB021 on the expression of phospho-IkBa and totalIkBa, IKKa/b and NEMO, and NF-kB target proteins A20 and XIAP1. D, PathScan ELISA for phospho-IKKa (Ser176/180) and phospho-IKKb (Ser177/181)showing inhibition of IKKa/b phosphorylation by BIIB021 (200 nmol/L for 48 hours) in BC-1 and BC-3 cells. A representative of 2 independent experimentscarried out in triplicate. Statistically significant differences are shown by asterisks (��) at a level of P � 0.001 and (��) at level of P � 0.0001.






activity of IKKa/b, which in turn results in inhibition ofIkBa phosphorylation and degradation.

BIIB021 blocks vFLIP K13 expressionTo examine the role of vFLIP K13 in the inhibitory effect

of BIIB021 on NF-kB pathway, we examined its expression

in BIIB021-treated cells. We observed a reduction in K13protein level upon BIIB021 treatment (Fig. 4A). However,reduction in K13 was evident only at the highest dose (200nmol/L) of BIIB021 andwas not seen at the lower doses (50and 100 nmol/L). BIIB021 also downregulated the expres-sions of LANA and vCyclin (Fig. 4A), 2 other KSHV latent

Figure 4. BIIB021 downregulatesvFLIP K13 expression and blocksK13-induced NF-kB activation. A,left, Western blot analyses showinga reduction in K13, LANA, andvCyclin protein levels in BC-1 andBC-3 cells treated for 48 hours withthe indicated concentrations ofBIIB021. Right, qRT-PCR analysisshowing a decline in K13, LANA,and vCyclin mRNA expression inBC-1 and BC-3 cells followingtreatment with 200 nmol/L BIIB021for 24 hours. Real-time PCRreactions were carried out intriplicate and the data are presentedas fold change in target geneexpression (mean � SEM) from arepresentative of 2 independentexperiments. Statistically significantdifferences are shown by asterisks(��) at a level of p�0.01, (��) at alevel of p�0.001 and (��) at levelof p� 0.0001. B, effect of BIIB021on the protein stability. BC-1 andBC-3 cells were treated with vehicleor BIIB021 in the presence of5 mg/mL cycloheximide (CHX)for 0, 3, 6, 12, 18, and 24 hours,respectively. Whole-cell lysateswere immunoblotted for indicatedproteins. Semiquantitative analysisof the immunoblots is presented inSupplementary Fig. S1. C, top,luciferase-based reporter assayshowing inhibition of K13-inducedNF-kB transcriptional activity byHSP90 inhibitors. 293-pSLIK-TO-K13/NF-kB-Luc cells were treatedwith increasing concentrations ofBIIB021, 17-DMAG, and NVP-AUY922 for 2 hours beforeinduction of K13 expression byaddition of doxycycline (DOX, 500ng/mL). After 15 hours, cell lysateswere prepared to measure theNF-kB luciferase activity. Thevalues (mean � SEM) shown arefrom a representative of 3independent experiments carriedout in triplicate. Statisticallysignificant differences are shown byasterisks (��) at level ofp� 0.0001.ns, not significant. Bottom,Westernblot analysis from the cell lysatesconfirmed that inhibition ofNF-kB activity by BIIB021 isaccompanied by inhibitionof IkBa phosphorylation anddownregulation of NF-kB targetgene (i.e., A20) expression but is notdue to a block in K13 expression.






proteins (25). A real-time quantitative reverse transcript-polymerase chain reaction (qRT-PCR) analysis revealed thatBIIB021 led to robust suppression of K13 and vCyclin andmodest suppression of LANA at the mRNA levels (Fig. 4A).HSP90 inhibitors are known to promote degradation ofHSP90 client proteins (26). As we observed a much greaterreduction in the levels of LANA, and to a lesser extent K13and vCyclin, at the protein level as compared with themRNA level, we examined the effect of BIIB021 on thestability of these proteins. For this purpose, we examinedthe effect of BIIB021 on the expression of K13, LANA, andvCyclin in BC-1 and BC-3 cells after blocking proteinsynthesis with cycloheximide (CHX). We used AKT, aknown HSP90 client protein, as a positive control andCOX-2 as a negative control (27). BIIB021 reduced thehalf-life of LANA by several hours in cycloheximide-treatedBC-1 and BC-3 cell lines (Fig. 4B and Supplementary Fig.S1), confirming the results of a recent study showing thatLANA is an HSP90 client protein (26). However, BIIB021did not have any significant effect on the half-lives of K13and vCyclin (Fig. 4B and Supplementary Fig. S1), indicatingthat these proteins are not clients of HSP90 (Fig. 4B).Finally, BIIB021 reduced the half-life of AKT but did notsignificantly affect COX-2 and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) stability.

BIIB021 blocks vFLIP K13 induced NF-kB activityAs the inhibition of K13 expression by BIIB021 was

evident only at its highest dose, we next asked whetherinhibition of K13 activity could also contribute to its inhib-itory effect on the NF-kB pathway. For this purpose, wegenerated a clone of 293 cells, designated 293A-pSLIK-TO-K13/NF-kB-Luc, stably expressing FLAG-tagged K13 from atetracycline-inducible promoter along with a stably inte-grated copy of an NF-kB–driven luciferase reporter con-struct. As K13 is being expressed from an ectopic promoter,this cell line afforded us the opportunity to examine theeffect of BIIB021 on K13 activity independent of its effecton K13 expression. As shown in Fig. 4C, induction ofK13 expression with doxycycline resulted in a significantincrease in NF-kB-Luc activity that was accompanied by anincrease in phosphorylation and degradation of IkBa andupregulation of A20 expression. However, all the aboveeffects were effectively blocked by BIIB021 in a dose-depen-dent manner (Fig. 4C). A Western blot analysis confirmedthat inhibition of NF-kB activity by BIIB021 is not due to ablock in K13 expression (Fig. 4C). Essentially similar resultswere obtained upon treatment with 17-DMAG and NVP-AUY922 (Fig. 4C).

HSP90 inhibitors disrupt the interaction between K13and the IKK complex

To understand the mechanism by which HSP90 inhi-bitors block K13-induced NF-kB, we examined their effecton K13/IKK complex interaction in 293-pSLIK-TO-K13/NF-kB-Luc cells using a co-immunoprecipitation (co-IP)assay. BIIB021 reduced the amounts of HSP90, IKKa/b,and IKKg/NEMO that co-immunoprecipitated with K13,

suggesting that HSP90 function is essential for the struc-tural integrity of the K13/IKK complex (Fig. 5A). Essen-tially similar results were obtained upon treatment with17-DMAG (Fig. 5A). Finally, treatment with NVP-AUY922resulted in a near-complete disappearance of HSP90,IKKa/b, and IKKg/NEMO bands in the co-IP samples,although a reduction in the expression of these proteinsprobably also contributed to this effect (Fig. 5A). Simi-larly, we observed a reduction in the levels of IKKa/b andIKKg/NEMO that co-immunoprecipitated with K13 in theBIIB021-treated BCBL-1 cells expressing FLAG-tagged K13cells as compared with the control-treated cells (Fig. 5A).Thus, HSP90 activity is required for the assembly of theK13/IKK complex and BIIB021 blocks K13-induced NF-kB activity by disrupting this complex.

K13 interacts with HSP90 via NEMONEMO is essential for the interaction of K13 with the IKK

complex (5, 12). To check whether NEMO is required forthe interaction of K13 with HSP90, we used FLAG-taggedK13-expressing wild-type and NEMO-deficient Jurkat cells.While we readily detected an interaction between K13 andHSP90 in the wild-type Jurkat cells using the co-IP assay, wefailed to observe this interaction in the NEMO-deficientcells (Fig. 5B). Thus,NEMO is essential for the interaction ofK13 with HSP90.

BIIB021 blocks alternate NF-kB pathway in PELPEL cells also show constitutively active alternate NF-kB

pathway that involves proteasome-mediated processingof p100/NF-kB2 into the active p52 subunit (28). Treat-ment of BC-3 cells with 50 nmol/L BIIB021 blocked theprocessing of p100 into p52, suggesting inhibition of thealternate NF-kB pathway (Fig. 5C). The expression of bothp100/NF-kB2 and RELB subunits of the alternative NF-kBpathway is under the transcriptional control of the classicalNF-kB pathway (29, 30). Consistent with above, treatmentwith higher doses of BIIB021 (100 and 200 nmol/L)resulted in reduced expression of both p100 and RELB (Fig.5C). Thus, BIIB021 blocks alternate NF-kB pathway in PELdirectly by downregulating IKK1/IKKa expression and indi-rectly by blocking the classical NF-kB pathway.

BIIB021 does not induce expression of KSHV lyticgenes

K13 is required for maintenance of KSHV latency andinhibition of K13-induced NF-kB results in KSHV lyticreactivation through the induction of expression of KSHVreplication and transcription activator (RTA), the masterregulator of KSHV lytic replication (31, 32). BIIB021 notonly failed to induce the expression of RTA in BCBL-1 cellsbut also blocked TPA-induced RTA expression in a dose-dependentmanner (Fig. 5D). KSHV-encoded viral IL6 (vIL6)not only is an autocrine growth factor for KSHV-infected PELcells but also contributes to immune evasion and angiogen-esis (33). Similar to its effect on RTA, BIIB021 not onlyfailed to induce vIL6 expression in BCBL-1 cells but alsoblocked vIL6 expression induced by treatment with TPA.






Essentially similar results were observed in BC-1 and BC-3cell lines (Supplementary Fig. S2). The effect of BIIB021 onthe transcripts of other KSHV genes is presented in Supple-mentary Fig. S3. Thus, the cytotoxicity of BIIB021 against PELis not accompanied by lytic reactivation of KSHV.

BIIB021 exhibits in vivo growth inhibitory potentialagainst PEL in a mouse xenograft modelTo check whether the antiproliferative effect of BIIB021

observed in vitro can be translated in vivo, we injected the

BC-1 cells subcutaneously in to the right flank of athymicNCr-nu/numice. The mice were monitored daily for devel-opment of palpable tumors and treatment with BIIB021 (orcontrol vehicle) was initiated when the tumors reachedapproximately 75 mm3. BIIB021 resulted in a significantreduction in tumor volume (Fig. 6A), which was accompa-nied by a corresponding decline in the levels of circulatinghuman IL-6 (Fig. 6A). BIIB021 also prevented the develop-ment of splenomegaly, which was seen in 6 of 6 vehicle-treated mice but in only 2 of 7 BIIB021-treated mice,

Figure 5. Effect of HSP90 inhibitors on K13/IKK complex interaction and KSHV lytic proteins. A, left, co-IP assay showing disruption of K13/IKKcomplex interaction byHSP90 inhibitors. 293-pSLIK-TO-K13/NF-kB-Luc cells were treatedwithBIIB021 (200 nmol/L), 17-DMAG (200 nmol/L), NVP-AUY922(100 nmol/L), and DMSO control for 2 hours followed by treatment with doxycycline (DOX, 500 ng/mL for 15 hours) to induce K13 expression.Whole-cell lysates (WCL)were immunoprecipitated (IP) using a control antibody (C) or FLAGantibody (F) and subjected toSDS-PAGEand immunoblotting (IB)with the indicated antibodies. UT, Untreated. Right, a co-IP assay showing reduced interaction between K13 and IKK complex in FLAG-tagged K13expressingBCBL-1cells upon treatmentwith 200nmol/LBIIB021 for 24hours. B, a co-IPassay showingK13 interactswithHSP90 inwild-typeJurkat cells butnot in NEMO-deficient Jurkat cells. WCLs were prepared from wild-type Jurkat cells and NEMO-deficient Jurkat cells expressing FLAG-K13 andimmunoprecipitated (IP) using a control antibody (C) or FLAG antibody (F) and immunoblotted (IB) with the indicated antibodies. C, Western blot analysesshowing downregulation of RELB and p100 expression and p100 to p52 processing in PEL cells upon treatment with BIIB021 for 48 hours. D, BIIB021fails to induce expression of KSHV lytic proteins RTA and vIL6 and blocks their expression induced by TPA (12-O-tetradecanoylphorbol-13-acetate).BCBL-1 cellswere treatedwith the indicateddosesofBIIB021 for 2 hours followedby treatmentwith TPA (100 ng/mL) for 48 hours.Cell lysateswere subjectedto Western blot analysis using the indicated antibodies. NS, nonspecific.






respectively (Fig. 6B). However, we failed to detect LANA-positive cells in the spleens of vehicle or BIIB021-treatedmice, suggesting that the observed splenomegaly may bedue to the stimulatory effect of cytokines produced by PELcells on splenic cells rather than direct infiltration of spleenby PEL cells. Tumor tissues from the animals treated withBIIB021 also exhibited reduced expression of IKKa/b andLANA (Fig. 6C) and increased apoptosis, as assessed by thestaining for cleaved caspase-3 and the number of TUNEL-positive nuclei (Fig. 6C and D). Taken collectively, theabove results indicate that BIIB021 exerts a potent in vivoinhibitory effect on PEL by inducing tumor cell apoptosis.

DiscussionA number of preclinical studies have documented the

activity of geldanamycin-based HSP90 inhibitors, such as17-AAG and 17-DMAG, against PEL cells (12, 34). Howev-er, several issues intrinsic to the chemical structure ofgeldanamycin-based HSP90 inhibitors have limited theirfull clinical development (35). 17-AAG contains a benzo-quinone moiety that is believed to be responsible forelevation of liver enzymes and liver toxicity observed inclinical trials (35, 36). Intrinsic and acquired resistance to17-AAG has been associated with low expression or aninactivating polymorphism of the NQO1 (NAD(P)H/qui-noneoxidoreductase I) gene that encodes for an enzymerequired for bioreduction of 17-AAG to a more potenthydroquinone (37). 17-AAG and 17-DMAG are also sub-

strates of P-glycoprotein/multidrug resistance protein 1(MDR1) and multidrug resistance–associated protein 1(MRP-1), and their cytotoxicity is dramatically reduced inMDR1 and/or MRP-1–overexpressing cells (14, 38). Over-expression of BCL-2 has been also linked to resistance to 17-AAG (38). Finally, for unclear reasons, 17-AAG is also lesspotent in several types of tumor sensitive to other HSP90inhibitors (14).

Because of the above limitations of the geldanamycin-based HSP90 inhibitors, we compared the 3main classes ofHSP90 inhibitors currently under development. We showthat while all 3 classes of HSP90 inhibitors have activityagainst PEL, BIIB021, a novel purine-based HSP90 inhib-itor, is preferentially toxic to PEL as compared with otherlymphoma subtypes. BIIB021 holds several additionaladvantages over geldanamycin-based HSP90 inhibitors.BIIB021 has improved pharmacologic profile as comparedwith 17-AAG especially with regard to availability throughchemical synthesis, metabolic stability, water solubility,and ease of administration via both oral and intravenousroutes (14, 15). BIIB021 does not require activation byNQO1 and is not a substrate of P-glycoprotein and MRP-1 drug efflux pumps (38). Indeed, no case of acquiredresistance to BIIB021 has been reported in the literature sofar and it has shown activity against a wider panel of tumors(37). BIIB021 has also been tested in a number of phase Iclinical trials and at least 2 phase II clinical trials, where itappears to be well-tolerated (10).

Figure 6. BIIB021 impairs in vivogrowth of PEL in amouse xenograftmodel. A, athymic NCr-nu/nu micewith established subcutaneous BC-1 tumors were treated with BIIB021(100 mg/kg body weight;n¼ 7)or vehicle control (n¼6) for 14days. Tumor volumes are presentedas mean � SEM from each group.Asterisks indicate significance atlevels of P � 0.05. Tumor weight (ingrams)of animals treatedwith eithervehicle control or BIIB021 on day14. Asterisks indicate significanceat levels of P � 0.05. Circulatinglevels of hIL-6 on day 14 in theplasma of animals treated witheither vehicle control or BIIB021.Asterisks indicate significance atlevels of P � 0.05. B, grossrepresentative images of mice,tumor, and spleen from groups ofanimals treated with either vehiclecontrol or BIIB021. C,immunohistochemical stainingshowing decreased expressions ofIKKa/b, LANA along with positivestaining for cleaved caspase-3,indicative of apoptosis, in thetumors of mice treated withBIIB021. D, TUNEL stainingshowing increased apoptosis intumor tissues extracted fromanimals treated with BIIB021. ��,significance at levels of P � 0.001.






Treatment of PEL cell lines with BIIB021 resulted in cell-cycle arrest and induction of apoptosis. As NF-kB pathwayhas been shown to be essential for the survival and prolif-eration of PEL cells (4, 6), we examined the effect of BIIB021on the status of this pathway in detail. Our results suggestthat BIIB021 blocks both the classical and alternate NF-kBpathways through multiple mechanisms. First, BIIB021reduced the levels of IKKa/IKKb, which is consistent witha previous report indicating that these are HSP90 clientproteins (39). Second, BIIB021 at higher doses resulted in amodest reduction in the mRNA and protein levels of vFLIPK13, a protein that is believed to be primarily responsiblefor activation of the classical and alternative NF-kB path-ways in PEL cells (6, 7). Most importantly, BIIB021 blockedthe activity of K13, which was associated with a disruptionof the K13-IKK complex. It is important to point out that theinhibition of K13 activity by BIIB021was independent of itseffect on K13 expression as it was also observed in the 293-pSLIK-K13 cell line in which K13 expression was unaffectedby the BIIB021 treatment. AsHSP90 is a known componentof the K13/IKK complex (12), the inhibitory effect ofBIIB021 on K13-induced NF-kB activity probably reflectsthe key role played by HSP90 in the assembly and functionof this complex. Taken collectively, our results suggest thatBIIB021 blocks NF-kB activity in PEL by downregulatingthe expression of IKKa/IKKb and K13 and by interferingwith the assembly and function of the K13/IKK complex. Itis important to point out, however, that inhibition of theNF-kB pathway is unlikely to be the sole mechanism ofaction of BIIB021 against PEL as several other cellularand viral proteins are dependent on the chaperone functionof HSP90. For example, HSP90 is known to enhance AKTsignaling (40) and, consistent with these results, weobserved downregulation of AKT expression in BIIB021-treated cells. HSP90 has been also shown to be required forthe expression of KSHV-encoded K1 protein and for itsapoptotic activity (34). Thus, we favor the hypothesis thatthe inhibition ofNF-kB pathwayworks in conjunctionwiththe inhibition of other signaling pathways to contribute tothe antiproliferative and cytotoxic effects of BIIB021 againstPEL.The NF-kB pathway has been shown to keep KSHV in a

latent state (41). As lytic replication of KSHV and expressionof lytic genes is believed to promote KSHV tumorigenesis(42), lytic reactivation represents a potential safety concernwith the use of HSP90 inhibitors. However, we observed

that treatment with BIIB021 not only failed to induce theexpression of lytic genes but also blocked their expressioninduced by TPA, thereby allaying these safety concerns.Essentially similar results have been reported recently using17-AAG (43).While the exactmechanism for the inhibitionof lytic reactivation by HSP90 inhibitors is not clear at thepresent, modulation of NF-kB–independent signalingpathways may be responsible for this effect.

In summary, we provide strong in vitro and in vivo datashowing the promising activity of BIIB021 against PEL.The mean Cmax for BIIB021 observed in clinical trial is3.6 mmol/L (44) which is 60-fold greater than the IC50

values of this compound for PEL cell lines observed in thepresent study. As PEL are relatively refractory to conventionalchemotherapy, the observed preclinical activity of BIIB021against PEL cells at lownanomolar doses suggests that itmaybe a promising compound for the treatment of PEL.

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

Authors' ContributionsConception and design: R. Gopalakrishnan, H. Matta, P.M. ChaudharyDevelopment of methodology: R. Gopalakrishnan, H. Matta, P.M.ChaudharyAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): R. Gopalakrishnan, H. Matta, P.M. ChaudharyAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): R. Gopalakrishnan, H. Matta, P.M.ChaudharyWriting, review, and/or revision of the manuscript: R. Gopalakrishnan,H. Matta, P.M. ChaudharyAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R. Gopalakrishnan, H. MattaStudy supervision: P.M. Chaudhary

AcknowledgmentsThe authors thank Dr. Jae Jung for his generous gift of PEL cell lines, Dr.

Izidore S. Lossos for UMPEL-1 cells, and Dr. Ellen Vitetta for myeloma celllines.

Grant SupportThis workwas supported by grants from theNIH (CA139119, DE019811,

SC CTSI UL1TR000130 and P30CA014089) and Stop Cancer Foundation.Flow cytometry was conducted in the USC Flow Cytometry Core Facility thatis supported in part by the National Cancer Institute Cancer Center SharedGrant award P30CA014089 and the USC Provost Office Dean’s Develop-ment Funds.

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 November 14, 2012; revised June 28, 2013; accepted July 17,2013; published OnlineFirst July 23, 2013.

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2013;19:5016-5026. Published OnlineFirst July 23, 2013.Clin Cancer Res Ramakrishnan Gopalakrishnan, Hittu Matta and Preet M. Chaudhary

BκvFLIP K13-Induced NF-against KSHV-Associated Primary Effusion Lymphoma and Blocks A Purine Scaffold HSP90 Inhibitor BIIB021 Has Selective Activity

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A Purine Scaffold HSP90 Inhibitor BIIB021 Has Selective ... · Lymphoma and Blocks vFLIP K13-Induced NF-kB Ramakrishnan Gopalakrishnan, Hittu Matta, and Preet M. Chaudhary Abstract