NVP-BEZ235 as a New Therapeutic Option for …...NVP-BEZ235 as a New Therapeutic Option for Sarcomas Maria C. Manara 1 , Giordano Nicoletti 1,3 , Diana Zambelli 1 , Selena Ventura

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Published OnlineFirst January 12, 2010; DOI: 10.1158/1078-0432.CCR-09-0816

Cancer Therapy: Preclinical Clinical
Cancer
Research
NVP-BEZ235 as a New Therapeutic Option for Sarcomas
Maria C. Manara1, Giordano Nicoletti1,3, Diana Zambelli1, Selena Ventura1, Clara Guerzoni1,Lorena Landuzzi1, Pier-Luigi Lollini3,4, Saveur-Michel Maira6, Carlos García-Echeverría6,Mario Mercuri2,3, Piero Picci1,3, and Katia Scotlandi1,3,5

Abstract

Authors'Div is ioneInterdipartiEmatologiadi RiferimeOrtopedicBioMedical

Note: SuppResearch O

Correspondi Barbiano39-051636

doi: 10.115

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Purpose: To evaluate the in vitro and in vivo effects of NVP-BEZ235, a dual pan-phosphoinositide3-kinase–mammalian target of rapamycin inhibitor in the three most common musculoskeletal tumors(osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma).Experimental Design: Antiproliferative activity as well as the effects on migration and metastasis were

evaluated in a panel of osteosarcoma, Ewing's sarcoma, as well as rhabdomyosarcoma cell lines. More-over, simultaneous and sequential treatments were done in association with two of the most importantconventional drugs in the treatment of sarcoma, doxorubicin and vincristine.Results: NVPBEZ235 effectively blocked the pathway in in vitro and in vivo settings. Under the exper-

imental conditions tested, the compound induced disease stasis, by arresting cells in G1 phase of cellcycle, without remarkable effects on apoptosis. As a consequence, to obtain the maximum exploitationof its therapeutic potential, NVP-BEZ235 has been evaluated in combination with conventional cytotoxicagents, thus showing promising efficacy with either doxorubicin and vincristine. Inhibition of the phos-phoinositide 3-kinase/mammalian target of rapamycin pathway increased activation of extracellularsignal-regulated kinase 1/2, likely due to the presence of autocrine circuits shifting growth factor signalingtoward the mitogen-activated protein kinase pathway. This supports the combined use of NVP-BEZ235with other small signaling inhibitors. Here, we showed synergistic effects when the compound wasassociated with a anti–insulin-like growth factor-I receptor tyrosine kinase inhibitor. NVP-BEZ235 alsoinhibited cell migration and metastasis. Combination with vincristine further potentiated the antime-tastatic effects.Conclusions: NVP-BEZ235 displays the features to be considered for sarcoma therapy to potentiate the

activity of other anticancer agents. The drug is currently undergoing phase I/II clinical trials in advancedcancer patients. Clin Cancer Res; 16(2); 530–40. ©2010 AACR.

Rhabdomyosarcoma, Ewing's sarcoma, and osteosar-coma, here collectively called musculoskeletal sarcomas,share several fundamental biological and clinical features.Unlike carcinomas, which affect the elderly and are pre-ceded by a long natural history of preneoplastic lesions,musculoskeletal sarcomas arise abruptly in infants andadolescents (1, 2). The progression of carcinomas througha sequence of preneoplastic and neoplastic stages allowedthe molecular definition of multiple carcinogenic events,

Affiliations: 1Laboratorio di Ricerca Oncologica and 2VOrtoped ica , Is t i tu to Ortoped ico Rizzo l i ; 3Cent ro

mentale di Ricerche sul Cancro “G. Prodi”; 4Dipartimento die Scienze Oncologiche, Universita di Bologna; and 5Centronto Specialistico Sviluppo di Terapie Biomolecolari, Istitutoo Rizzoli, Bologna, Italy; and 6Novartis Institutes forResearch, Novartis Pharma AG, Basel, Switzerland

lementary data for this article are available at Clinical Cancernline (http://clincancerres.aacrjournals.org/).

ding Author: Katia Scotlandi, Istituto Ortopedico Rizzoli, Via1/10, 40136 Bologna, Italy. Phone: 39-0516366760; Fax:

6761; E-mail: [email protected].

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whereas the natural history of musculoskeletal sarcomasis still mostly unknown. Genes causally involved in canceronset and progression are being progressively turned intotherapeutic targets, and a bonanza of selective inhibitors,ranging from small molecules to monoclonal antibodies,is revolutionizing the therapeutic approach to carcinomaswhile therapy of musculoskeletal sarcomas is substantiallystill firmly entrenched in conventional cytotoxic drugs.This is partly the consequence of an insufficient knowl-edge of relevant therapeutic targets. However, in the lastyears, with an increasing number of molecular pathwaysbeing actively investigated, new systemic treatments havebeen proposed and targeted therapies have been success-fully proved for some soft tissue sarcomas (for a review,see ref. 3) or are on the horizon for others. Regarding rhab-domyosarcoma, Ewing's sarcoma and osteosarcoma pre-clinical data convincingly supported the development ofinsulin-like growth factor-I receptor (IGF-IR) inhibitorsin the therapy of sarcoma (for a review, see ref. 4, 5).The antitumor and antimetastatic effects against Ewing'ssarcoma and osteosarcoma xenograft models with anti-IGF-IR–targeted drugs have been shown (6–8). Recently,

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Translational Relevance

Data show a broad antiproliferative activity of thenew dual phosphoinositide 3-kinase/mammalian tar-get of rapamycin inhibitor NVP-BEZ235 on a panelof sarcoma cell lines. The inhibitor induced cytostaticeffects, with no induction of apoptosis. As a conse-quence, to obtain the maximum exploitation of itstherapeutic potential, NVP-BEZ235 has been evaluatedin combination with conventional cytotoxic agents,thus demonstrating promising efficacy with eitherdoxorubicin and vincristine. NVP-BEZ235 also inhib-ited cell migration and metastasis. Combination withvincristine further potentiates the antimetastatic ef-fects. In addition, the drug may be of interest also inprevention of resistance to some new targeted drugs,such as the anti– insulin-like growth factor-IR HAbsand the allosteric and specific mTORC1 inhibitor(e.g., rapamycin and its analogues).

NVP-BEZ235 an Effective New Drug for Sarcomas


phase I clinical studies with human neutralizing antibo-dies to IGF-IR showed stabilization of disease (9, 10),and three of eight patients with Ewing's sarcoma treatedwith R1507, a humanized monoclonal antibody to IGF-IR, developed partial response to treatment in a phase Itrial.7 Thus, phase II trials of IGF-IR inhibitors in Ewing'ssarcoma, osteosarcoma, rhabdomyosarcoma, and othersarcomas are under way. In these trials, neutralizing anti-bodies to IGF-IR have been generally preferred. However,the small molecular mass inhibitors maintain several po-tential advantages, including easier administration andpotential higher flexibility in combination therapy. Be-cause most of the IGF-IR actions are mediated by the acti-vation of phosphatidylinositol-3 kinase 10-Akt andmitogen-activated protein kinase (MAPK) pathways, apossible alternative to the use of specific small molecularmass tyrosine kinase IGF-IR inhibitors is represented bythe new generation of phosphoinositide 3-kinase (PI3K)or MAPK pathway modulators that mostly overcome ear-lier problems of poor selectivity, unfavorable pharmacoki-netic profiles, and unacceptable toxicity (for a review, seerefs. 11–13). A number of these agents have entered early-phase clinical trials. NVP-BEZ235 is a synthetic smallmolecular mass compound belonging to the class of imi-dazoquinolines that potently and reversibly inhibits class1 PI3K catalytic activity by competing at its ATP-bindingsite. NVP-BEZ235 also inhibits mammalian target of rapa-mycin (mTOR) catalytic activity (14), and this makes thecompound particularly interesting for sarcomas (15). The

7 Benjamin R., et al., CTOS 13th annual meeting, Seattle, WA, abstract 932.

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mTOR signaling pathway is abnormally activated in manyhuman tumors that have multiple mTOR alterations bothupstream and downstream, leading to its dysregulation. Inrhabdomyosarcoma, high levels of phosphorylation inseveral components of the pathway, such as Akt, 4E-BP1,S6K1, and eIF-4G, and low levels of 4E-BP1 expression areassociated with poor overall and disease-free survival (16).Thus, the mTOR pathway is considered an important ther-apeutic target. Currently, the allosteric and specificmTORC1 inhibitor rapamycin and its derivatives CCI-779, RAD001, AP23573 are being evaluated in cancer clin-ical trials. For targeted therapies of sarcomas, AP23573 hasshown promising clinical efficacy and low toxicity profilesin patients (17). However, resistance to rapamycin is a fre-quent event and involves cross-talk with IGF-IR signalingand Akt activation (18–20). Some patients treated with ra-pamycin analogues showed an increase in p-Akt in tumorsand this effect is thought to be one of the reasons of theirstill limited clinical activity (19, 21). Therefore, a dualPI3K/mTOR catalytic inhibitor, such as NVP-BEZ235,may be of great help. In this report, we show that NVP-BEZ235 is active against the three most common muscu-loskeletal sarcomas by inhibiting tumor cell growth, mi-gration, and metastasis.

Materials and Methods

Cell lines. A panel of nine osteosarcoma, nine Ewing'ssarcoma, and five rhabdomyosarcoma cell lines was ana-lyzed. The osteosarcoma cell lines Saos-2, U-2OS, andMG-63 and the Ewing's sarcoma cell lines SK-ES-1, SK-N-MC, and RD-ES were all obtained from the AmericanType Culture Collection. The alveolar rhabdomyosarcomacell lines SJ-RH30 and SJ-RH4 were provided by Dr. A.Rosolen (University of Padua, Padua, Italy) and Dr. D.N.Shapiro (St. Jude Children's Hospital, Memphis, TN). Ew-ing's sarcoma cell lines TC-71 and 6647 were kindly pro-vided by T.J. Triche (Children's Hospital, Los Angeles, CA).All other osteosarcoma and Ewing's sarcoma cell lineswere obtained from the Rizzoli laboratories and were pre-viously described (22). The alveolar rhabdomyosarcomacell line RMZ-RC2 (named RC2) and the embryonal rhab-domyosarcoma cell line CCA were established in the Can-cer Research Section, Department of ExperimentalPathology, University of Bologna, Bologna, Italy (23,24). The RD/18 cell line is a clone, obtained at the CancerResearch Section, University of Bologna, Bologna, Italy, ofthe commercially available human embryonal rhabdo-myosarcoma cell line RD (Flow Laboratories). Cells wereroutinely cultured in Iscove's modified Dulbecco's medi-um supplemented with 20 U/mL penicillin, 100 μg/mLstreptomycin (Sigma), and 10% heat-inactivated fetalbovine serum (Lonza).Drugs. NVP-BEZ235 and NVP-AEW541 were kindly pro-

vided by Novartis Pharma. Stock solution of this drug wasprepared in DMSO and was stored at −20°C. Doxorubicinand vincristine were purchased from Sigma.

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In vitro treatments with NVP-BEZ235 alone or in combi-nation with other drugs. Cells were plated into 6-well(200,000 cells per well) or 96-well plates (2,500 cellsper well) in Iscove’s modified Dulbecco’s medium plus10% fetal bovine serum. After 24 h, various concentrationsof NVP-BEZ235 (1 nmol/L to 10 μmol/L) were added andcells were exposed up to 72 h. Cell proliferation was eval-uated either by trypan blue vital cell count or by MTT assay(Roche) according to the manufacturer's instructions. Cellswere also treated with DMSO-containing medium as acontrol. The final concentration of DMSO in the mediumwas <0.001%, and it had no effect on cell growth inhibi-tion. For combined treatments, cells were treated for 72h with varying concentrations of doxorubicin (0.3-100ng/mL) or vincristine (0.01-30 ng/mL), or NVP-AEW541(30 nmol/L to 3 μmol/L), a selective tyrosine kinase inhib-itor against IGF-IR (6), without (control) or with NVP-BEZ235 (50 nmol/L for TC-71 and 100 nmol/L for U-2OS, corresponding to the dose that gives ∼30% growth in-hibition in these cell lines). In sequential treatments, cellswere exposed to doxorubicin or vincristine for 12 h (a timethat corresponds to the in vitro doubling time of these celllines) and then to NVP-BEZ235 for 72 h. Otherwise, cellswere treated for 6 h with or without NVP-BEZ235 andthen with doxorubicin or vincristine for 72 h.Analysis of cell cycle and apoptosis was done on cells

treated with NVP-BEZ235 for 24 or 48 h accordingly toprocedures previously described (6).Parameters of in vitro malignancy. Anchorage-indepen-

dent growth was determined as previously described (6) in0.33% agarose (SeaPlaque, FMC BioProducts) with a 0.5%agarose underlay. The number of cells per dish was as fol-lows: 3,300 for TC-71, SK-N-MC, 6647, RD-ES, and SKES-1; 10,000 for U-2OS and Saos-2; and 33,000 to 100,000for the other cell lines. Motility assay was done usingTrans-well chambers (Costar) as previously described (7).In addition, cells were pretreated with NVP-BEZ235 for 12h, were counted, and 100,000 viable cells were seeded in theupper chamber for migration analysis to rule out possibleeffects of drug treatment on cell vitality that might affect cellmigration. The wound healing assay was done using a pi-pette tip on confluent cells. Cell migration was visualizedat regular intervals of time (1-24 h) at ×100 magnificationusing an inverted microscope (Nikon Diaphot).Western blotting. Semiconfluent cells were treated with

increasing doses of NVP-BEZ235 for 1 to 24 h in stan-dard medium. Cell lysates were prepared and processedas previously described (6). Membranes were incubatedovernight with the following primary antibodies: anti–phospho-Akt (Ser473; Cell Signaling Technology, Inc.; di-lution, 1:1,000), anti-Akt (Cell Signaling Technology;dilution, 1:1,000), anti–phospho-mTOR (Ser2448; CellSignaling Technology; dilution, 1:1,000), anti-mTOR(Cell Signaling Technology, dilution, 1:1,000), anti–extracellular signal-regulated kinase (ERK; Cell SignalingTechnology; dilution, 1:2,000), anti–phospho-S6(Ser240/244; Cell Signaling Technology; dilution,1:3,000), anti-S6 (Cell Signaling Technology; dilution,

Clin Cancer Res; 16(2) January 15, 2010


1:2,000), and anti–phospho-ERK (Tyr202/Tyr204; Cov-ance, Princeton; dilution, 1:1,000). Anti-rabbit andanti-mouse antibody conjugated to horseradish peroxi-dase (GE Healthcare) was used as secondary antibody.Akt phosphorylation by cell-based ELISA. A cell-based

ELISA kit (SABiosciences) was used tomeasure Akt phosphor-ylation. Briefly, cells were seeded into 96-well microplatesat the density of 5 × 103 per well in Iscove’s modified Dul-becco’s medium plus 10% fetal bovine serum. After 48 h,cells were fixed in 4% formaldehyde at room temperatureand were processed according to the manufacturer's in-structions. Absorbance readings were normalized to rela-tive cell number as determined by a cell staining solutionand to the amount of total protein.In vivo treatments with NVP-BEZ235 alone or in combi-

nation with vincristine. Athymic Crl:CD1-Foxn1nu (nude)mice were purchased from Charles River Italy. BALB-Rag2−/−;γc

−/− breeders were kindly provided by the CentralInstitute for Experimental Animals. Mice were bred inthe Animal Care Facility of Section of Cancer Research,Department of Experimental Pathology, University ofBologna (Prof. Pier-Luigi Lollini), under sterile condi-tions. Seventeen- to 34-wk-old female BALB-Rag2−/−;γc

−/−

mice or 4- to 5-wk-old female nudemice were used. Tumor-igenicity was determined after the s.c. injection of 5 × 106

cells in nude mice; metastatic ability was evaluated afterthe i.v. injection of 2 × 106 cells in BALB-Rag2−/−;γc

−/−

mice. For the evaluation of treatment effectiveness, micewere randomized into controls (seven animals) and trea-ted groups (five animals/group) when tumors started tobe measurable (7 d after cell inoculation). In the grouptreated with NVP-BEZ235 alone, each mouse orally re-ceived 25 or 50 mg/kg dissolved in NMP 10% (1-methyl-2-pyrrolidone)/PEG300 90% (Fluka) daily, 5 d weekly,for 3 to 4 wk. A group received vincristine alone i.p.(1 mg/kg/d) on days 0 and 1 of treatment. Another groupreceived either NVP-BEZ235 and vincristine following thetime schedule mentioned above. The control group wastreated with NMP10%/PEG300 90% orally only. Tumorvolume was calculated as π[√(a·b)]3/6 where a is the max-imal tumor diameter and b is the tumor diameter perpen-dicular to a. Lung, kidney/adrenal, and liver metastaseswere counted using a dissection microscope. All animalexperiments were authorized by the local ethics commit-tee, and mice were treated according to institutional andEuropean Union guidelines.Immunohistochemistry. Avidin-biotin-peroxidase proce-

dure was used for immunostaining, as previously de-scribed (7). For immunohistochemical detection of allthe antigens, sections were pretreated with a citrate buffersolution [0.01 mol/L citric acid and 0.01 mol/L sodiumcitrate (pH 6.0)] in a microwave oven at 750 W for threecycles of 5 min each. The primary antibodies used wereas follows: anti-phosphorylated Akt (Ser473; Cell Signal-ing Technology; dilution, 1:10), anti–phospho-ERK1/2(Tyr202/Tyr204; Covance; dilution, 1:10), and anti-KI67(DAKO; dilution, 1: 100).

Clinical Cancer Research





Statistical analysis. Differences among means wereanalyzed using a two-sided Student's t test. When datawere not normally distributed, the nonparametric Mann-Whitney rank-sum test was used. IC50 values were cal-



culated from linear transformation of dose-responsecurves. To define drug-drug interactions (in terms ofsynergism, additivity, or antagonism), the combination in-dex (CI) of each two-drug treatment was calculated with

Fig. 1. NVP-BEZ235 inhibits PI3K/mTOR pathway mediators. A, dose-dependent effects on PI3K/mTOR family members in TC-71 Ewing's sarcoma, U-2OSosteosarcoma, and RD/18 rhabdomyosarcoma cells after 24 h of exposure to NVP-BEZ235. B, time course inhibitory effects of NVP-BEZ235 on PI3K/mTOR pathway members in U-2 OS cells. In A and B, phosphorylation level of Akt, mTOR, S6, and Erk1/2 was revealed by Western blotting. Total proteinswere detected as controls. C, absorbance readings related to the amount of p-Akt in time course experiments. Once normalized to the amount of totalprotein, the phosphorylation level is directly related to the extent of activation. Data from two separate cell-based ELISA tests (three wells per test).




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Fig. 2. NVP-BEZ235 inhibits proliferation but it does not induce apoptosis in sarcoma cells, independently from the basal level of p-Akt or p-mTOR.A, in vitro sensitivity of nine Ewing's sarcoma, nine osteosarcoma, and five rhabdomyosarcoma cell lines to NVP-BEZ235. Cells were exposed for 72 h todifferent doses of the compound and IC50 doses were calculated. Columns, mean of three independent experiments; bars, SEM. B, constitutiveactivation of PI3K/mTOR pathway on a representative panel of sarcoma cell lines maintained in standard culture conditions. C, cell cycle analysis ofNVP-BEZ235 effects after 24 h of treatment in three representative cell lines. Data as percentages of mean of two independent experiments. Percentage ofcells in the G1 phase are significantly different after treatments compared with controls (P < 0.01 by Student's t test). D, Annexin V Fluorescent testwas used to identify cells in apoptosis or necrosis after 24 h of NVP-BEZ235 treatment. Data as percentages of mean of two independent experiments.

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Fig. 3. NVP-BEZ235 inhibits tumor cell growth in anchorage-independent conditions and cell migration. A, effects of NVP-BEZ235 on sarcoma cells inanchorage-independent conditions. Columns, mean of three experiments; bars, SEM. *, P < 0.05; **, P < 0.01 by Student's t test. Representative pictures ofNVP-BEZ235 effects on colony formation from RD/18 rhabdomyosarcoma cells. B, migration ability of TC-71 and U-2OS cells after treatments withNVP-BEZ235 for 18 h. Columns, mean of three experiments; bars, SEM. *, P < 0.05, Student's t test. C, effects of NVP-BEZ235 on cell migration, asdetermined by wound assay. Representative pictures after 6 and 12 h of treatments are shown.

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the isobologram equation (25) by using the CalcuSynsoftware (Biosoft).

Results

NVP-BEZ235 inhibits PI3K/mTOR signaling andefficiently affect growth, migration, and adhesion ofsarcoma cells. Dose-response experiments showed that



NVP-BEZ235 was able to inhibit the phosphorylationof Ser473 Akt, Ser2448 mTOR, and Ser240/244 S6 in tworepresentative cell lines, without significant differencesamong them (Fig. 1A). Time course experiments showedtransient inhibition of pAkt, which progressively regainedits basal phosphorylation levels, whereas mTOR and pS6seemed tobemore sensitive to the drug and remained inhib-ited for at least 24 h starting from the dose of 100 nmol/L

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Fig. 4. NVP-BEZ235 showspositive association withconventional and anti–IGF-IRdrugs. A, effects of simultaneouscombined or sequential treatmentsof NVP-BEZ235 in association withdoxorubicin (DXR) or vincristine(VCR). In sequential treatments,cells were first exposed todoxorubicin and vincristine for12 h and then to NVP-BEZ235.Isobologram analysis isshown. The individual doses ofNVP-NEZ235, and doxorubicin orvincristine to achieve 90% (straightline) growth inhibition (ED90),75% (dotted line) growthinhibition (ED75), and50% (hyphenated line) growthinhibition (ED50) were plotted onthe X- and Y-axes. CI valuescalculated using Calcusynsoftware are represented by pointsabove, on, or below the lines,indicating antagonism, additive,or synergy, respectively. B,isobologram analysis ofcombination of NVP-BEZ235with the anti–tyrosine kinaseIGF-IR inhibitor, NVP-AEW541.CI values are represented by pointsabove, on, or below the lines,indicating antagonism, additive,or synergy, respectively.


ssociation for Cancer




(Fig. 1B). The transitory effects on pAkt were also confirmedby cell-based ELISA assay (Fig. 1C). Inhibition of PI3Kpathway induced an increased activation of pERK1/2(Fig. 1B).



Effects of NVP-BEZ235 on cell proliferation wereexamined in a panel of nine Ewing's sarcoma, nineosteosarcoma, and five rhabdomyosarcoma cell lines (Sup-plementary Fig. S1; Fig. 2A). Rhabdomyosarcoma was the

Fig. 5. NVP-BEZ235 inhibits tumor growth and increases the effects of vincristine (VCR). A, inhibition of TC-71 tumor growth in nude mice by NVP-BEZ235in association or not with vincristine. Mice were orally treated for 5 d weekly, once per d with NVP-BEZ235 (25 or 50 mg/kg) or with vehicle alone(controls). In combined treatments, vincristine (1 mg/kg/d i.p.) was added on days 0 and 1 of treatment [P < 0.05, Student's t test compared with controls(CTR)]. Point, tumor size mean; bars, SEM. B, immunohistochemical evaluation of p-Akt, p-mTOR, p-Erk-1/2, and Ki-67 in controls or in tumors derivedfrom mice treated with NVP-BEZ235 (50 mg/kg orally). Representative figures are shown (magnification, ×200). C, representative H&E-stained lung sectionsof controls and mice treated with NVP-BEZ235 plus vincristine (magnification, ×100 or ×200 for inserted small figures; lungs were previously perfusedwith black India ink to count metastases). D, representative livers and kidneys from controls and mice treated with NVP-BEZ235 plus vincristine.




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most sensitive tumor (IC50 mean values: rhabdomyosarco-ma versus Ewing's sarcoma or versus osteosarcoma,P < 0.05; Student's t test). However, all cells tested were po-tently inhibited by NVP-BEZ235, with IC50 values rangingfrom 6 nmol/L to approximately 500 nmol/L. The differ-ences in sensitivity did not correlate with the basal levelof phosphorylation of Akt or mTOR (Spearman's corre-lation test; Fig. 2B). Cell cycle progression was inhibitedand the proportion of cells in the G1 phase was substan-tially increased after treatment with the inhibitor (P <0.01, Student's t test; Fig. 2C). In contrast, no inductionof apoptosis was observed (Fig. 2D).Growth inhibitory effects were confirmed in anchorage-

independent conditions. NVP-BEZ235 treatment re-duced both the number and the size of cell colonies insoft-agar as shown in Fig. 3A. Cell migration was also in-hibited by NVP-BEZ235 (Fig. 3B and C). Effects weredose- and time-dependent.NVP-BEZ235 effects in combination therapy and against

chemoresistant cells. Simultaneous and sequential treat-ments were made in association with two of the most im-portant conventional drugs in the treatment of sarcomas,doxorubicin and vincristine. Synergistic or additive effectswere observed with both drugs (synergism: CI < 0.9; addi-tive: 0.90 ≤ CI ≤ 1.10 according to ref. 25; Fig. 4A). In se-quential treatments, chemotherapeutic agents should beadministered before NVP-BEZ235 to obtain advantageouseffects (Supplementary Fig. S2; Fig. 4A). The administra-tion of NVP-BEZ235 before the chemotherapeutic agentsinduced subadditive effects (CI ≥ 1.1, according toref. 25; Supplementary Fig. S3), likely because of theNVP-BEZ235–induced G1 blockade that reduced the in-hibitory cell cycle dependent effects of vincristine anddoxorubicin. Considering that Akt is only a part of theIGF-IR signaling and that inhibition of PI3K pathway in-duces compensatory activation of p-ERK1/2 in sarcomacells (Fig. 1), we evaluated the possible combination ofNVP-BEZ235 with more specific anti-IGF-IR–targeteddrugs. Systematic analysis of relationship between NVP-BEZ235 and components of different signaling pathways,



including IGF-IR–mediated pathway, was done by Mairaet al. (14). In this report, we show synergistic (U-2 OScells) or additive (TC-71 cells) effects when NVP-BEZ235is combined with the TK inhibitor NVP-AEW541 (syner-gism: CI < 0.9; additive: 0.90 ≤ CI ≤ 1.10 according toref. 25; Supplementary Fig. S2; Fig. 4B). This advantageouseffect may also be due to the ability of IGF-I to revert p-Aktinhibitory activity of NVP-BEZ235 at doses as low as 50 to100 nmol/L (Supplementary Fig. S4). Because sarcomacells show autocrine activation of the IGF-IR pathway(26, 27) and are constantly exposed to high levels ofIGF-I stored in bone tissue, simultaneous inhibition ofboth MAPK and PI3K pathways may be required to obtaina significant antiproliferative effect.Activity of NVP-BEZ235 against tumor growth and metas-

tasis. The antitumor activity of NVP-BEZ235 was analyzedin a xenograft model from TC-71 Ewing's sarcoma cellline. The model is very aggressive with a latency time of7 days after s.c. injection of 5 × 106 cells. When tumorswere measurable and reached an average volume of0.1 cm3, the animals were treated with two doses ofNVP-BEZ235 (25 or 50 mg/kg). The inhibitor reduced tu-mor growth rate (Fig. 5A), but no statistical significancewas obtained when tumors were treated with NVP-BEZ235alone or with vincristine. The positive and necessary associa-tion of NVP-BEZ235 with vincristine was confirmed in vivobecause only combined treatments induced a significantshrink in tumor size. No significant changes in the weightof the tumor-bearing mice were observed after treatmentswith NVP-BEZ235 (data not shown). Specific effects ofNVP-BEZ235 on the PI3K/Akt pathway in vivo were con-firmed by immunohistochemistry on tumor tissues atthe end of treatments (Fig. 5B). Consistently with the cy-tostatic effects induced by NVP-BEZ235, KI-67 immunos-taining was also reduced following treatments with theinhibitor (Fig. 5B).Activity against metastasis was studied in RD/18 rhab-

domyosarcoma experimental model because this cell linegave the broadest spectrum of metastasis (lungs, liver, andkidneys) when injected in BALB-Rag2−/−;γc

−/− mice, which

Table 1. Activity of NVP-BEZ235 against RD/18 metastasis in Rag2−/−; γc−/− mice

Lungs metastases

h

Liver metastases

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Kidney/adrenal metastases

Incidence
Median Range Weight mean ± SEM Incidence
Clinica

ican Associat

Median

l Cancer R

ion for Can

Range

CTR
4/4 >200 72->200 4,172 ± 373 4/4 7 2-11 50 mg/kg NVP-BEZ235 5/5 38 9-191 1,166 ± 49 3/5 2 0-2 VCR 5/5 30 6-49 1,272 ± 66 3/5 1 0-4 50 mg/kg VCR-BEZ235 3/5 13 0-51 1,068 ± 26 2/5 0 0-1
NOTE: Number of lung and kidney/adrenal metastases were obtained using a dissection microscope. Liver metastases were un-countable so that liver weight was used as a measure of the treatment efficacy. All the treatments gave significant differencescompared with controls (P < 0.05, Mann-Whitney or Student's t test). Combined treatments gave additional benefits althoughno statistical significance was achieved with respect to single treatments.

esearch

cer




lack both the recombinase activating gene 2 (Rag2) and thecommon cytokine receptor γ chain (γc), and show acomplete absence of T, B, and natural killer response(28). NVP-BEZ235 significantly inhibited metastasis,either alone or in combination with vincristine (Table 1;Fig. 5C and D).

Discussion

In this study, we analyzed the therapeutic potential ofthe novel dual inhibitor of PI3K/mTOR NVP-BEZ235against the most common musculoskeletal tumors (Ew-ing's sarcoma, osteosarcoma, and rhabdomyosarcoma).NVP-BEZ235 is an ATP competitor that reversibly reducesthe kinase activity of both PI3K and mTOR in wild-typeand mutated p110 (29), as well as in cells with PTEN loss(12), thus offering a valid therapeutic agent for the treat-ment of solid tumors with mutated PI3K.In sarcomas, aberrant activation of PI3K is mainly due

to the existence of redundant autocrine circuits rather thanmutations. IGF-IR signaling is commonly activated (4),and constitutive activation of the two major signalingmediators, MAPK and PI3K, have been described (6, 30).Here, we confirmed that PI3K and mTOR are generally ac-tive in cell lines and showed the specificity of action ofNVP-BEZ235 to inhibit several PI3K pathway effectors, in-cluding Akt, mTOR, and the ribosomal protein S6. Consis-tent with the data recently obtained in carcinoma (29), wealso observed differential time course effects on Akt ormTOR and S6. Although cell exposure to NVP-BEZ235 re-sulted in a sustained inhibition of mTOR and S6 for atleast 24 hours, the effects on pAkt are transient and themediator regained its activity after 6 to 24 hours, depend-ing on the dose. This effect may be due, at least partly, tothe loss of negative p70 S6K-IRS1 feedback (15) on pAkt,as a consequence of the disruption of pS6 activity by NVP-BEZ235. In our hands, inhibition of the PI3K pathway in-duced the activation of ERK1/2 phosphorylation, likelydue to the presence of redundant autocrine circuits in sar-coma cells (26), mainly IGF-IR mediated, that may shiftgrowth factor receptor signaling on MAPK when PI3K isinhibited. Thus, concomitant inhibition of MAPK pathwayshould be emphasized. Accordingly, we observed synergis-tic effects when NVP-BEZ235 was combined with NVP-AEW541, presumably by concomitant inhibition of MAPKpathway and full suppression of IGF-I actions (6, 31), sug-gesting that one may achieve improved antitumor activityby targeting two components of the integrated receptor-signaling pathways.NVP-BEZ235 blocked cell proliferation, by inducing G1

arrest, in all sarcoma cell lines (23 cell lines), independ-ently of their basal levels of activation of the PI3K/mTORpathway. Among the three histotypes, rhabdomyosarcomawas the most sensitive (IC50 values between 1-30 nmol/L),likely due to its higher sensitivity to mTOR inhibitors (15).However, the drug was active at doses lower than 1 μmol/Lin all cell lines considered here and may find applicationin the treatment of osteosarcoma and Ewing's sarcoma



patients refractory to conventional drugs. In addition,NVP-BEZ235 may be of interest also in the preventionof resistance to some new targeted drugs, such as the al-losteric and specific mTORC1 inhibitor rapamycin andits analogues or anti–IGF-IR HAbs. In fact, recent studieshave shown that rapamycin and its analogues inducedfeedback activation of Akt in several human cancers, in-cluding rhabdomyosarcoma (20), thus weakening anti-tumor activity. Cao et coworkers (30) have recentlyshown how the neutralizing anti–IGF-IR antibodyh7C10 was initially capable of downregulating IGF-IRand inhibiting AKT pathway in sarcoma cells with elevatedIGF-IR but p-AKT levels eventually recovered after tumorsresumed growth in the presence of h7C10. Accordingly,the use of NVP-BEZ235 may result in a dual benefit of pre-venting the tumor escape seen after anti–IGF-IR treatmentalone, and limiting the potential problem of mTOR-induced upregulation of AKT.In our hands, the inhibitor induced cytostatic effects, with

no induction of apoptosis. As a consequence, to obtain themaximum exploitation of its therapeutic potential, NVP-BEZ235 should be combined with conventional cytotoxicagents. Advantageous drug-drug combinations were ob-tained either with doxorubicin or vincristine. In combiningtreatments, NVP-BEZ235 should be administered simulta-neously or after the chemotherapeutics to obtain maximumadvantageswith respect to single agents. The associationwithvincristine was found to be effective also in vivo, both againsttumor growth and metastasis. NVP-BEZ235 alone reducedthe rate of tumor growth and the number of experimentalmetastasis. Both effects were potentiated by vincristine. Re-cently, another dual PI3K/mTOR inhibitor, PI-103, wasfound to be active against T-cell acute lymphoblastic leuke-mia cells and was found to synergize with vincristine (32).The positive drug-drug combination may find a rationalein previous reports indicating that mTOR activation specifi-cally confers resistance to chemotherapeutic agents thattarget the microtubules, including vincristine (33).In conclusion, our investigation could pave the way for

using dual PI3K/mTOR inhibitors to improve the thera-peutic outcome of sarcoma patients. Activation of PI3K/mTOR signaling appears as a common feature of Ewing'ssarcoma, osteosarcoma, and rhabdomyosarcoma celllines. Its inhibition reduces cell proliferation, migration,and metastasis. However, as a single agent, the drug in-duces only disease stasis, due to the lack of apoptotic ef-fects and increased activation of MAPK pathway. Thus,association with conventional drugs (vincristine or doxo-rubicin) or with other signaling inhibitors (anti–IGF-IRdrugs or other specific MAPK inhibitors) is highly recom-mended to obtain therapeutic improvements in the cure ofsarcoma patients.

Disclosure of Potential Conflicts of Interest

S.-M. Maira and C. Garcia-Echexverria, employment andownership interest, Novartis Pharma AG. The otherauthors disclosed no potential conflicts of interest.




Manara et al.

540


Acknowledgments

We thank Sarah Nosari for her invaluable technical helpand Cristina Ghinelli and Alba Balladelli for their help inediting the manuscript.

Grant Support

EU project Eurobonet (#018814), the Italian Associationfor Cancer Research (K. Scotlandi), the Italian Ministry of



Health (Strategico Oncologia RFPS-2006-3-340280; Alli-ance against Cancer), and the Italian Ministry of Researchand Instruction (PRIN 2006).The costs of publication of this article were defrayed

in part by the payment of page charges. This articlemust therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicatethis fact.Received 4/1/09; revised 9/30/09; accepted 11/1/09;

published OnlineFirst 1/12/10.

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2010;16:530-540. Published OnlineFirst January 12, 2010.Clin Cancer Res Maria C. Manara, Giordano Nicoletti, Diana Zambelli, et al. NVP-BEZ235 as a New Therapeutic Option for Sarcomas

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NVP-BEZ235 as a New Therapeutic Option for …...NVP-BEZ235 as a New Therapeutic Option for Sarcomas Maria C. Manara 1 , Giordano Nicoletti 1,3 , Diana Zambelli 1 , Selena Ventura