Inhibition of Hsp90 Suppresses PI3K/AKT/mTOR Signaling and ... · Small Molecule Therapeutics Inhibition of Hsp90 Suppresses PI3K/AKT/mTOR Signaling and Has Antitumor Activity in

Small Molecule Therapeutics

Inhibition of Hsp90 Suppresses PI3K/AKT/mTORSignaling and Has Antitumor Activity in BurkittLymphomaLisa Giulino-Roth1,2, Herman J. van Besien2, Tanner Dalton2, Jennifer E. Totonchy2,Anna Rodina3, Tony Taldone3, Alexander Bolaender3, Hediye Erdjument-Bromage4,Jouliana Sadek2, Amy Chadburn2, Matthew J. Barth5, Filemon S. Dela Cruz6,Allison Rainey6, Andrew L. Kung6, Gabriela Chiosis3, and Ethel Cesarman2

Abstract

Hsp90 is amolecular chaperone that protects proteins, includ-ing oncogenic signaling complexes, from proteolytic degrada-tion. PU-H71 is a next-generation Hsp90 inhibitor that prefer-entially targets the functionally distinct pool of Hsp90 present intumor cells. Tumors that are driven by theMYConcoproteinmaybe particularly sensitive to PU-H71 due to the essential role ofHsp90 in the epichaperome, which maintains the malignantphenotype in the setting of MYC. Burkitt lymphoma (BL) is anaggressive B-cell lymphoma characterized by MYC dysregula-tion. In this study, we evaluated Hsp90 as a potential therapeutictarget in BL. We found that primary BL tumors overexpressHsp90 and that Hsp90 inhibition has antitumor activity in vitro

and in vivo, including potent activity in a patient-derived xeno-graft model of BL. To evaluate the targets of PU-H71 in BL, weperformed high-affinity capture followed by proteomic analysisusing mass spectrometry. We found that Hsp90 inhibitiontargets multiple components of PI3K/AKT/mTOR signaling,highlighting the importance of this pathway in BL. Finally, wefound that the anti-lymphoma activity of PU-H71 is synergisticwith dual PI3K/mTOR inhibition in vitro and in vivo. Overall, thiswork provides support for Hsp90 as a therapeutic target in BLand suggests the potential for combination therapywith PU-H71and inhibitors of PI3K/mTOR. Mol Cancer Ther; 16(9); 1779–90.�2017 AACR.

IntroductionBurkitt lymphoma (BL) is a germinal center B-cell-derived

malignancy that is molecularly characterized by the translocationof theMYC proto-oncogene to either the immunoglobulin heavyor the k or l light chains. Beyond the MYC translocation, BL isheterogeneous from a genomic standpoint. Recurrent somaticalterations have recently been described in TP53 (approximately50%of cases); TCF3or its negative regular ID3 (34–70%of cases);CCND3 (38% of cases); and the SWI/SNF family of chromatinremodeling genes (17–43% of cases; refs. 1–4). Novel therapies

are needed in BL, where the survival for patients with relapseddisease is <20% (5, 6). One approach to therapeutic targeting inBL given the molecular heterogeneity is to focus on globalmechanisms of lymphomagenesis, which are independent ofgenetic subtype.

Hsp90 has emerged as a desirable target in cancer due to itsrole in the regulation of pathways required for malignant growth(7). Hsp90 is an ATP-dependent molecular chaperone that pro-tects proteins from proteolytic degradation including oncogenicsignaling complexes. Inhibitors that broadly target Hsp90, how-ever, have been limited in their clinical development due tosuboptimal target inhibition and off-target toxicities. More recentstudies have elucidated a biochemically distinct Hsp90 complexin cancer cells that is unique from the fraction of Hsp90 engagedin housekeeping functions (8). This "tumor-enriched" Hsp90 isamenable to small molecule inhibition. PU-H71 is a purinescaffold Hsp90 inhibitor with preferential uptake in tumortissue and selective binding to the tumor-enriched fraction ofHsp90 (9). PU-H71 specifically targets oncogenic Hsp90 clients,for example: Bcr-Abl but not cellular Abl; mutated, but not WTmB-Raf; and the aberrantly activated signaling complex contain-ing Bcl6 (8, 10). This agent, as well as other inhibitors of Hsp90,are currently being evaluated in phase I and phase II clinicaltrials (clinicaltrials.gov ID: NCT01393509, NCT02261805,NCT02474173).

More recently, a chaperome network (coined the epichaper-ome) was identified in a subset of cancers, and found to bespecifically targeted by PU-H71 (11). This epichaperome appearsto rely on dysregulated MYC, and is present in tumors that

1Department of Pediatrics, Weill Cornell Medical College, New York, New York.2Department of Pathology and Laboratory Medicine, Weill Cornell MedicalCollege, New York, New York. 3Program in Chemical Biology, Memorial SloanKettering Cancer Center, New York, New York. 4Molecular Biology Program,Memorial Sloan Kettering Cancer Center, New York, New York. 5Department ofPediatrics, Roswell Park Cancer Institute, Buffalo, New York. 6Department ofPediatrics, Memorial Sloan Kettering Cancer Center, New York, New York.

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

Current address for H. Erdjument-Bromage: Department of Biochemistry andMolecular Pharmacology, New York University School of Medicine, SkirballInstitute, New York, New York.

Corresponding Author: Lisa Giulino Roth, Weill Cornell Medical College, 525East 68th Street, Payson 695, New York, NY 10065. Phone: 212-746-3494; Fax:212-746-8609; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0848

�2017 American Association for Cancer Research.

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overexpress the MYC oncoprotein. Although BL is characterizedby MYC translocation and deregulation, the Hsp90 chaperomeand the effects of PU-H71 have not been studied in BL. In thisstudy we evaluated Hsp90 as a therapeutic target in BL. Wehypothesized that Hsp90 inhibition would disrupt oncogenicclient proteins required for BL survival. We found that Hsp90inhibitionhas anti-lymphoma effects in vitro and in vivo, includingactivity in a patient-derived xenograft (PDX)model of BL. In orderto determine the relevant Hsp90 clients in BL and thereby definethe Hsp90 oncoproteome, we used PU-H71 affinity capture andproteomics (8, 12). We identified multiple components in thePI3K/AKT/mTOR signaling pathway as Hsp90 clients in BL. Inaddition, we found that Hsp90 inhibition was synergistic withPI3K/mTOR inhibition. Collectively, these results support Hsp90as a rational therapeutic target in BL and identify a novel strategyfor combination therapy.

Materials and MethodsCell culture, immunoblot, and reagents

Ramos, Raji, Namalwa, Daudi, CA-46, Ly10, and Ly18 celllines were purchased from ATCC in 2013. Jiyoye and DG-75 werepurchased fromATCCin2014.Raji 2RandRaji 4RHwereprovidedby Roswell Park Cancer Institute (M.J.B.). All cells were used forexperimentswithin2monthsof thawing.Cell line characterizationwas performed by Idex Bioresearch using short tandem repeatprofiling and multiplex PCR to report cell line characterizationand interspecies contamination. Testing was last performed inFebruary 2014. Cells were cultured in RPMI1640 media (Invitro-gen) supplementedwith 10%heat inactivated FBS and gentamicin50 mg/mL (Sigma-Aldrich). PU-H71, PU-H71-beads, and controlbeads were synthesized and prepared at Memorial Sloan KetteringCancer Center as previously reported (13, 14). Idelalisib (CAL-101), IPI-145, BKM120 (15), BEZ-235 (16), ganetespib (17), andBIIB-021 (18) were purchased from Selleck Chemicals. MPC-3100(19) and CUDC-305 (20) were purchased from Santa Cruz andChemieTek, respectively. The chemical structures for PU-H71 andBEZ-235 are presented in Supplementary Fig. S1.

Immunoblot, IHC, and tissue microarray constructionCell lysis and immunoblot were performed by standard pro-

cedure using the following antibodies: GAPDH (Genetex); p110dand p110a (Santa Cruz); p110b and p85a (Abcam); PARP(BD Pharmingen); AKT, p110d, p110b, eIF4E (Cell SignalingTechnology); PARP (Santa Cruz); RB and pRB (Cell SignalingTechnology). IHC was performed using the following antibodies:Hsp90 (Enzo Life Sciences) p-4EBP1 (BD Pharmingen), BCL2(Cell Marque), BCL6 (Dako), Ki67 (Dako), TUNEL (prepared aspreviously described; ref. 21). A TMA was constructed using 0.6mm cores obtained from formalin-fixed paraffin embedded BLtumor samples. Each case was represented on the TMA in dupli-cate. Quantification of IHC was performed using the Halo imageanalysis software program (Indica Labs).

Cell viability assays, IC50 calculation, and combination studiesCell viability was determined using trypan blue staining and an

ATP-based luminescent assay (CellTiter-Glo, Promega). Lumines-cence was measured using the GloMax Multiþ Detection System(Promega). IC50 values were calculated using Prism 6 software.For combination treatments, cells were exposed to increasingdoses of each drug alone or the combination at a constant ratio

in 96-well plates. Cell viability was measured and normalized tovehicle control. Compusyn software (Biosoft) was used to plotdose effect curves and calculate combination index (CI) value. Toevaluate cell viability in germinal center (GC)B cells, lymphocyteswere extracted from de-identified primary human tonsil speci-mens which were obtained after routine tonsillectomy. Totallymphocytes were treated with PU-H71 in RPMI containing20% FBS and 100 mg/mL Primocin and cultured for 24 hours ongamma-irradiatedmouse LCDw32 feeder cells. At indicated timesposttreatment lymphocyte cultures were incubated with fixableviability stain (FVS, BD Catalog No. 564406). Cells were pelletedand resuspended in 100 mL cold PBS without calcium and mag-nesium containing 5% FBS, and 0.1% Sodium Azide (FACSBlock) and incubated on ice for 15 minutes after which 100 mLcold PBS containing 0.5% FBS and 0.1% Sodium Azide (FACSWash) was added. Cells were pelleted and resuspended in FACSWash containing B cell phenotype panel as follows: Ig LambdaCD19-PE (BD Catalog No. 240720), CD38-PECy7 (BD CatalogNo. 560667), IgD-PerCP Cy5.5 (BD Catalog No. 561315), andCD27-APC H7 (BD Catalog No. 560222). Parallel cultures ofRamos treated with PU-H71 and stained only with FVS wereincluded as positive control. Data were acquired on a BD LSR2Flow Cytometer and analyzed using FlowJo software. Datareported for primary tonsil specimens are the percentage ofCD19þ lymphocytes, which were both germinal center cells(CD38hi, IgDlow) and viable (FVS-).

Flow cytometry for apoptosis and cell-cycle analysisCells were treated as indicated and 5� 105 cells were harvested

by centrifugation at indicated timepoints,washedonce inPBS andresuspended in 1� AnnexinV staining buffer (BD CatalogNo. 556454) containing 3 mL/test AnnexinV-Alexa Fluor 647(ThermoFisher A23204) and 5 mL/test 7-AAD (BD Catalog No.559925) and incubated at room temperature for 15minutes.Datawere acquired with a BD LSR2 analytical flow cytometer andanalyzed using FlowJo software. Necrotic cells were defined asannexin Vþ/7AADþ or annexinV�/AADþ and early apoptoticcells were defined as annexin Vþ/7AAD�.

Cell-cycle analysisCells were treated as indicated and 5� 105 cells were harvested

by centrifugation, washed once in PBS, and resuspended in 1�Transcription Factor Fix/Perm buffer and incubated for 50 min-utes on ice. An equal volume of 1� Perm/Wash buffer was addedand cells were pelleted and washed once more in 1� Perm/Wash.Buffers for this procedure were purchased from BD (Catalog No.562725). Ki67 staining was performed overnight at 4�C in 1�Perm/Wash using 0.5 mL Ki67-BV510/test (BD Catalog No.563462). After staining cells were washed twice with 1� Perm/Wash and resuspended in PBS containing 5 mL/test propidiumiodide (BD Catalog No. 556463). Data were acquired with a BDLSR2 analytical flow cytometer, analyzed using FlowJo software,and plotted using the ggplot2 package in R.

PU-H71 affinity captureAffinity capture and proteomics analysis were performed as

previously described (8). Briefly, cells were lysed by collectingthem in Felts buffer [20 mmol/L HEPES, 50 mmol/L KCl,5 mmol/L MgCl2, 0.01% (w/v) NP-40, freshly prepared20 mmol/L Na2MoO4 (pH 7.2–7.3)] with added 1 mg/mLprotease inhibitors (leupeptin and aprotinin), followed by

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three successive freeze (in dry ice) and thaw steps. Total proteinconcentration was determined using the BCA Kit (Pierce)according to the manufacturer's instructions. The PU-H71 con-jugated beads were added at a volume of 80 mL to the cell lysate(1 mg), and the mixture incubated at 4�C for 4 hours. The beadswere washed five times with Felts lysis buffer and separatedby SDS-PAGE, followed by a standard Western blotting proce-dure. Proteins identification was then performed by LC/MS-MSas follows: purified PU-H71 interacting protein complexeswere resolved using SDS-PAGE, followed by brief staining withSimply Blue (Life Technologies) and excision of the separatedprotein bands. In situ trypsin digestion of polypeptides ineach gel slice was performed and the tryptic peptides wereresolved on a nano-capillary reverse phase column. Peptideswere directly infused into a linear ion-trap mass spectrometer(LTQ Orbitrap XL, Thermo Electron Corp.). The mass spec-trometer was set to collect one survey scan (MS1), followed byMS/MS spectra on the nine most intense ions observed in theMS1 scan. Proteins were identified by searching the tandemmass spectra against a human segment of Uniprot proteindatabase (20,273 sequences; European Bioinformatics Insti-tute, Swiss Institute of Bioinformatics and Protein InformationResource) using Mascot search engine (Matrix Science; version2.5.0; http://www.matrixscience.com/) Decoy database searchwas always activated and, in general, for merged LC/MS-MSanalysis of a gel lane with P < 0.01, FDR averaged around lessthan 1%. Scaffold (Proteome Software, Inc.) version 4_4_1 wasused to further validate and cross-tabulate tandem mass spec-trometry (MS-MS) based protein and peptide identifications.

Cell line xenograftsSix- to eight-week-old male NOD-SCID mice obtained from

Jackson Laboratory were injected in the subcutaneous flank with1�107NamalwaorRamos cells inPBS.Micewere evaluateddailyfor weight and tumor measurement. A 7.5 mg/mL PU-H71solution was prepared in a 10 mmol/L PBS and delivered viaintraperitoneal injection. A 5 mg/mL BEZ-235 solution wasprepared in 1:9 N-methylpyrrolidone:polyethylene glycol anddelivered via oral gavage. Tumor volumes were measured dailywith calipers and tumor volume was calculated as 0.5 length �width2. AUC (22) was used to measure tumor volume over time(GraphPad Prism software). Mice were sacrificed when tumorsreached a volume of 3,000 mm3 or at the completion of theobservation period (15 days). Tumors were harvested after ani-mals were sacrificed and sectioned for IHC staining.

Patient-derived xenograftPDXs were established by implanting fresh tumor fragments in

the subcutaneous flanks of NOD-SCID interleukin-2R gammanull (NSG)mice (n¼3). Tumor formationwasmonitoredweeklyby inspection and measurement using calipers. When the tumorsreached a volume of 1,000 to 1,500 mm3, the PDX tumors werecollected after humane euthanasia. The tumors were sectionedinto 2 mm fragments and implanted into additional animals forsubsequent drug testing.

Statistical analysisTwo-tailed unpaired t test was used unless otherwise specified.

Survival estimates were calculated using Kaplan–Meier analysis.All statistical analyses were carried out using Prism software(GraphPad).

Study approvalDe-identified patient samples were obtained under approval

from the Institutional Review Board (23) of Weill CornellMedical College. The Research Animal Resource Center of WeillCornell Medical College approved cell line xenograft studies.PDX studies were conducted at Columbia University under theapproval of the Columbia University Institutional Animal Careand Use Committee.

ResultsHsp90 is overexpressed in primary BL tumors

Hsp90 is ubiquitously expressed in human cells and is upre-gulated in conditions of chronic cellular stress including diseasestates and cancer (24). Hsp90 is overexpressed in a variety ofcancer subtypes and overexpression has been linked to moreaggressive disease and resistance to chemotherapy (25, 26). Toassess the potential for Hsp90 as a therapeutic target in BL, we firstevaluated Hsp90 expression in primary human BL tumors. IHCfor Hsp90was performed on a BL tissuemicroarray containing 59primary tumors and tonsil tissue for control. Fifty-three of 59 cases(90%) demonstrated high levels of Hsp90 expression defined as>90% tumor cell positivity. Quantification of Hsp90 by meanoptical density demonstrated a significant increase in expressionover normal tonsil (0.673 vs. 0.275, P¼ 0.001; Fig. 1A and B). InHsp90 þ tumors, nonmalignant infiltrating cells were negativefor Hsp90 (representative images shown in Fig 1B). Because thecell of origin for BL is the germinal center B-cell (27), we alsoexamined Hsp90 expression in normal GC cells in tonsil andlymph node specimens where GCs could be visualized andstaining of both tumor and GC cells was performed on thesame slide. GC B cells demonstrated moderate Hsp90 expres-sion relative to high expression seen in BL tumors (Supple-mentary Fig. S2).

Hsp90 inhibition induces growth arrest in human BL cell linesAlthough BL tumors were found to express high levels of

Hsp90, total amount of this chaperone protein does not neces-sarily reflect cellular dependency on this protein or sensitivity toHsp90 inhibitors (11). To determine the sensitivity of BL toHsp90 inhibition, we performed in vitro viability assays with theATP-based CellTiter-Glo assay in nine human BL cell lines(Ramos, DG-75, Raji, Namalwa, Daudi, Jiyoye, CA-46, Raji 2R,and Raji 4RH) and two DLBCL cell lines known to be sensitive toPU-H71 (Ly10, Ly18; Fig. 1C and Supplementary Fig. S3A). Cellswere exposed to increasing concentrations of PU-H71 and via-bility was assessed at 24, 48, and 72 hours. All cell lines demon-strated time and dose-dependent growth inhibition. Results wereconfirmed by evaluating cell viability with trypan blue (Supple-mentary Fig. S3B). The concentration of PU-H71 that inhibitedviability by 50% (IC50) was in the nanomolar range (171–337nmol/L) and was consistent across cell lines. The Raji 2R and Raji4RH cell lines which have acquired resistance to rituximab andmultiple chemotherapeutic agents (28) were equally sensitive toPU-H71 (IC50 175–181 nmol/L). To determine if PU-H71 wasbroadly toxic to GC B cells, we exposed primary human tonsiltissue to PU-H71 and evaluated viability in GC B cells using flowcytometry. Ramos cells but not GC B cells demonstrated substan-tial reduction in viability upon exposure to PU-H71 (Supplemen-tary Fig. S3C). To determine if the effect on BL growth was uniqueto PU-H71, we tested four additional small molecule Hsp90

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inhibitors in BL cells: BIIB-021, CUDC-305, ganetespib, andMPC-3100 (refs. 17–20; Fig. 1D). Cells were sensitive to all fourHsp90 inhibitors with IC50 ranging from 3.8 to 149.5 nmol/L.This work demonstrates that BL is broadly sensitive to Hsp90inhibition.

Given data from our group that Hsp90 inhibition may prefer-entially target gamma herpesvirus-associated malignancies (29),we included both EBV-positive (n ¼ 4) and EBV-negative (n ¼ 3)subtypes of BL in our PU-H71 screen including both the sporadicand endemic forms of BL (Fig. 1C). There was no difference insensitivity in EBV-positive and EBV-negative cells (IC50 290 and217 nmol/L, respectively, P ¼ 0.239). This suggests that themechanism of action of PU-H71 is independent of EBV-mediatedoncogenesis in BL. This may be explained by the restricted EBVlatency observed in BL where few viral proteins are expressed andare not major oncogenic drivers (30).

To determine the mechanism of growth inhibition, we evalu-ated the effect of treatment on cell cycle using flow cytometry andPI/Ki67 staining (Fig. 2A). Cell-cycle analysis revealed an accu-mulation of cells in G0 upon treatment with PU-H71, suggestingthat PU-H71 inhibits the growthof BLprimarily through cell-cyclearrest. An evaluation of apoptosis using flow cytometry andAnnexin V/7AAD staining revealed an increase in necrotic cells(annexin Vþ/7AADþ or annexinV�/7AADþ) by 72 hours oftreatment, but no corresponding increase in early apoptotic cells(annexinVþ/7AAD�) at any timepoint (Fig. 2B). Moreover, wedid not observe PARP cleavage as measured by Western blotanalysis (Fig. 2C) or increases in caspase 3 or 7 activity asmeasured by the fluorescent assay Caspase-Glo (Fig. 2D) consis-tent with a nonapoptotic mechanism of cell death. This is alsosupported by a lack of increase in the sub-G0 population in thecell-cycle analysis, which reflects DNA fragmentation and is

Figure 1.

BLs overexpresses Hsp90 and are sensitive to inhibition with Hsp90 inhibitors in vitro. A and B, Hsp90 protein expression as determined by IHC in primary BLtumors (n¼ 59) and control tonsil tissue (n¼ 3). Optical density wasmeasured (A) and is shown as average� SEM. B shows representative samples of tonsil and BLtumor, original magnification 600� with 60� objective lens. Microscope: Olympus BX 43; camera: Jenoptik ProgRes CF; software: ProgRes Mac CapturePro2.7.6. C and D, IC50 of a panel of EBV-positive and EBV-negative BL cell lines after exposure to PU-H71 or other Hsp90 inhibitors. Cells were exposed to increasingdoses of PU-H71, MPC-3100, ganetespib, CUDC-305, BIIB-021, or vehicle control (water þ DMSO) and evaluated for viability at 48 hours using theCellTiter-Glo assay. IC50 was calculated using Prism software (Graphpad). Results represent the mean of three biological replicates, each of which was performedin experimental triplicates. Error bars represent SEM. DLBCL cell lines known to be sensitive to PU-H71 (Ly10, Ly18) were used as positive controls.

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characteristic of apoptotic cell death. Based on this we concludethat PU-H71 induces cell-cycle arrest in BL followed by nona-poptotic cell death.

Hsp90 inhibition delays tumor growth and prolongs survivalin vivo

To determine the anti-lymphoma effect of Hsp90 inhibition invivo, we established Namalwa xenografts in NOD-SCID mice bysubcutaneous injection. After tumors reached 150 to 200 mm3,mice were randomized to receive PU-H71 or vehicle control

(4 mice/cohort). PU-H71 was administered daily by intraperito-neal injection at a dose of 50 mg/kg for 10 days. This dose waschosen based on a pilot experiment where we evaluated BLxenografts treated at 75 mg/kg i.p. daily, as previously reported(31, 32), and 50 mg/kg i.p. daily and found similar efficacy withboth doses. Mice were monitored daily for evidence of toxicityand measurement of tumor volume. Mice were sacrificed oncetumors reached 3,000mm3. To evaluate tumor growth over time,we calculated both tumor volume and tumor AUC as previouslydescribed (31).

Figure 2.

PU-H71 inhibits the growth of BL through cell-cycle arrest. A, Cells were treated with PU-H71 or vehicle control for 24 hours, harvested, fixed, and stainedwith Ki67-BV510 antibody followed by propidium iodide (PI) and 1� 105 cells were analyzed by flow cytometry. The average of three independent experiments arepresented with bars representing SEM. B, Cells were treated with 1 mmol/L PU-H71 or vehicle control for 2, 8, 24, 48, or 72 hours, stained with 7-AAD andAnnexin V-Alexa Fluor 647 and analyzed by flow cytometry. The average of three independent experiments is shown.C,Cells were treatedwith 1 mmol/L PU-H71 andevaluated for PARP cleavage at 2 hours. BC3 cells treated with 5 mmol/L of Bay11, which is known to induce apoptosis, were used as a positive control. D, Cellswere treated with 1 mmol/L PU-H71 at the indicated timepoints. Thirty minutes before harvesting, cells were stained with CAS-MAP Green (IntracellularTechnologies) working solution. After harvesting, cells were washed and resuspended in PBS for flow cytometry analysis. Cells undergoing apoptosis weredetected via fluorescence at 488 nm.






PU-H71 significantly decreased tumor growth relative tovehicle control in Namalwa bearing xenografts (P ¼ 0.0117; Fig.3A and B). Similar results were seen in a Ramos xenograft model(P ¼ 0.0057; Supplementary Fig. S4). There was no evidence oftoxicity from PU-H71 based on behavioral evaluation and serialweight measurements. More extensive toxicity profiling has beenperformed in previous studies including complete blood count,blood chemistry, liver function tests, and thyroid hormone testingwith no significant toxicity noted (10, 33). This safety profilefinding is concordant with that seen in the phase I clinical study ofPU-H71 in patients with previously treated solid tumors, lym-phoma, and myeloproliferative neoplasms (34).

Because cell line xenografts may not fully recapitulate diseasecharacteristics found in patients, we also evaluated the effect ofHsp90 inhibition in a BL PDX (Fig. 3C and D). Tumor for PDXstudies was obtained from a pediatric patient with an abdom-inal presentation of BL who underwent a biopsy prior to theinitiation of therapy. After written informed consent for PDXgeneration was obtained, surplus primary BL tumor sample wasobtained fresh. The tumor was implanted into the subcutane-ous flank space of NSG mice. Once tumors reached an approx-imate volume of 1,500 mm3, the animals (P ¼ 0) were eutha-nized and the tumor removed. This tumor was then separatedinto 2 mm fragments and implanted into (P ¼ 1) animals forsubsequent drug testing.

We first evaluated PDX tumors to determine if theywere similarto the patient tumor phenotype. PDX BL tumors resembledprimary BL by histology, immunophenotype, and cytogeneticanalysis. The PDX tumors demonstrated the classic starry sky

appearance on H&E, had a Ki67 proliferative index >90%, andwere positive for CD20 and BCL6 and showed only very weakstaining for BCL2, consistent with a BL immunophenotype. Inaddition, both the PDX and the primary tumor harbored thecharacteristic MYC translocation (Supplementary Fig. S5).

To study the effect of Hsp90 inhibition, PDXmice were treatedwith PU-H71 (n¼ 6) or vehicle (n¼ 6) once tumors reached 150to 200 mm3. PU-H71 was given at a dose of 50 mg/kg daily �15days. Mice were monitored daily for weight, tumor measure-ments, and toxicity. Treatment with PU-H71 resulted in a signif-icant decrease in tumor volume compared to control (P ¼0.0074; Fig. 3C). PDX mice did not demonstrate any evidenceof PU-H71 induced toxicity. The antitumor effect of PU-H71 wasmore pronounced in the PDX model than in either cell linexenograft model, highlighting the differences between cell linesand patient-derived tumors.

Proteomic screen reveals the Hsp90 oncoproteome in BLGiven the promising anti-lymphoma activity of PU-H71 in BL

models, we wanted to further explore the mechanism of action tobetter understandHsp90-dependent signalingnetworks inBL andto develop rational therapeutic combinations. To evaluate thetargets of PU-H71 in BL, we performed high-affinity capturefollowed by proteomic analysis (8). Cellular lysates from two BLcell lines (Ramos, Daudi) were incubated with PU-H71 conju-gated agarose beads to chemically precipitate Hsp90 complexes.The protein cargo was then subject to SDS-PAGE and proteinswere analyzed using LC/MS-MS. A total of 752 and 1,783 Hsp90client proteins were identified in Ramos and Daudi, respectively

Figure 3.

PU-H71 has anti-lymphoma effect invivo:A,NOD-SCIDmicewere injectedin the subcutaneous flank with 1� 107

Namalwa cells. When tumors reached150 to 200 mm3 mice were treatedwith PU-H71 or vehicle control. Tumorgrowth plot of mice treated with PU-H71 or vehicle control. Error barsrepresent SEM. B,Growth of tumor asmeasured by AUC. Average tumorgrowth is represented on the y-axis,which represents tumor volume(mm3)/time (days). Error barsrepresent SEM. C, PDX mice weregenerated using fresh tumor tissuefrom a patient with an abdominalpresentation of BL. Cells wereimplanted into NSG mice (P0) andonce palpable tumors developed,they were fragmented and implantedinto additional mice (P1) for furtherstudies. Once tumors reached 150 to200mm3,micewere treatedwith PU-H71 or vehicle control � 10 days. Atthe completion of treatment, all micewere humanely sacrificed for biologystudies. Tumor growth plot of micetreatedwithPU-H71orvehicle control.Error bars represent SEM. D, Growthof tumor as measured by AUC.Average tumor growth is representedon the y-axis, which represents tumorvolume (mm3)/time (days). Error barsrepresent SEM.

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(Supplementary Table S1). A total of 701 proteins were commonto both cell lines.

Ingenuity Pathway Analysis was used to determine the sig-naling networks represented by Hsp90 clients in BL. In both celllines, the PI3K/AKT/mTOR pathway was identified among themost highly represented canonical pathways including "PI3K/AKT signaling"; "p70S6K signaling"; "regulation of eIF4 andp70S6K signaling"; and "mTOR signaling" (SupplementaryTable S2). Other top hits which are relevant to lymphocytebiology, included "MYC mediated apoptosis," "EIF2 signaling,"and "protein ubiquitination pathway." To validate the IPAfindings, we performed an independent analysis of the commonproteins identified in both Ramos and Daudi using the SearchTool for the Retrieval of Interacting Genes/proteins (STRING)with a CI of 0.99. PI3K proteins formed one of the primaryclusters identified in this analysis (Fig. 4A). Other clustersincluded the proteasome, the ribosome, replication machinery,ribonucleoproteins, translation machinery, and the COP9 sig-nalosome. Both the STRING and the IPA analyses demonstrate

that PU-H71 is targeting PI3K signaling among other oncogenicpathways in BL.

PI3K/AKT/mTOR signaling pathway proteins are chaperonedby Hsp90 and require Hsp90 to maintain expression

Recent genomic and functional studies in BL indicate that PI3Ksignaling contributes to lymphomagenesis (4, 35). Given thatPI3K, AKT, and mTOR signaling were among the top networksidentified in our proteomic screen, we explored the relationshipbetween these pathways and Hsp90. We interrogated our prote-omic data and found significant enrichment for PI3K proteinsincluding: multiple isoforms of the PI3K catalytic subunit(PIK3CB, PIK3CD, PIK3CG), the PI3K regulatory subunit(PIK3R1, PIK3R4), and downstream elements (MTOR, S6K1, andEIF4E; Fig. 4B). These findings were validated using chemicalprecipitation with PU-H71-conjugated beads (Fig. 4C). Cellularlysates from Ramos and Daudi were incubated with PU-H71-conjugated beads. The protein cargo was then evaluated usingimmunoblot analysis probing for PI3K pathway proteins. In both

Figure 4.

PI3K signaling pathway proteins areHsp90 clients in BL. A, STRINGrepresentation of the union of proteinsidentified in Ramos and Daudichemical precipitations at 0.99confidence. B, A representation of thePI3K signaling pathway is shown.Proteins with red borders wereidentified as Hsp90 chaperone clientproteins by LC/MS-MS. C, Chemicalprecipitation with PU-H71 conjugatedbeads: lysates from Ramos and Daudiwere subjected to chemicalprecipitation with PU-H71 conjugatedbeads or Hsp90-inert control beads(CO) followed by immunoblotting forthe indicated PI3K pathway proteins.Hsp90 andMCL1 are used as a positiveand negative controls, respectively.






Ramos and Daudi, p110b, p110g , p110d, p85a, AKT, and eIF4Ewere precipitated with PU-H71 beads, confirming their role asHsp90 clients. In contrast, MCL1, which was not identified as anHsp90 client in our proteomic screen, was not precipitated by thePU-H71 beads.

To determine if PI3K pathway proteins require Hsp90chaperoning to maintain expression, we exposed Ramos andNamalwa cells to increasing doses of PU-H71 and then analyzedprotein abundance by immunoblot (Fig. 5A). P-mTOR, p70S6K,and AKT demonstrated both time- and dose-dependent decreaseupon exposure to PU-H71. To determine if this effect was uniqueto PU-H71,we also tested other smallmoleculeHsp90 inhibitors.TreatmentwithCUDC-305or ganetespib resulted in similar dose-dependent downregulation of PI3K signaling, confirming thedependence of these proteins on Hsp90 (Fig. 5B).

To determine if Hsp90 inhibition impacts PI3K/AKT/mTORsignaling in vivo, we performed IHC on Namalwa xenografttumors after exposure to PU-H71. NOD-SCIDmice were injectedwith 1� 107Namalwa cells andmonitored for tumor progression(n ¼ 9). Once tumors reached 500 mm3, mice were randomized1:2 to vehicle or a single dose of PU-H71 at 100 mg/kg. Tumors

were harvested 8 or 24 hours after treatment and evaluated byIHC. IHC for p-mTOR and p-p70S6K were negative in all casesincluding control, reflecting the transient nature of phosphory-lation of these proteins. 4EBP1, however, which is downstream inPI3K signaling was moderately decreased in the PU-H71-treatedmice at the 8-hour timepoint andmarkedly decreased by24hours(Fig. 5C). Of note, we also evaluated Ki67 and TUNEL in thismodel to determine if the mechanisms of cell death in vivo wereconsistent with what we observed in vitro. Similar to our in vitroexperiments, we observed a decrease in proliferation as evidencedby Ki67 upon treatment with PU-H71, which was statisticallysignificant at 8 hours (Supplementary Fig. S6A) and no increase inTUNEL (Supplementary Fig. S6B) consistent with a nonapoptoticmechanism for cell death.

Hsp90 inhibition is synergistic with PI3K/mTOR inhibitionin BL

Our data suggest that Hsp90 inhibition is effective in BL in partdue to the dependence of PI3K pathway proteins on Hsp90chaperoning. Given this, we were interested in evaluating theeffect of isolated PI3K inhibition on BL. PI3K inhibitors can

Figure 5.

PI3K pathway proteins depend on Hsp90 to maintain expression in vitro and in vivo. A and B, Ramos and Namalwa cells were exposed to PU-H71, CUDC-305,or ganetespib for 24 hours at the doses indicated and evaluated by immunoblot using the indicated antibodies. GAPDH was used as a loading control.Numerical quantification relative toGAPDH and expression in untreated cells is shown below each band (C). Namalwa xenograftswere generated by injecting 1� 107

cells into NOD-SCID mice. 10 to 15 days after injection, mice were treated with a single dose of PU-H71 at 100 mg/kg i.p. or vehicle control. Mice were humanelysacrificed at 8 or 24 hours after treatment to harvest tumors for IHC as indicated. Tumors from three mice in each condition (untreated, 8 hours, and 24 hours) areshown: original magnification 600� with 60� objective lens. Microscope: Olympus BX 41; camera: Olympus Q-COLOR3; software: QCapture Version 2.9.8.0(Quantitative Imaging).

Giulino-Roth et al.





broadly be categorized into class I PI3K isoform-specific inhibi-tors, pan-class I PI3K inhibitors, and dual class I PI3K/mTORinhibitors (36). We tested all three classes of PI3K inhibitors in apanel of BL cell lines. Cells were broadly resistant to isoformspecific PI3K inhibition with idelalisib/CAL101 (p110d inhibi-tor) and IPI-145 (p110d/g inhibitor). Cells demonstrated mod-erate sensitivity to pan-PI3K inhibition with the pan-class I PI3Kinhibitor BKM-120 (15). Cells were most sensitive to BEZ235,which targets both mTOR and all class I isoforms of PI3K(ref. 16; Table 1).

We suspected that PI3K inhibition would be most effective inBLwhenmultiple components of the pathway are targeted, whichcan be achieved by PU-H71 or BEZ235. Based on this, wehypothesized that PU-H71 might synergize with PI3K inhibitorsthrough more robust dual targeting of PI3K signaling and inhi-bition of possible feedbackmechanisms. To test this we evaluatedthe combination of PU-H71 and PI3K inhibitors in four BL celllines (Ramos, Namalwa, Daudi, DG-75). Cells were exposed to

increasing doses of each drug alone and the combination. Thecombined response at 48 and 72 hours was evaluated using theChou-Talalay method (37). A combination index (CI) of <1 wasconsidered synergistic. CI of 1 was considered additive and >1infra-additive. Because cells were resistant to idelalisib and IPI-145, CI could not be calculated for these combinations. Thecombination of PU-H71 and BKM-120was additive or synergisticin three of four cell lines. The combination of PU-H71 and BEZ235 was additive or synergistic in all four cell lines (Fig. 6A).

To further evaluate combination therapy with PU-H71 andBEZ235, we established Namalwa bearing xenografts. Oncetumors reached 150 to 200 mm3, mice were randomized toreceive PU-H71, BEZ235, the combination, or vehicle (6 mice/cohort). Mice were treated daily with PU-H71 at 50 mg/kgdelivered by intraperitoneal injection, BEZ235 at 50 mg/kg deliv-ered by oral gavage, the combination, or vehicle for 15 days. Assingle agents both PU-H71 and BEZ235 reduced tumor growthcompared to vehicle (P ¼ 0.0196 and 0.0004, respectively). The

Table 1. IC50 (nmol/L) of inhibitors of PI3K including isoform specific inhibitors (CAL-101, IPI-145), pan class I inhibition (BKM120), and dual PI3K/mTOR inhibition(BEZ-235)

IC50 (nmol/L)Cell line CAL-101 p110d IPI-145 p110d, g BKM-120 BEZ-235 PU-H71

Ramos R R 1,033 57 181DG-75 R R 2,009 41 238Daudi R R 2,215 35 260Namalwa R R 2,602 61 337

R, resistant.

Figure 6.

Hsp90 inhibition is synergistic withPI3K/mTOR inhibition in BL. A,Namalwa, Daudi, DG-75, and Ramoscells were treated with increasingdoses of PU-H71 alone, a PI3Kinhibitor as indicated alone, or thecombination. Cell viability wasmeasured using CellTiter-Glo.Synergy was calculated usingCompusyn. CI values <0.9 aresynergistic. Results represent themean of the experiment performed intriplicate � SEM. B, NOD-SCID micewere injected with 1 � 107 Namalwacells. When tumors reached 150 to200mm3mice were treated with PU-H71, BEZ235, the combination, orvehicle control. Tumor growth plot ofmice treated with PU-H71, BEZ235,the combination, or vehicle control.Error bars represent SEM. C, Growthof tumor as measured by AUC.Average tumor growth is representedon the y-axis, which represents tumorvolume (mm3)/time (days). Errorbars represent SEM.






combination of PU-H71 and BEZ235 resulted in marked tumorgrowth suppressionwithminimal or no growth inmost cases. Thecombination of PU-H71 and BEZ235 suppressed BL tumorgrowth more than vehicle (P � 0.001), PU-H71 alone (P ¼0.0008), or BEZ235 alone (P ¼ 0.0386; Fig. 6B and C).

DiscussionOur work has identified Hsp90 as a novel therapeutic target in

BL and has expanded our understanding of the function of Hsp90in BL. We found that BL tumors overexpress Hsp90 and that BLcells are sensitive to inhibition with small molecule Hsp90inhibitors. Hsp90 plays a crucial role in normal cellular functionby supporting the proteome machinery, which is involved in anumber of housekeeping activities. In states of cellular stressHsp90 exists in an active binding conformation, which is distinctfrom the "housekeeping" Hsp90 species and is uniquely sensitiveto small molecule inhibition (8, 38). This unique sensitivityallows for specific targeting of oncogenic signaling pathways withminimal disruption of "housekeeping" Hsp90. Immunoprecipi-tationwith PU-H71 conjugated beads allows specific pulldown ofHsp90-dependent oncogenic protein clients which are requiredby tumor cells to maintain growth (8, 10, 29, 31). This pulldownlikely includes not only proteins that bind directly to Hsp90 butalso those in the epichaperome network (co-chaperones, adap-tors, and folding enzymes) that are dependent on Hsp90 tomaintain the malignant phenotype (11). In this work we utilizedthe specificity of PU-H71 as an unbiased approach to characterizethe Hsp90-dependent chaperome in BL. We identified multiplecomponents of the PI3K/AKT/mTOR pathway as Hsp90 clients.We also found other essential pathways in BL to interact withHsp90 including MYC-mediated apoptosis, B-cell receptor sig-naling, and protein ubiquitination. Our findings highlight theimportance of these pathways in BL and provide a unique oppor-tunity to target multiple oncogenic pathways with a single agent.

The PI3K pathway is thought to play a role in BL pathogenesisbased on recent genomic and functional studies. BL tumors harborrecurrent alterations in TCF3 and its negative regulator ID3 whichresult in tonicB-cell receptor signaling andactivationofPI3K (2–4).In addition, a mouse model in which MYC and PI3K are consti-tutively active in germinal center B cells recapitulates the histomor-phology andclinical phenotype (35).Despite the apparent biologicdependence on PI3K, we found isolated PI3K inhibition to haveonly a modest effect in BL. We suspect that the improved anti-lymphoma effect of PU-H71over isolated PI3K inhibition is due totargeting of not just PI3K but multiple cellular pathways in BL thatdependonHsp90. It is also likely that isolatedPI3K inhibition inBLresults in activation of feedback loops and alternative survivalpathways, which facilitate escape. In contrast, Hsp90 inhibitionprevents escape by also inhibiting these feedback and alternativepathways, which are likely to also be Hsp90 dependent.

In aggressive lymphomas such as BL, single-agent therapy isunlikely to be curative. Therefore, the use of targeted therapieswillrequire rational combinatorial treatment. Here we demonstratesynergy between PU-H71 and dual PI3K/mTOR inhibition in BL.This treatment combination is particularly well suited for clinicalapplication as both categories of drugs are being evaluated inclinical trials in lymphoma and the drugs are not expected to haveoverlapping toxicities. A phase I study of PU-H71 is nearingcompletion and phase II studies are being developed. In addition,other next-generation Hsp90 inhibitors, which we also found to

be effective in BL, are moving toward clinical implementationwith minimal toxicity reported to date (39). Dual PI3K/mTORagents are also in phase I/II trials in lymphoma (NCT02669511,NCT02249429, NCT01991938).

Although the majority of patients with BL in resource richsettings are cured of their disease (>85%), there remain distinctpatient subsets for whom outcomes are poor and targeted ther-apies are needed (40). Specifically, patients with relapsed diseasehave a poor response to therapy and an expected overall survivalof <20%. In addition, patients with endemic BL have an OSranging from 25% to 62% due to the inability to safely deliverintensive chemotherapy in resource-limited settings (41). Ourwork suggests that Hsp90 inhibition-based treatment might beeffective in both settings. The sensitivity of chemotherapy andimmunotherapy resistant BL cells to Hsp90 inhibition (Raji 2R,Raji 4RH) suggests that this approachmay be effective in relapseddisease. Because we were only able to evaluate two resistant celllines, additional work on other models of resistant BL (such asPDX mice from refractory cases) will be needed to determine therole of PU-H71 in chemotherapy-resistant disease. With regard touse of this agent in the endemic subtype of BL, we observed ananti-tumor effect in endemic BL cell lines in-vitro (Raji, Daudi,Namalwa, Jiyoye) and in-vivo (Namalwa) suggesting that Hsp90inhibition may be a rational approach in sub-Saharan Africa.

In summary, our work demonstrates that small moleculeinhibition of Hsp90 suppresses PI3K/AKT/mTOR signaling andhas robust antitumor activity in BL. We describe, for the first time,the Hsp90-dependent oncoproteome in BL and identify multiplecomponents of PI3K/AKT/mTOR signaling as Hsp90 clients inthis malignancy. Finally, we identify a novel treatment strategy inBL through dual targeting of Hsp90 and PI3K/mTOR. Thisapproach may offer improved efficacy and reduced toxicity overstandard chemo-immunotherapy for patients with BL.

Disclosure of Potential Conflicts of InterestA. Rodina is a consultant/advisory board member for Samus Therapeutics.

T. Taldone is a consultant and a consultant/advisory board member for SamusTherapeutics. G.Chiosis is aDirector, boardmember at Samus Therapeutics; hasownership interest (including patents) of Samus Therapeutics. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: L. Giulino-Roth, E. CesarmanDevelopment of methodology: L. Giulino-Roth, H.J. van BesienAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Giulino-Roth, H.J. van Besien, T. Dalton,J.E. Totonchy, A. Rodina, H. Erdjument-Bromage, J. Sadek, A. Chadburn,F.S.D. Cruz, A. Rainey, A.L. KungAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Giulino-Roth, H.J. van Besien, T. Dalton,J.E. Totonchy, H. Erdjument-Bromage, J. Sadek, F.S.D. Cruz, A.L. KungWriting, review, and/or revision of the manuscript: T. Dalton, J. Sadek,A. Chadburn, M.J. Barth, E. Cesarman, Giulino-RothAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Giulino-Roth, H.J. van Besien, A. Bolaender,M.J. BarthStudy supervision: E. CesarmanOther (prepared and provided reagents): T. TaldoneOther (provided reagents and methods, and know-how for their use):G. Chiosis

AcknowledgmentsWe thank Susan Mathew, MD, for performing FISH analysis on PDX tumor

samples for the MYC translocation.

Giulino-Roth et al.





Grant SupportThis work was funded by grants from the NIH (R01-CA103646 and

R01-CA154288; to E. Cesarman), Lymphoma Research Foundation (toL. Giulino-Roth), Hyundai Hope on Wheels (to L. Giulino-Roth), St.Baldrick's Foundation (to L. Giulino-Roth), and the Leukemia and Lym-phoma Society (SCOR 7012-16; to E. Cesarman). G. Chiosis is funded byR01 CA172546 and R01 CA155226.

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

Received December 7, 2016; revised May 8, 2017; accepted June 8, 2017;published OnlineFirst June 15, 2017.

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2017;16:1779-1790. Published OnlineFirst June 15, 2017.Mol Cancer Ther Lisa Giulino-Roth, Herman J. van Besien, Tanner Dalton, et al. Antitumor Activity in Burkitt LymphomaInhibition of Hsp90 Suppresses PI3K/AKT/mTOR Signaling and Has

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