Targeting the Hsp90-associated viral oncoproteome in

doi:10.1182/blood-2013-01-479972Prepublished online August 13, 2013;

Chiosis, Y. Lynn Wang and Ethel CesarmanHediye Erdjument-Bromage, Max Chomet, Ronald Blasberg, Ari Melnick, Leandro Cerchietti, Gabriela Utthara Nayar, Pin Lu, Rebecca L. Goldstein, Jelena Vider, Gianna Ballon, Anna Rodina, Tony Taldone, gammaherpesvirus-associated malignanciesTargeting the Hsp90-associated viral oncoproteome in

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1

Targeting the Hsp90-associated viral oncoproteome

in gammaherpesvirus-associated malignancies

Utthara Nayar1, Pin Lu1, Rebecca L. Goldstein2, Jelena Vider3*, Gianna Ballon1, Anna Rodina4, Tony Taldone 4, Hediye Erdjument-Bromage5, Max Chomet1, Ronald Blasberg3,6, Ari Melnick2, Leandro Cerchietti2, Gabriela Chiosis4, Y. Lynn Wang1, Ethel Cesarman1

1Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA 2Department of Medicine, Weill Cornell Medical College, New York, NY, USA 3Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 4Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 5Molecular Biology, Memorial Sloan-Kettering Cancer Center 6Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

*Present affiliation for JV: Host Response to Cancer Laboratory, School of Medical Science, Griffith University, Parklands Dr, Gold Coast, QLD 4215, Australia Corresponding authors: Ethel Cesarman, MD, PhD Department of Pathology and Laboratory Medicine Weill Cornell Medical College 1300 York Avenue, Room C410 New York, NY 10065 Phone: (212)746-8838; Fax: (212)746-4483 e-mail: [email protected] Y. Lynn Wang, MD, PhD Department of Pathology and Laboratory Medicine Weill Cornell Medical College 1300 York Ave, New York, NY 10065 Phone: (212)746-6402; Fax: (212)746-8345 e-mail: [email protected] Running title: Targeting KSHV oncoproteins by HSP90 inhibition

Blood First Edition Paper, prepublished online August 13, 2013; DOI 10.1182/blood-2013-01-479972

Copyright © 2013 American Society of Hematology




2

KEY POINTS 1. Hsp90 oncoproteome analysis identifies relevant pathways in KSHV-associated PEL that can

inform novel combinatorial therapies.

2. The Hsp90 inhibitor PU-H71 affects chaperoning of KSHV viral proteins, blocking latent and

lytic viral functions.

ABSTRACT

PU-H71 is a purine-scaffold Hsp90 inhibitor that in contrast to other Hsp90

inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is

preferentially associated with oncogenic client proteins and enriched in tumor cells

(teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing

toxicity in normal cells. We found that lymphoma cells infected by EBV or KSHV are

exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an

unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in

KSHV+ primary effusion lymphoma cells. Viral and cellular proteins were identified,

including many involved in NF-κB signaling, apoptosis and autophagy. KSHV vFLIP is a viral

oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We

show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced

degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro,

and more importantly, tumor responses in mice. Analysis of the interactome revealed

apoptosis as a central pathway, we thus tested a BCL2 family inhibitor in PEL cells. We

found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity

capture identifies actionable networks that may help design rational combinations of

effective therapies.




3

INTRODUCTION

Hsp90 is a molecular chaperone that plays a crucial role in the proper folding and

function of a number of proteins with critical roles in cell survival signal transduction. Hsp90 is

considered a promising target for cancer therapy since it is highly expressed in a number of

malignancies, and acts as a chaperone for oncogenic signaling complexes1. A role for Hsp90 in

virus-associated lymphomas has been proposed, including those caused by infection with

Epstein-Barr virus (EBV/HHV-4) and Kaposi’s sarcoma herpesvirus (KSHV/HHV-8) 2,3.

KSHV is a γ-herpesvirus and the etiologic agent of Kaposi sarcoma (KS), multicentric

Castleman disease (MCD), and primary effusion lymphoma (PEL)4-6. KS is a tumor of

endothelial origin, while PEL is a rare non-Hodgkin B cell lymphoma commonly manifesting as

lymphomatous effusions. Combined antiretroviral therapy is effective in some patients with KS,

and other non-curative approaches such as radiation, surgery, and chemotherapy are helpful.

However, a large percentage of patients are refractory to these treatments and there is a pressing

need for specific drug development.

Several studies have pointed to an essential role for Hsp90 in the survival of KSHV-

associated malignancies. Field et al. showed that the viral oncoprotein vFLIP is in complexes

containing IKKγ and Hsp90; inhibition of Hsp90 by geldanamycin decreased NF-κB activity

induced by vFLIP and killed PEL cells2. Inhibition of extracellular Hsp90 blocked viral gene

expression during de novo infection by KSHV7. Hsp90β was uncovered as a binding partner of

the KSHV K1 protein8, a viral protein that immortalizes endothelial cells in vitro9. Furthermore,

Hsp90 inhibition led to the degradation of LANA, an important viral protein expressed during

latency, in KSHV-infected endothelial cells10. Hsp90 also serves crucial functions in the

transformation and maintenance of malignant cells by EBV. Hsp90 inhibition blocked EBV-




4

induced transformation of B cells3 and was toxic to EBV-infected lymphoblastoid cell lines

(LCLs), in part by directly repressing the transcription of EBV EBNA1, the functional equivalent

of KSHV LANA in terms of viral episome maintenance11. Additionally, Hsp90 inhibition by

geldanamycin or 17-AAG induced apoptosis of EBV-associated NK/T cell lymphoma cell

lines12.

As an alternative to classical toxic quinone inhibitors of Hsp9013, Chiosis et al developed

a new class of purine scaffold non-quinone Hsp90 inhibitors that inhibit Hsp90 function by

competing for the ATP-binding pocket14, preventing the completion of the chaperone cycle.

Among these is PU-H71, which has been well characterized in multiple models of solid and

lymphoid malignancy with low toxicity15. Significantly, PU-H71 accumulates at levels up to 25X

higher in malignant cells compared to primary cells16. Hsp90 inhibition by PU-H71 leads

specifically to the disruption of oncoprotein-containing complexes over normal signaling

complexes containing Hsp9017. This observation was demonstrated in cancer cell lines such as

CML and AML, where PU-H71 pulldown revealed preferential association with BCR-ABL,

rather than the normal ABL protein18. Affinity capture thus demonstrated that this compound

specifically binds the fraction of Hsp90 that is associated with oncogenic proteins and enriched

in tumor cells, designated tumor-enriched Hsp90 (teHsp90). A possible mechanism for this

preferential association to proteins of particular importance to tumor cells is that these proteins

require active chaperoning, and are preferentially bound to Hsp90 in complex with co-

chaperones, which in turn are preferentially identified by PU-H7118.

Based on the reported importance of Hsp90 in viral lymphomagenesis, we evaluated the

effect of teHsp90 inhibition using PU-H71 in virus infected lymphoma cell lines, and performed

a global analysis of the teHsp90 interactome using PEL as a model. We found that PU-H71 was




5

highly effective against EBV and KSHV-infected lymphoma cells. Treatment caused viral onco-

signaling disruption that led to cell death and these results were translatable to a mouse xenograft

model of PEL. Based on the teHsp90 interactome, we validated the hypothesis that inhibition of

antiapoptotic proteins would lead to PEL cell death and synergize with PU-H71. These results

indicate that characterization of the teHsp90 interactome can identify vulnerable pathways in

tumor cells, and specific inhibitor combinations that merit consideration for clinical

development.

MATERIALS AND METHODS

Cell culture, viability and apoptosis assays

Cell were cultured in RPMI-1640 medium (Invitrogen) supplemented with 50mg/ml

Gentamicin (Sigma-Aldrich), and 10% or 20% heat-inactivated fetal bovine serum. Cell viability

was determined using the Cell Proliferation Kit (MTT) (Roche, Indianapolis, IN). Apotosis was

evaluated by flow cytometry for Annexin V. Further details are provided in the Supplemental

Materials.

Autophagy detection: Western blotting for LC3B

Rabbit Anti-LC3B monoclonal antibody (Cell Signaling Technology Inc., Danvers, MA)

was used for detection of cleavage of LC3 in BC3 cells after PU-H71 treatment. The western

blotting was conducted as described previously19.

Immunoblotting and co-immunoprecipitation

For all intracellular signaling studies, BC3 cells were treated with 2μM PU-H71 at a

plating concentration of 2X105 cells/ml for time periods ranging from 30 minutes to 48 hours as

indicated. Immunoblotting was performed as previously described19.




6

PU-H71 affinity capture and proteomics

Affinity capture and proteomics analysis were performed as described previously18.

Experimental details are provided in the Supplemental Materials. Data were analyzed through

the use of IPA (Ingenuity® Systems, www.ingenuity.com).

In vivo xenograft model

The in vivo xenograft model was set up as previously described20. Details are provided in

the Supplemental Materials. Essentially, NOD-SCID mice (Jackson Labs) were injected

intraperitoneally (I.P.) with 1X107 BC3-NF-κB-luc cells and followed by in vivo imaging. For

assessment of disease-free survival and tumor burden, Kaplan-Meier analysis was performed

using GraphPad Prism software, and p values determined by 2-tailed analysis with log-rank tests.

Combinatorial cell viability assays

We determined cell viability by using a luminescent ATP-content method (CellTiter-Glo,

Promega, Madison, Wisconsin) and Trypan blue dye-exclusion. Details are provided in the

Supplemental Materials.

Ethics Statement. These studies were approved by the Weill Cornell Medical College

Institutional Animal Care and Use Committee (IACUC).

RESULTS

KSHV-positive PEL cell lines are highly sensitive to teHsp90 inhibition by PU-H71

Previous studies had shown that KSHV-infected PEL cell lines are sensitive to several

Hsp90 inhibitors, including geldanamycin, 17-AAG and 17-DMAG8. We extended these studies

by testing the efficacy of the two major classes of Hsp90 inhibitors (quinone and protein

scaffold) in a wide range of lymphoma cell lines, including three KSHV+ PEL, three

KSHV+/EBV+ PEL, one EBV+ lymphoblastoid cell line, one EBV+ immunoblastic lymphoma




7

line, one EBV+ Hodgkin lymphoma cell line, and three virus-negative DLBCL cell lines. We

performed MTT assays and determined concentration of compound that inhibited viability of the

cell lines by 50% (growth inhibition, GI50), confirming that KSHV and EBV-infected lymphoma

cell lines were sensitive to Hsp90 inhibition by 17-AAG (Table 1). We found that the infected

cells were exquisitely sensitive to PU-H71. GI50s ranged from 27 to 337 nM, with no obvious

explanation for the variability within this range. For all virus-positive cell lines the GI50 was

lower with PU-H71 than with 17-AAG.

Identification of the Hsp90 interactome in PEL cells

We performed unbiased affinity-capture using PU-H71-conjugated agarose beads to determine

the teHsp90 interacting proteins in the KSHV-infected BC3 PEL cell line. The entire cargo

isolated by the beads was subjected to proteomic analysis by mass spectrometry. A total of 1756

proteins were identified (Supplemental Table 1). One of the top signaling networks identified by

Ingenuity Pathway (IPA) analysis was “NF-κB Activation by Viruses” (Supplemental Figure 1).

We found a remarkable number of proteins in the NF-κB pathway, including TRADD, TRAF2,

IKKα, IKKβ, IKKγ, and four REL/NF-κB proteins (p65/RELA, RELB, p50 and p52) (Figure

1A). Additionally, there was a particular enrichment of molecules involved in apoptosis and

autophagy, a feature not previously seen in other tumor types18. To validate these findings, we

used a targeted approach, consisting of pulldown by PU-H71-conjugated beads followed by

immunoblot analysis. We focused on representative proteins in the apoptosis (MCL1 and

cIAP1), autophagy (ATG3), and NF-κB pathways (p65), and tested additional PEL cell lines

(BC3, BC1, BC2, BC5 and BCBL1) as well as one EBV+ immunoblastic lymphoma cell line

(IBL1) (Figure 1B). We found that in almost all PEL cells and in IBL1, teHsp90 binds these




8

representative proteins, validating the unbiased proteomic approach. The only exceptions were

an absence of p65 in BCBL1 cells, and low levels of MCL1 in IBL1 cells.

Other top networks identified have been shown to be functionally significant in PEL

cells, including AKT/PI3K/mTOR signaling (Supplemental Figure 2), and IL-6 signaling

(Supplemental Figure 3). Interestingly, among the top pathways were those associated with

angiogenesis, such as RAC (Supplemental Figure 4) and VEGF signaling (Supplemental Figure

5).

Several KSHV viral proteins were also pulled down (Table 2). With the exception of

vCyclin and vIL-6, the viral proteins identified as bound to PU-H71 are known to be only

expressed or upregulated during lytic replication. While most PEL cells have latent KSHV in

culture, a small fraction express lytic viral proteins, so it is likely that the lytic proteins detected

derive from these scarce lytic cells, as these cells express lytic genes at much higher levels.

An examination of the major pathways enriched in the pulldown analysis (i.e., NF-κB

activation, apoptosis, and autophagy) suggested KSHV vFLIP as a common viral link, since this

protein has previously been shown to be essential for the enhanced survival and NF-κB-induced

antiapoptotic signaling in PEL cells21 and to inhibit autophagy22. Thus, we validated our

findings by directly examining the binding and effect of PU-H71 on vFLIP. In PEL cells, vFLIP

has been found to exist primarily as part of the IKK signalosome, comprising IKKs α, β, γ, and

Hsp90. While vFLIP was not directly identified by targeted pulldown proteomic analysis, this

method favors proteins expressed abundantly and may fail to identify relevant interactions.

Therefore, we assessed the presence of vFLIP in complex with teHsp90 by performing pulldown

analysis in four different PEL cell lines using PU-H71-conjugated beads. Both vFLIP and

IKKγ were identified, implying their presence in complexes with teHsp90 (Figure 2A).




9

To determine whether cFLIP also binds teHsp90 (directly or indirectly), we performed

pulldown analysis of BC3 cell extracts, followed by immunoblotting with an antibody to cFLIP,

which recognizes the long and short form in PEL cells. We identified only cFLIPL (long isoform)

and not cFLIPS (short isoform) in association with PU-H71 (Figure 2B), in spite of vFLIP being

most closely related structurally to cFLIPS. This confirms a selective targeting of onco-

complexes, since vFLIP and cFLIPL to a lesser extent, are both active in their ability to induce

NF-κB signaling (Figure 2C), leading to downstream anti-apoptotic and pro-proliferative

signaling in PEL cells21. In contrast, the ability of cFLIPS to activate NF-κB is minor23.

PU-H71 induces PEL cell death by apoptosis and autophagy

We used Annexin V and 7-AAD staining of BC3 cells to assess the mechanisms involved

in cellular cytotoxicity of PU-H71 and found extensive apoptosis, with expansion of both early

and late apoptotic compartments, by 72 hours at 1μM PU-H71 (Figure 3A). Treatment with 1μM

PU-H71 led to a six-fold increase in apoptosis. Interestingly, 100 nM PU-H71 did not lead to

significant apoptosis, although this dose is higher than the GI50 (63.4 nM, Table 1). This

discrepancy indicates that the metabolic inhibition as determined by MTT assay (which measures

mitochondrial respiratory chain activity) does not accurately reflect cell death, although it is a

standard method for determining drug sensitivity. Of note, the GI50 obtained for BC3 cells using

a luminescent ATP content method was 290 nM (described below), indicating that this method

may be a better indicator of cell viability. PARP cleavage was used as a second method for

testing apoptosis, and it was observed by 24 hours post-treatment in BC3 cell lines, as well as in

all of four additional PEL cell lines examined at 24 hours post-treatment with PU-H71 (Fig 3B).

Additionally, a large increase in the sub-G1 population, which is indicative of DNA fractionation

accompanying apoptosis, was also found using DNA content cell cycle analysis (Figure 3C).




10

Unlike 17-AAG which has been shown to induce cell cycle arrest in PEL cells8, PU-H71 treated

cells did not reveal such arrest.

KSHV vFLIP has been shown to prevent autophagosome biogenesis by competing with

the ubiquitin-like LC3 for binding to the E2-like ATG3 protein22. Commonly used markers of

autophagy are the appearance of the LC3-IIb cleavage product, as well as appearance of

autophagosomes, both of which were observed 48 hours post-treatment (Figure 3D and 3E).

PU-H71 leads to the elimination of the vFLIP-IKKγ signaling complex and inhibition of

NF-κB signaling in PEL cells

Classical Hsp90-regulated clients depend on Hsp90 for their stability and their steady-

state concentration decreases upon Hsp90 inhibition18,24,25. Thus, we speculated that this

chaperone might play a role in the stabilization of the vFLIP signalosome. Immunoblotting

showed that inhibition of teHsp90 led to the decrease of IKKγ, as well as the known Hsp90

target AKT (both total and phosphorylated forms) by 6h (Figure 4A). In addition, vFLIP protein

levels were also reduced by 24h. Co-immunoprecipitation using anti-IKKγ to pull down

associated vFLIP and Hsp90 revealed a decrease of signalosome components (Supplemental

Figure 6).

Since KS is a tumor of endothelial cell origin, we evaluated whether PU-H71 is also

cytotoxic to KSHV-infected endothelial cells. We used a long-term telomerase-immortalized

endothelial cell line as an in vitro model for KS26. The infected cell line was approximately 14-

fold more sensitive than the uninfected control (Table 1). In these cells, PU-H71 also led to a

decrease in both IKKγ and vFLIP protein levels (Figure 4B).




11

Since the vFLIP-IKKγ-Hsp90 signaling complex is directly upstream of NF-κB

activation, the effect of teHsp90 inhibition by PU-H71 on NF-κB signaling was studied in an

NF-κB-reporter luciferase BC3 cell line (BC3-NF-κB-luc)20. A time course of treatment revealed

decrease of NF-κB reporter luciferase activity within 4 hours (Figure 4C).

PU-H71 does not reduce the levels of other latent viral proteins and inhibits lytic

reactivation in PEL cells

We evaluated the effect of PU-H71 on the other KSHV latent proteins, LANA, vCyclin,

and vIL-6 and found a partial decrease in vCyclin and no consistent changes in LANA and vIL-6

upon treatment (Figure 5A). Although both vCyclin and vIL-6 were identified in the PU-H71

pull-downs (Table 2), some Hsp90 binding proteins are not destabilized upon inhibition of

Hsp9018, so vCyclin and vIL-6 appear to fall into this category. Similarly, binding to Hsp90 by

LANA, with a decrease of the viral protein following treatment with Hsp90 inhibitors, has been

reported10, but we did not observe this decrease with PU-H71.

Induction of lytic reactivation has been reported as a consequence of NF-κB inhibition27.

To assess effects on the lytic viral cycle, we used the modified recombinant KSHV(rKSHV.219)-

infected PEL cell line JSC-1 which expresses GFP under the control of the cellular EF-1 alpha

promoter and RFP under the control of the early lytic PAN promoter of KSHV 28. Treatment of

these cells with PU-H71 turned on RFP expression in only a very small fraction of cells (3%), in

contrast to treatment with the lytic phase inducer TPA (42%) (Figure 5C).

We analyzed PU-H71 treated cells for levels of latent and lytic viral transcripts. Real time

qRT-PCR analysis of whole cell extracts from PU-H71 treated cells showed a variable reduction

of the latent transcripts evaluated in BC3 cells (Figure 5D) and other PEL cell lines

(Supplemental Figure 7A). Note that LANA, vFLIP and vCYC are controlled by the same




12

promoter, and vFLIP and vCYC are encoded in the same transcript, but the decrease in RNA

level did not translate into consistently decreased LANA or vCYC (Figure 5A and 5B).

Similarly, vIL-6 protein levels did not change, although transcriptional levels were variable

(Figure 5A and 5B). There were also variable changes in the two lytic transcripts examined in

other PEL cell lines. The global decrease in the major latent viral transcripts can be explained by

the inhibition of several transcription factors by PU-H71as well by effects on chromatin

remodeling proteins and ribonucleoproteins, also identified by PU-H71 affinity capture (Table

1). For example, the shared vFLIP/vCYC/LANA promoter is highly dependent on SP129, a

transcription factor that is an established Hsp90 client protein30. Indeed, immunoblotting

revealed a significant decrease in SP1 levels after PU-H71 treatment in PEL cells (Supplemental

Figure 7B). The reduction of vFLIP mRNA is partial and transient, so it may be only partially

responsible for the dramatic decrease in vFLIP protein levels, and there is probably also post-

translational down-regulation of vFLIP.

We assessed the role of Hsp90 in lytic reactivation. There was a reduction of the late lytic

transcripts K8.1 and ORF59 and a minor induction of the immediate-early transcript ORF50

(Figure 5C), with similar but variable trends observed in other PELs (Supplemental Figure 7A),

confirming that this compound induces only minimal lytic reactivation. Furthermore, we found

that PU-H71 inhibits TPA-induced lytic gene expression (Figure 5D).

PU-H71 confers a significant survival advantage to PEL-xenografted mice

To evaluate PU-H71 in a pre-clinical model, we used the BC3-NF-κB-luc xenograft

model in NOD-SCID mice in 3 independent trials. Mice were randomized after xenograft

engraftment into vehicle (n=10) and treatment cohorts (n=13). The PU-H71 cohort was treated

on a daily basis by IP injection at 75mg/kg/day for two 10-day intervals separated by a week.




13

Representative in vivo imaging results are shown in Figure 6A. A pattern of protection

conferred by PU-H71 treatment was obvious from quantification of tumor burden in these mice.

Kaplan-Meier analysis indicated that PU-H71 treatment conferred a significant survival

advantage (median survival time=90 days for treated, 26 days for vehicle) to xenografted mice

(Figure 6B; p=0.0008).

We performed ex vivo analysis to verify PU-H71 activity in the PEL xenografts. Whole

cell extracts of ascites from vehicle cohort mice treated with PU-H71 12 hours pre-sacrifice were

compared to ascites from untreated mice. We confirmed target inhibition ex vivo by

immunoblotting for AKT (Figure 6C), due to the lack of a robust commercial antibody for vFLIP

in xenograft analysis.

PU-H71 synergizes with a pan-BCL2 family inhibitor in PEL cells

Based on the large number of proteins involved in apoptosis by PU-H71 affinity-capture

in PEL cells, we hypothesized that inhibition of apoptosis is critically important and that

targeting antiapototic proteins could be a viable approach for the treatment of KSHV-associated

malignancies. We used obatoclax (GX15-070), a small-molecule BH-3 mimetic that antagonizes

BCL2, BCLXL, BCLW and MCL131, to treat PEL cells. This compound was shown to

successfully inhibit viability of a panel of five PEL cell lines with submicromolar GI50 (Figure

7A). In order to assess synergy, cell lines were treated with a range of concentrations of both PU-

H71 and obatoclax. We chose doses for each drug that had partial toxicity. We used an ATP-

content assay to assess viability rather than the MTT assay shown in Table 1, which gave

somewhat higher GI50. As shown in Figure 7B, some cell lines (BC3 and BC2) had much

greater synergy. We further assessed synergy for BC3, by treating with six concentrations of PU-

H71, obatoclax, the combination, or vehicle, for 48 and 72 h. We determined the combinatorial




14

index32 and found that it was synergistic at both time points (Figure 7C and 7D). The Fa-CI

(fraction affected–combinatorial index) plot showed that the combination is highly synergistic

for all the effect points at 72 h (Figure 7D) and 48 h (not shown).

DISCUSSION

This study is the first to identify the entire teHsp90 interactome in PEL cells and

demonstrates specific targeting of a viral oncoprotein by an oncocomplex-specific Hsp90

inhibitor. We show that a teHsp90 inhibitor can successfully be used to block pathogenic

pathways in viral malignancies, and reveal synergistic interactions through teHsp90 interactome

analysis. Our results are consistent with previous studies showing that Hsp90 exists in a specific

conformation in tumor cells, described here as teHsp90, which is selectively bound and inhibited

by PU-H71, and that is enriched in complexes containing dysregulated oncoproteins18.

Specifically, using PU-H71 affinity capture and proteomic analysis, we were able to identify

proteins in pathways known to be essential for KSHV-mediated oncogenesis, including NF-κB,

apoptosis and autophagy. This analysis also revealed other proteins and additional pathways that

may play a role in KSHV oncogenesis.

vFLIP is a critical viral oncoprotein and Hsp90 has been shown to be present in vFLIP

and IKKγ containing complexes. While we did not find vFLIP in the unbiased proteomic

approach, it was previously shown18 that Hsp90 interacts with nodal proteins in signaling

pathways, and affinity purification may identify only key components of an Hsp90-addicted

protein pathway. Nevertheless, we confirmed the interaction of teHsp90 and vFLIP, and

demonstrated that teHsp90 inhibition effectively abolishes the oncogenic vFLIP-IKKγ signaling

complex. Complex elimination decreased downstream signaling and inducted autophagy and

apoptosis. Notably, while the closest cellular homolog of vFLIP is cFLIPS, we were only able to




15

document teHsp90 binding to vFLIP and cFLIPL. Since vFLIP and cFLIPL can induce NF-κB

activity in PEL cells, these results confirm the selectivity of binding of teHsp90 to active proteins

in particular cellular contexts.

The number of proteins involved in apoptosis, including proapoptotic, antiapoptotic, and

effector molecules (Figure 1), that were found to be associated with teHsp90 was remarkable,

and not seen in other tumors so far18. Since the presence of a virus is likely to be an important

apoptotic trigger, it follows that teHsp90 in virus-associated malignancies maintains proteins

involved in apoptosis in complexes or configurations that prevent cell death. These observations

explain the exquisite sensitivity of KSHV and EBV infected lymphoma cell lines to treatment

with PU-H71. We hypothesized that based on the large number of apoptosis pathway proteins

bound to teHsp90, we could also kill PEL cells with an inhibitor of antiapoptotic proteins. In

fact, we found that all PEL cell lines tested were highly sensitive to treatment with a pan-BCL2

inhibitor obatoclax, and that, at least in BC3 cells, there is dramatic synergy when treating cells

with PU-H71 and obatoclax. This result indicates that an understanding of the PU-H71/teHsp90

interactome in cancer cells can inform future therapeutic choices and inhibitor combinations.

The inhibition of autophagy is a major function for vFLIP22. vFLIP directly binds ATG3

and prevents it from processing LC322. We identified ATG3 by PU-H71 affinity capture, as well

as additional proteins involved in autophagy. The PI3K pathway has also been shown to inhibit

starvation-induced autophagy in macrophages33, suggesting that the AKT/PI3K pathway may

contribute to induction of autophagy in PEL cells by PU-H71. We observed a decrease of Akt

protein levels in cells treated with PU-H71, both in vitro and in vivo. Other proteins in this

pathway were also identified in the teHsp90 interactome.




16

KS is a tumor of endothelial cell origin where angiogenesis plays a critical role.

Remarkably, among the pathways that were identified in the teHsp90 interactome are those

associated with VEGF and RAC. These two pathways have been implicated to be central in

angiogenesis and KS pathogenesis34,35. PU-H71 was also found to bind several lytic proteins.

This is most likely a result of the extremely high levels of lytic proteins in the few lytic cells in

cell culture. It is also likely that binding by teHsp90 to complexes containing lytic proteins is

very robust, and possibly direct, which could be tested using recombinant proteins for direct

binding to Hsp90 and dissociation in vitro. PU-H71 also inhibits expression of lytic viral

transcripts. Consistent with this, another study found inhibition of KSHV viral replication by

Hsp90 inhibitors36. This antiviral activity could provide further benefit for the treatment of

individuals with KS, where lytic genes may contribute to disease pathogenesis37.

Analysis in mice demonstrated that PU-H71 can be used successfully to prolong disease-

free survival. While post-treatment relapses were observed, they may be overcome by extending

treatment38. Thus, treatment with PU-H71 in combination with other specific inhibitors may be

required for an effective clinical response. PU-H71 has demonstrated efficacy in several in vivo

models of malignancy39, with Phase I clinical trials ongoing.

In summary, we used PU-H71 affinity capture and proteomics to elucidate the Hsp90-

associated oncoproteome of a KSHV-associated PEL cell line. This unbiased analysis revealed

many pathways associated with KSHV pathogenesis, lending support to the power of this

methodology. This approach demonstrated a novel mechanistic function for teHsp90 inhibitors,

wherein they can be successfully used to target viral oncoproteins. We demonstrated clear

potential for PU-H71 in the treatment of KSHV-associated diseases, and our analysis revealed a




17

number of teHsp90-interacting proteins and pathways that will provide the basis for exploring

additional new therapeutic approaches and combinations.

ACKNOWLEDGEMENTS This work was funded by NIH/NCI grant R01-CA68939 to EC, P30CA008748 to EC and GC,

P30CA008748 to the Microchemistry and Proteomics Core Laboratory of Memorial Sloan-

Kettering Cancer Center, and T32 AI007621 Immunology and Microbial Pathogenesis Program

Training Grant support for UN.

AUTHORSHIP CONTRIBUTIONS AND DISCLOSURE OF CONFLICTS OF

INTEREST

UN: designed and performed in vitro and mouse studies, analyzed data, wrote and edited paper,

PL: designed and performed GI50 and apoptosis experiments, RLG: performed validation

pulldowns and synergy experiments, JV: analyzed mouse data, GB: developed vFLIP transgenic

mice and isolated mouse B cells, AR: performed targeted pulldown, TT: prepared compound,

HE: analyzed proteomics data, MC: assisted with qRT-PCR for lytic genes, RB: provided

resources for mouse studies, AM: supervised synergy experiments, LC: designed and analyzed

synergy experiments, GC: supervised and designed targeted pulldown, YLW: supervised and

designed GI50 and apoptosis experiments, EC: supervised and designed in vitro and mouse

experiments, analyzed pulldown data, wrote and edited paper.

COMPETING INTERESTS STATEMENT: MSKCC holds the intellectual rights to the purine-

scaffold Hsp90 inhibitors. Samus Therapeutics, of which G.C. has partial ownership, has

licensed PU-H71.




18

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24

Table 1: GI50s of PU-H71 and 17-AAG in KSHV- and EBV-positive Cell Lines

Cell lines

PU-H71

GI50 (nM)

17-AAG

GI50 (nM) Lymphoma type Viral status

BC-1 36.9 50.9 PEL KSHV+/EBV+

BC-2 27.1 78.6 PEL KSHV+/EBV+

BC-5 122.7 650 PEL KSHV+/EBV+

BC-3 63.4 605.4 PEL KSHV+

BCBL-1 217.0 1009.2 PEL KSHV+

VG-1 281.5 608.9 PEL KSHV+

L591 337.0 952.7 HL EBV+

BCKN-1 70.9 548 Effusion DLBCL EBV+

IBL-1 87.0 833.4 DLBCL EBV+

Ly18 308.3 308.4 DLBCL Negative

Ly3 376.7 ND DLBCL Negative

Ly10 256.8 ND DLBCL Negative

TIVE 20,561 ND Endothelial Negative

TIVE-LTC 1,423 ND Endothelial KSHV+

Abbreviations: PEL: primary effusion lymphoma; DLBCL: diffuse large B cell lymphoma; HL:

Hodgkin’s lymphoma; ND: not done; nM: nanomolar.




25

Table 2: Viral proteins identified by proteomic analysis of PU-H71 pull-downs Identified

proteins

Molecular

weight Classification Known function(s)

ORF20 28 kDa Late UL24 family: induces cell cycle arrest and apoptosis

ORF18 29 kDa Late Function not known for KSHV- involved in late gene transcription in

other herpesviruses

ORF64 290 kDa Early Tegument protein with deubiqutinase (DUB) activity: suppresses RIG-I-

mediated IFN signaling by reducing the ubiquitination of RIG-I

ORF K2 23 kDa Early/ Latent* vIL-6: viral homologue of interleukin 6

ORF57 51 kDa Early mRNA biogenesis

ORF22 81 kDa Late Virion envelope glycoprotein

ORF72 28 kDa Latent vCyclin: viral homolog of cyclin D

ORF25 153 kDa Late Major capsid protein

ORK9 113 kDa Early DNA polymerase

ORF27 32 kDa Late Membrane glycoprotein that contributes to intercellular viral spread

ORF63 104 kDa Early viral homolog of human NLRP1 (blocks innate immune

responses/inflammasome)

ORF6 125 kDa Early multi-functional DNA binding proteins with roles central to viral DNA

replication

ORF56 96 kDa Early Primase

ORF4 61 kDa Late Complement inhibitor- KCP for complement control protein

ORF35 17 kDa Early Induced by RTA, required for in vitro infection

ORF21 65 kDa Early Thymidine kinase

ORF19 61 kDa Early Tegument protein

ORF K4.1 13 kDa Early vMIP-III/vCCL3- CCR4 agonist

ORF17.5 31 kDa Early Assembly/scaffolding protein

ORF62 36 kDa Late Triplex-1 structural protein

ORF2 23 kDa Early vDHFR; Viral homologue of dihydrofolate reductase

* vIL-6 is increased upon lytic reactivation, but is expressed in latently infected PEL 40




26

Figure 1: PU-H71-association based identification of proteins involved in NF-κB activation, apoptosis and autophagy in PEL cells. A) PU-H71 affinity capture of BC3 cell extracts was followed by proteomic analysis. Proteins identified in these pull downs are shown with a solid color as follows: those involved in NF-κB signaling are blue, those involved in apoptosis are green, and ATG3, involved in autophagy, in brown. vFLIP, a key nodal protein is shown in orange. Results shown are from one of two independent experiments. B) Immunoblot from PU-H71 pull-downs, but not control bead pull-downs, using extracts from the indicated cell lines, confirmed that Hsp90 interacts with selected proteins in key pathways, including NF-κB (p65), apoptosis (MCL1, cIAP1) and autophagy (ATG3)




27

Figure 2. PU-H71 recognizes Hsp90 bound to the vFLIP signalosome, and shows preferential binding to the long form of cFLIP. A) Immunoblot from PU-H71 pull-downs, but not control bead pull-downs, using extracts from five different KSHV+ PEL cell lines (BC3, BC1, BC2, BC5 and BCBL1), recognized vFLIP and IKKγ. Control extracts from a B cell

lymphoma cell line lacking KSHV and thereby vFLIP (IBL1) showed binding of Hsp90 to IKKγ, but no bands were seen with the vFLIP antibodies, indicating specificity of the vFLIP band. Results shown are representative of two independent experiments. B) Immunoblots from PU-H71 pull-downs were evaluated for Hsp90 binding to cFLIP, the cellular homolog of vFLIP. Although vFLIP is more closely structurally related to the short form (cFLIPS), PU-H71 failed to recognize this protein, and only the long form (cFLIPL) was identified. C) Schematic representation of cFLIP long and short forms is shown; the most active of these proteins in terms of ability to activate NF-κB is vFLIP, followed by cFLIPL, and lastly cFLIPS

21,23.




28

Figure 3. PU-H71 induces PEL cell death by apoptosis and autophagy. A) BC3 cells treated with 1µM PU-H71 for 72 hours were examined by flow cytometry after staining for 7-AAD and Annexin V. Results were quantified into percentages of live and apoptotic cells (Annexin V positive). Asterisks (***) indicate a statistical significance of P<0.001 as compared to the control (untreated) group. B) Top panel: Immunoblot analysis using extracts from BC3 cells treated at the indicated time points with 2µM PU-H71 for the indicated time points shows PARP cleavage by 24 hours as indication of apoptosis. Lower panel: PARP cleavage is apparent after PU-H71 treatment (2μM for 24 hours) in additional PEL cell lines. C) BC3 cells treated with 1µM PU-H71 for 72 hours were examined by flow cytometry for DNA content cell cycle analysis. Only the Sub-G0 fraction, indicative of apoptosis, was found to increase in treated cells. D) Autophagy induction was examined by immunoblot of PU-H71 treated cells. Appearance of the cleavage product of LC3B is indicative of autophagy. E) BC3 cells treated with 1µM PU-H71 were examined for the appearance of autophagosomes containing degraded cellular organelles using electron microscopy. Autophagosomes are clearly visible in the right panel at higher magnification (arrows).




29

Figure 4: PU-H71 leads to the loss of the vFLIP-IKKγ signaling complex. A) BC3 cells were treated with 2µM PU-H71 for the time course indicated and were subjected to immunoblot analysis. Treatment resulted in decrease of vFLIP, IKKγ, phospho-Akt, and total Akt. This result is representative of at least three independent experiments. B) TIVE-LTC (KSHV+) cells were treated with 2µM PU-H71 for 48 hours, followed by immunoblot with the indicated antibodies. Results are representative of three independent experiments. C) BC3-NFκB-luc reporter cells were treated with 2µM PU-H71, and luciferase assays performed at the indicated time points. Results shown are the average of three independent experiments.




30

Figure 5. PU-H71 inhibits NF-κB with minimal lytic reactivation. A) Immunoblot analysis of BC3 cells treated with 2µM PU-H71 at the indicated time points shows that vFLIP is eliminated by 48 hrs, while there is no consistent decrease in vCyclin, LANA or vIL-6, as determined by quantification of band intensity and normalized to β-actin and untreated control cells a time 0 (shown below each immunoblot lane). B) Immunoblot analysis for the panel of viral genes indicated was done using additional PEL cell lines, treated with 2μM PU-H71 for 24 hours. C) JSC-1 cells were treated with TPA or PU-H71, and the percentages of GFP+ and RFP+ cells determined by counting under a fluorescent microscope. D) Expression levels of latent (LANA, vFLIP, vIL-6) and lytic (ORF50, ORF59, and K8.1) transcripts was studied by qRT-PCR analysis of extracts from BC3 cells treated with PU-H71 for the indicated time points. The experiment was performed three times in triplicate, and representative results are shown. A variable but global decrease of these transcripts was observed (for analysis of other cell lines see Supplemental Figure 7A). E) Expression of lytic genes induced by TPA was inhibited by PU-H71, as shown after treatment with the compounds for 24 hours.




31

Figure 6. PU-H71 induces tumor responses in mice with PEL. A) In vivo imaging of mice that received an IP injection of BC3/NFκB-luc cells (Day 0) and subsequently were untreated (left) or mice that received PU-H71 intraperitoneally at a dose of 75 mg/kg/day on days 4-13 and 20-29. B) Kaplan-Meier survival curves for treated and control mice showed prolonged survival in the treatment arm; mice were euthanized when the developed ascites leading to a >10% increase of body weight in a one week period, and therefore survival is directly related to tumor growth and independent of possible reporter effects. C) Established tumors from 3 mice in the vehicle arm were collected 12 hours after administration of one 75 mg/kg dose of PU-H71 (right lanes), and protein extracts were compared to those from tumors in untreated controls (left lanes). Immunoblot analysis shows a decrease in vivo of a classic Hsp90 client.




32

Figure 7. PU-H71 synergizes with a pan-BCL2 family inhibitor. A) The indicated PEL cell lines were treated with pan-BCL2-family inhibitor obatoclax for 72 hrs, and the dose response curves are shown. The fraction of cells killed relative to the untreated control is represented on the y-axis, and the obatoclax dose is shown in the x-axis. Plots were calculated using Compusyn Software and represent best-fit models of the data point. The R value for the dose effect curves ranged between 0.94 and 0.97 in all the cell lines. B) The indicated panel of PEL cells were treated with PUH71, Obatoclax (Obx) and a combination of both for 72 hours. Doses were chosen based to the sensitivity of each cell line, and were as follows: BC3: PUH71 0.078125μM, Obx 0.15625μM, BC2: PUH71 and Obx 0.3125μM, BC5 and BCBL1: PUH71 and Obx 0.625μM. C) BC3 cells treated with PU-H71 or obatoclax for 48 hours were analyzed for synergy. The isobologram indicates GI50, GI75, and GI90 values (for PUH71 GI50, GI75, and GI90 are 0.29256, 0.83922, and 2.38530 μM, respectively, and for Obatoclax, 0.14949, 0.24479, and 0.40084 μM), indicating strong synergy with combination index values of ~0.2. D) Fa-CI plot for the combination of obatoclax and PU-H71 at 72 hours. The fraction of cells killed is represented on the x-axis (Fa) and the combinatorial index is represented on the y-axis (CI). The dotted line represents an additive effect and divides the antagonistic zone (above) and synergistic zone (below). The points represent actual combination data points and the blue line represents the computer simulation for the relationship.




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