Preclinical Characterization of OSI-027, a Potent and ...mct.aacrjournals.org/content/molcanther/10/8/1394.full.pdffurther support the important role of mTOR as a driver of tumor growth

Preclinical Development

Preclinical Characterization of OSI-027, a Potent and SelectiveInhibitor of mTORC1 and mTORC2: Distinct from Rapamycin

Shripad V. Bhagwat1, Prafulla C. Gokhale3, Andrew P. Crew2, Andy Cooke3, Yan Yao1, Christine Mantis3,Jennifer Kahler1, Jennifer Workman3, Mark Bittner3, Lorina Dudkin4, David M. Epstein1, Neil W. Gibson1,Robert Wild3, Lee D. Arnold2, Peter J. Houghton4, and Jonathan A. Pachter1

AbstractThe phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and

mTOR is a clinically validated target. mTOR forms two distinct multiprotein complexes, mTORC1 and

mTORC2, which regulate cell growth, metabolism, proliferation, and survival. Rapamycin and its analogues

partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clinical

utility in certain cancers. Here, we report the preclinical characterization of OSI-027, a selective and potent

dual inhibitor of mTORC1 and mTORC2 with biochemical IC50 values of 22 nmol/L and 65 nmol/L,

respectively. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Ka, PI3Kb, PI3Kg ,and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as

themTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 andOXA-01 (close analogue

of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and

engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K–AKT signaling.

OSI-027 shows concentration-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 andAKT in

tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several

different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO

colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results

further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a

potent anticancer agent. OSI-027 is currently in phase I clinical trials in cancer patients. Mol Cancer Ther;

10(8); 1394–406. �2011 AACR.

Introduction

mTOR is a serine/threonine protein kinase related tothe phosphoinositide 3-kinase (PI3K) family (1). ThemTOR signaling pathway integrates both extracellular

and intracellular signals and serves as a central regulatorof cell metabolism, growth, proliferation, and survival (2–6). Many cancer-driving mutations in genes encodingreceptor tyrosine kinases, Ras, PI3K, and PTEN stimulatecell proliferation, growth, and survival through activa-tion ofmTORkinase signaling.mTOR resides in 2 distinctmultiprotein complexes called mTORC1 and mTORC2(3). mTORC1 directly phosphorylates ribosomal proteinS6 kinase 1 (S6K1) and the eukaryotic translation initia-tion factor eIF4E-binding protein 1 (4E-BP1), bothinvolved in protein translation (7). The more recentlydiscovered mTORC2 phosphorylates the survival kinaseAKT on Ser 473, thus leading to activation of the AKTpathway, which drives several cancer-related cellularresponses, including increased cell growth, proliferation,and survival, increased cell migration, and a shift toglycolytic metabolism (8–12). Evidence also suggests thatmTORC2 phosphorylates PKCa and regulates the orga-nization of the actin cytoskeleton (9–10).

Rapamycin and rapamycin-related analogues (rapa-logs) are allosteric inhibitors of mTORC1 (13, 14). Inaddition to lacking a direct mTORC2 inhibitory effect,rapalogs do not inhibit the function of mTORC1 com-pletely (15, 16). Although rapalogs (e.g., temsirolimusand everolimus) have shown clinical benefits as single

Authors' Affiliations: 1Cancer Biology and 2Cancer Chemistry, OSI Phar-maceuticals Inc., Farmingdale, New York; 3In vivo Pharmacology, OSIPharmaceuticals Inc., Boulder, Colorado; and 4Center for ChildhoodCancer, Nationwide Children's Hospital, Columbus, Ohio

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

S.V. Bhagwat and P.C. Gokhale contributed equally to this work.

Current address for N.W. Gibson: Regulus Therapeutics, San Diego, CA92121.

Current address for R. Wild: Eli Lilly and Company, Indianapolis, Indiana.

Current address for L.D. Arnold: Discover Elucidations LLC, Mt Sinai, NewYork.

Current Address for J.A. Pachter: Verastem Inc., Cambridge, MA 02142

Corresponding Author: Shripad V. Bhagwat, Biochemical and CellularPharmacology, OSI Pharmaceuticals Inc., Farmingdale, NY 11735. Phone:631-962-0620; Fax: 631-845-5671; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-1099

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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agents in a limited number of tumor types (e.g., renal cellcarcinoma and mantle cell lymphoma), the mode ofaction of these drugs does not completely exploit theantitumor potential of fully inhibiting mTOR signaling(17). The emerging role of mTORC2 in the activation ofAKT in cancer is now considered important to tumormaintenance and progression, but this complex is gen-erally resistant to rapalogs. In addition, inhibition ofmTORC1 by rapamycin and its analogues has beenshown to result in hyperactivation of AKT through therelease of negative feedback loop between S6K1 and IRS-1(18). Indeed, Cloughesy and colleagues have recentlyshown that hyperactivation of AKT following rapamycintreatment was associated with shorter time to progres-sion in PTEN-deficient glioblastoma patients, suggestingthat TORC1-specific inhibition and associated AKT acti-vation limits anticancer activity (19). Consequently,intense efforts are now underway to develop inhibitorsof the PI3K/AKT/mTOR pathway, including ATP-com-petitive small molecule mTOR kinase inhibitors targetingboth mTORC1 and mTORC2 (20).Several recent reports have described biochemical and

cellular properties of selective ATP-competitive inhibi-tors of mTORC1 and mTORC2 (21–26). These chemicallydistinct compounds all show suppression of bothmTORC1- and mTORC2-mediated downstream signal-ing in various tumor cell lines. These inhibitors have beenshown to induce a stronger suppression of protein synth-esis and cell growth than rapamycin. More recently,antitumor activity and tumor pharmacodynamic effectswere reported with the selective mTORC1 and mTORC2inhibitors, AZD8055 and WYE-132. In this article, wedescribe the preclinical characterization of OSI-027, anorally bioavailable, potent, and specific dual inhibitor ofmTORC1 and mTORC2. We show broad-spectrum anti-tumor activity in several xenograft models, in vivo differ-entiation from rapamycin, PK/PD/efficacy correlationsin PI3K-wt versus mutant models and tumor growthinhibition (TGI) by intermittent and once weekly dosingschedules. OSI-027 is currently in phase I clinical devel-opment in cancer patients.

Materials and Methods

Synthesis of OSI-027 and OXA-01OSI-027 is a 4,5,7-trisubstituted imidazo[5,1-f]triazine

and OXA-01 is a 1,3,8-trisubstituted imidazo[1,5-a]pyrazine. These were synthesized by the methodsdescribed in patent application US-20070112005 (27).Compound identity and purity (>99%) were verifiedby 1H and 13C nuclear magnetic resonance, mass spectro-metry, and high-performance liquid chromatography byusing Bruker Avance 400, Waters Micromass ZQ, andWaters LC Module I Plus instruments, respectively, aswell as by elemental analysis. OSI-027 was dissolved indimethyl sulfoxide (DMSO) at 10 mmol/L for use inbiochemical or cellular in vitro assays. For in vivo studies,OSI-027 was dissolved in 20% Trappsol at an appropriate

concentration to deliver the described dose in 10 mL/kgby oral gavage.

Cell linesCancer cell lines were obtained from the American

Type Culture Collection or other sources, as indicatedin Supplementary Materials and Methods, banked afterreceipt, and passaged for less than 6 months before use inexperiments. All cell lines were cultured as directed. Rh1,Rh1mTORrr, Rh30, Rh30/mTORrr, and Rh30/Rapa10Kcells were grown in culture medium previouslydescribed (28).

Western blot analysisWhole-cell lysates were prepared in radioimmunopre-

cipitation assay buffer supplemented with protease inhi-bitor cocktail, phosphatase inhibitor cocktail, 1 mmol/Lsodium orthovanadate, and 10 mmol/L sodium fluoride(all reagents purchased from Sigma). Lysates were clearedby centrifugation and 20 mg protein was loaded per well.Lysates were fractionated on 4% to 12% tris-glycine poly-acrylamide gels and transferred to nitrocellulose mem-branes by using a semidry apparatus. Membranes wereblocked with 5% nonfat dry milk in TBS and incubatedovernight with primary antibody in 3% bovine serumalbumin. The following primary antibodies from Cell Sig-naling Technology were used at 1:1,000 dilution: phospho-4E-BP1[T37/46], phospho-4E-BP1[S65], 4E-BP1, phospho-AKT[S473], phospho-AKT[T308], AKT, phospho-PRAS40(T246), PRAS40, phospho-S6K1[T389], S6K1, phospho-S6[S235/236], S6, cyclinD1, andglyceraldehyde-3-phosphatedehydrogenase (GAPDH). b-Actin antibody was pur-chased from Sigma. Horseradish peroxidase-conjugatedsecondary antibodies were obtained from Jackson Labsor GEHealthcare. Horseradish peroxidase-conjugated sec-ondary antibodies were incubated in nonfat dry milk for 1hour. SuperSignal chemiluminescent reagent (Pierce Bio-technology)was used according to the directions and blotswere imaged by using the Alpha Innotech image analyzer.

AnimalsFemale nu/nu CD-1 mice (6–8 weeks, 22–25 g) were

purchased from Charles River Laboratories and allowedto acclimate for a minimum of 1 week before initiation ofa study. Throughout the studies, animals were allowedsterile rodent chow and water ad libitum. All in vivostudies were conducted at OSI facilities with the approvalof the Institutional Animal Care andUse Committee in anAmerican Association for Accreditation of LaboratoryAnimal Care accredited vivarium and in accordancewith guidelines from the Institute of Laboratory AnimalResearch.

Cell proliferation assays, apoptosis assays, andcell-cycle analysis

For assays of cell proliferation, cells were seeded into96-well plates and incubated for 3 days in the presence ofOSI-027 or OXA-01 at various concentrations. Inhibition

Preclinical Profile of Dual mTORC1/2 Inhibitor OSI-027

www.aacrjournals.org Mol Cancer Ther; 10(8) August 2011 1395




of cell growth was determined by luminescent quantifi-cation of intracellular ATP content by using CellTiterGlo(Promega). Proliferation on day 0 versus 72 hours wasused to plot dose–response curves for IC50 calculationsand to determine cell death. For apoptosis induction(caspase-3/7) assay, cells were seeded into 96-well platesand incubated for 48 hours in the presence of OSI-027 atvarious concentrations. Induction of caspase-3/7 activitywas determined by luminescent quantification by usingCaspaseGlo reagent (Promega). Fold induction of apop-tosis was calculated as compared with DMSO-treatedcontrols. For cell-cycle analysis, cells were treated withDMSO, rapamycin, or OSI-027 for 48 hours and stainedwith propidium iodide for analysis by flow cytometry(BD FACS Caliber). Percent of total cells in subG1, G0–G1,S, and G2–M were calculated by using fluorescence-activated cell sorting (FACS) analysis software.

Pharmacokinetic analysisBlood from mice dosed with OSI-027 was collected in

EDTA tubes and separated plasma was extracted byprotein precipitation with methanol followed by centri-fugation (10,000 � g for 10 minutes at 4�C). Extractedplasma samples were analyzed by high-performanceliquid chromatography–tandem mass spectrometry. Allpharmacokinetic parameters were obtained by noncom-partmental analysis.

In vivo pharmacodynamic analysisTo assess the ability of OSI-027 to inhibit phosphor-

ylation of 4E-BP1 (T37/T46) or AKT (S473) in tumortissue, female nu/nu CD-1 mice were implanted in themammary fat pad with MDA-MB-231 tumor cells har-vested from cell culture flasks. Animals with estab-lished tumors of 300 � 50 mm3 size were dosedorally with OSI-027 dissolved in 20% Trappsol (CTDInc.). Tumor samples were collected at specified timepoints and snap frozen in liquid nitrogen. Tumor lysateswere prepared by homogenizing samples in a Precellys24 homogenizer (MO Bio Laboratories, Inc.) with tumorlysis buffer [50 mmol/L HEPES (pH 7.4) containing 1%Triton X-100, 10% glycerol, 50 mmol/L, 150 mmol/LNaCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA supple-mented with fresh protease inhibitor cocktail (Sigma),phosphatase inhibitor cocktail (Sigma), 10 mmol/LNaF, and 1 mmol/L sodium orthovanadate]. Tissuehomogenates were clarified by centrifugation at14,000 � g for 5 minutes at 4�C and supernatants werethen analyzed by Western blot as indicated. Plasmasamples were also collected at specified time pointsfor OSI-027 concentration analysis. Phopsho-4E-BP1(T37/46), total 4E-BP1, phospho-AKT (S473), totalAKT, or b-actin were quantified from Western blotsby using Multi Gauge software program (FujiFilm).Ki67 nuclear staining was carried out by using forma-lin-fixed, paraffin-embedded SKOV-3 xenografts. Ki67immunohistochemical nuclear staining was quantifiedby using ACIS II imager scoring ten fields.

In vivo antitumor efficacy studiesCells were harvested and implanted s.c. in the right

flank of nu/nu CD-1 mice as described previously (29).Tumors were allowed to establish to 200� 50 mm3 in sizebefore randomization into various treatment groups with8 mice per group. OSI-027 was administered orally dis-solved in 20% Trappsol or rapamycin administered intra-peritoneally (i.p.) in an aqueous solutionof 4%ethanol, 5%Tween 80, and 5% PEG400 at indicated doses. Rapamycinwas purchased from LC Laboratories. Tumor volumeswere determined from calipermeasurements by using theformula V ¼ (length � width2)/2. Tumor sizes and bodyweights were measured twice weekly. TGI was deter-mined at different time points for each animal by thefollowing formula: %TGI ¼ {1 � [(Tt/T0)/(Ct/C0)]/1 �[C0/Ct]} � 100, in which Tt ¼ tumor volume of treatedanimal� at time t, T0¼ tumor volume of treated animal�at time 0, Ct ¼ median tumor volume of control group attime t, andC0¼median tumor volume of control group attime 0. Median% TGI was calculated and reported for theentire dosing period for each group. Significant antitumoractivity is defined as achievement of a median% TGI of atleast 50%. Regressions in tumor volume were calculatedas: % regression ¼ 100 (V0 � Vt)/V0, in which V0 ¼meantumor volume of treated group at time 0, and Vt ¼ meantumor volume of that group at time t. Rank ANOVAwithDunnett’s comparison was used to compare treatmentgroups with the control group. All comparisons weredeemed statistically significant if P � 0.05.

Results

OSI-027 is a selective inhibitor of mTORC1 andmTORC2

To establish the potency and selectivity of OSI-027against mTOR and related kinases, native protein com-plexes were immunoprecipitated from HeLa cell lysatesand assayed for inhibition of mTOR by OSI-027. TheIC50 value for OSI-027 was 4 nmol/L (SupplementaryFig. S1). Immunoprecipitation with anti-Raptor anti-bodies to evaluate mTORC1 activity in the presenceof drug, or anti-Rictor antibodies to assess mTORC2activity indicated that OSI-027 is equipotent in theinhibition of both functional complexes. Greater than100-fold selectivity was observed for mTOR relative toother PI3K-related kinases in biochemical assays. Todetermine the selectivity profile, OSI-027 (1 mmol/L)was tested against 101 kinases by using Caliper kinaseprofiling assays and the data are shown in Supplemen-tary Table S1. None of those kinases were inhibited bymore than 50%. In addition, OSI-027 activity was testedagainst 37 kinases at Invitrogen at 100 mmol/L ATP andnone of them were significantly inhibited (Supplemen-tary Table S1). Additional selectivity data for OSI-027 incell assays and an Ambit KinomeScan has been pub-lished recently by our colleagues (30). These data indi-cate that OSI-027 is a potent and selective inhibitor ofmTORC1/mTORC2.

Bhagwat et al.

Mol Cancer Ther; 10(8) August 2011 Molecular Cancer Therapeutics1396




OSI-027 potently inhibits mTORC1 and mTORC2activity in cellsStructure of OSI-027, OXA-01, and rapamycin is shown

in Fig. 1A. Inhibition of mTORC1 and mTORC2 substratephosphorylation and downstream signaling by OSI-027in BT-474 (PIK3CA-mut), IGR-OV1 (PTEN-null), andMDA-MB-231 (PIK3CA-wt) cell lines were determinedby immunoblot analysis. OSI-027 inhibited phosphoryla-tion of AKT at S473 and T308 in a concentration-depen-dent manner in BT-474 and IGR-OV1 cells under normalculture conditions (Fig. 1B) and in MDA-MB-231 cellsupon insulin stimulation (Supplementary Fig. S2). TheIC50 of OSI-027 in BT-474 cells was determined with apAKT S473 sandwich ELISA and was found to be0.3 mmol/L (Supplementary Fig. S3). In addition, OSI-027 inhibited phosphorylation of mTORC2-mediatedPRAS40 in these 3 cell lines in a dose-dependent manner.

In BT-474, IGR-OV1, and MDA-MB-231 cells, OSI-027inhibited phosphorylation of the mTORC1 substrateS6K1 at T389 in a concentration-dependent manner withpotency similar to pAKT [S473] inhibition, suggestingequipotent cellular inhibition of both mTORC1 andmTORC2 by OSI-027 (Fig. 1B). Similarly, phosphoryla-tion of S6 at S235/236 downstream of S6K1 was inhibitedby OSI-027 in these 3 cell lines. Moreover, OSI-027 inhib-ited phosphorylation of the mTORC1 and mTORC2substrate 4E-BP1 at T37/T46 and S65 in a concentra-tion-dependent manner in these 3 cell lines (Fig. 1B) withIC50 of 0.4 mmol/L in BT-474 cells (SupplementaryFig. S3). In addition, treatment with OSI-027 inhibitedcyclin D1 expression (translation known to be regulatedin a cap-dependent manner) in a concentration-depen-dent fashion in IGR-OV1, BT-474, and MDA-MB-231cells (Fig. 1B) and shows reasonable correlation with

Figure 1. OSI-027 is a potentinhibitor of mTORC1 andmTORC2 in cells. A, structures ofrapamycin, OXA-01, and OSI-027.B, BT-474, IGR-OV1, and MDA-MB-231 cells were treated withOSI-027 in a dose-dependentmanner for 2 hours and total celllysates were analyzed by Westernblotting. In MDA-MB-231 cells,pAKT (T308) was not detected innormal culture conditions, but itwas detected after IGF-1stimulation (see SupplementaryFig. S2).

A

B

OSI-027OXA-01Rapamycin

HOO

OH

O

HO

O

H

O

N

H O

O

OHO

H O

O

N

NN

NHNH2

HOO

Cl

N

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NHNH2

HOO

O

BT-474 IGR-OV1 MDA-MB-231

DM

SO

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0.1

1 10

OSI-027 (µmol/L)

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SO

0.01

0.1

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OSI-027 (µmol/L)

DM

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0.1

1 10

OSI-027 (µmol/L)

pAKT (S473)

pAKT (T308)

AKT

pPRAS40 (T246)

PRAS40

p4E-BP1 (T37/46)

4E-BP1

pS6K1 (T389)

S6K1

pS6 (S235/236)

S6

Cyclin D1

GAPDH

p4E-BP1 (S65)






p4E-BP1 (both T37/36 and S65) inhibition. These datasuggest that OSI-027 inhibits both mTORC1 andmTORC2 potently in cancer cell lines with diverse geneticbackgrounds.

OSI-027 inhibits 4E-BP1 and AKT phosphorylationand prevents feedback to AKT in majority of cancercell lines

To survey the ability of OSI-027 to inhibit mTORC1-and mTORC2-mediated downstream signaling, we usedWestern blot analysis to assess the inhibition of 4E-BP1(T37/46) and AKT (S473) phosphorylation by OSI-027 (20mmol/L) and rapamycin (20 mmol/L) after 24 hours oftreatment in 12 or 24 cancer cell lines representing varioustumor types, respectively. These concentrations werechosen on the basis of the plasma exposure of OSI-027at efficacious doses in preclinical models. OSI-027 sig-nificantly inhibited 4E-BP1 (T37/46) phosphorylation inthe majority of cell lines tested and representative dataare shown in Fig. 1C. In contrast, rapamycin failed tosignificantly inhibit p4E-BP1 in the majority of cell lines

even at 20 mmol/L, which is more than 200-fold higherthan clinically relevant plasma concentrations. Moreover,OSI-027 strongly inhibited pAKT (S473) in 83% of the 12cell lines tested, whereas rapamycin inhibited pAKT(S473) in only 13% of the 24 cell lines tested (Fig. 1Cand Table 1). More importantly, rapamycin stimulatedpAKT (S473) in 62% of total 24 cell lines (Fig. 1C andTable 1) or in 58% of same 12 cancer cell lines tested forOSI-027. These data suggest that OSI-027 can effectivelyinhibit pAKT and prevent feedback activation of AKT inthe majority of tumor cell lines tested, which stands inclear contrast to the observed effects of rapamycin.Furthermore, OSI-027 inhibited IGF-1–induced pAKT(S473), whereas rapamycin was ineffective (Supplemen-tary Fig. S4). We also determined whether OSI-027 caninhibit pAKT (S473) induced by rapamycin treatment inthe NCI-H23 NSCLC cell line and, indeed, OSI-027 fullyinhibited pAKT (S473) induced by rapamycin treatment(Fig. 1D), suggesting the likely role of mTORC2 activationin the stimulation of pAKT by rapamycin. Overall, thesedata suggest that OSI-027 can inhibit both mTORC1 and

C

D

pAKT (S473)

AKT

U-87 MG

RL95-2

NCI-H2122

20 µmol/L,24 h

p4E-BP1 (T37/46)

4E-BP1

GAPDH

pAKT (S473)

AKT

p4E-BP1 (T37/46)

4E-BP1

GAPDH

pAKT (S473)

AKT

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AKT

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-027

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a

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-027

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C-33A

IGR-OV1

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Rapamycin, 20 µmol/LOSI-027, 20 µmol/L

––

–+

+–

++

pAKT (S473)

AKT

GAPDH

24h Treatment

Figure 1. (Continued ) C, U-87MG, RL95-2, NCI-H2122, C-33A,and IGR-OV1 cells were treatedwith OSI-027 (20 mmol/L) orrapamycin (20 mmol/L) for 24hours and total lysates wereanalyzed by Western blotting forp4E-BP1 (T37/46), 4E-BP1, pAKT(S473), AKT, and GAPDH. D,NCI-H23 NSCLC cells treated withDMSO, rapamycin (20 mmol/L),OSI-027 (20 mmol/L), andrapamycin þ OSI-027 (20 mmol/Leach) for 24 hours and totallysates were analyzed by Westernblotting for pAKT (S473).

Bhagwat et al.





mTORC2 activity in the majority of tumor cell lines,whereas rapamycin only inhibits mTORC1 and generallystimulates or has no effect on mTORC2-mediatedsignaling.

OSI-027 and OXA-01 inhibits cell proliferationand induces cell death: superiority versusrapamycinmTOR is important in the control of cell proliferation.

On the basis of the potency of rapamycin in the inhibitionof proliferation, we defined cell lines as either rapamycinsensitive (IC50 < 0.075 mmol/L) or rapamycin insensitive(<30% inhibition at 20 mmol/L). We then examined theantiproliferative potency of OSI-027 among these rapa-mycin-sensitive and -insensitive subgroups. Inhibition ofcell proliferation was determined by quantifying cellularATP levels after 72 hours of exposure to increasingconcentrations of OSI-027. Cell proliferation on day 0was used as a background to calculate IC50 for inhibitionof cell proliferation and to determine whether additionalcell death occurred. OSI-027 potently inhibited cell pro-liferation in rapamycin-insensitive cell lines, with IC50

values ranging from 0.4 to 4.5 mmol/L (Fig. 2A, Supple-mentary Table S2). Moreover, OSI-027 induced 10% to50% cell death in majority of rapamycin-sensitive celllines (Fig. 2B) but not rapamycin (Supplementary Fig. S5).Taken together, these data suggest that OSI-027 is super-ior to rapamycin in inhibiting cell proliferation and indu-cing cell death in a large panel of tumor cell lines. Wefurther tested sensitivity to rapamycin and themTORC1/mTORC2 inhibitor OXA-01 (a close analogue of OSI-027)in parental Rh1 and an Rh1/mTORrr cell line in whichS2035I mutation was introduced in FRB domain of mTORto prevent rapamycin-FKBP12 binding. Rh1 cells arehighly sensitive to rapamycin, whereas Rh1/mTORrrcells show a decrease in rapamycin potency of at least3 orders of magnitude (28). In contrast to rapamycin, bothRh1 and Rh1/mTORrr cells show similar sensitivity tothe mTOR kinase inhibitor OXA-01, consistent with thedirect action of this inhibitor on the mTOR catalyticprotein rather than the FRB domain (Fig. 2C). Similarly,

OXA-01 inhibited proliferation of Rh30 (rapamycin-sensitive parental), Rh30/mTORrr (FRB domainmutant), and Rh30/Rapa10K (selected in vitro for resis-tance to 10 mmol/L rapamycin) cells with similar IC50

values, whereas rapamycin showed a significantpotency shift of more than 10,000-fold (Fig. 2C andTable 2). These data suggest that ATP-competitivemTORC1/2 inhibitors, such as OXA-01 maintainpotency in rapamycin-insensitive cell lines and inhibitmTOR by a different mechanism as compared withrapamycin. Furthermore, these data predict that somepatients with rapamycin-refractory cancers might stillrespond to mTOR kinase inhibitors such as OXA-01 orOSI-027.

OSI-027, but not rapamycin, strongly inducesapoptosis in a PTEN-null cell line

To study proapoptotic effects more directly, we nextinvestigated whether OSI-027 and rapamycin can inducecaspase-3/7 activation in multiple cancer cell lines withor without PIK3CA or PTEN mutation. OSI-027 inducedcaspase-3/7 activity by more than 2-fold in 3 of 22 celllines, whereas rapamycin failed to induce caspase-3/7activity in all cell lines tested (Supplementary Table S3).Caspase-3/7 induction was concentration dependent inIGR-OV1 and BT-474 cells (Fig. 3A). Furthermore, wequantified sub-G1 cells following treatment with OSI-027(20 mmol/L), rapamycin (20 mmol/L), or DMSO (control;Fig. 3B). OSI-027, but not rapamycin, induced a signifi-cant sub-G1 fraction (40%) indicating apoptotic cell death(Fig. 3B).

Pharmacodynamic effects of OSI-027 on p4E-BP1and pAKT in MDA-MB-231 xenografts

Pharmacokinetic analysis following oral administra-tion of OSI-027 to mice revealed that plasma levels ofOSI-027 (Cmax and AUC) increased approximately line-arly between 20 and 200 mg/kg doses with oral bioavail-ability of 65% to 95% (Supplementary Fig. S6).

Pharmacodynamic studies were carried out in theMDA-MB-231 breast cancer xenograft model to evaluateeffects on phosphorylation of the mTORC1/mTORC2substrate 4E-BP1 and the mTORC2 substrate AKT inrelation to plasma drug concentrations after a single oraldose of 25 or 65mg/kg of OSI-027. A single oral dose of 65mg/kg resulted in considerable inhibition (>75%) of 4E-BP1 phosphorylation that was evident for at least 16hours (Fig. 4A, Supplementary Fig. S7). At 24 hourspostdose in the 65 mg/kg dose group, 47% inhibitionof p4E-BP1 was still maintained with a correspondingplasma OSI-027 concentration of 2.2 mmol/L. Suchextended target suppression was associated with signifi-cant efficacy corresponding to 100% median TGI in theMDA-MB-231 xenograft model (Fig. 4C). In comparison,once daily administration of OSI-027 at 25 mg/kg for14 days resulted in only moderate antitumor activitycorresponding to 64% median inhibition of MDA-MB-231 tumor growth (TGI) compared with control vehicle

Table 1. Summary table showing effect of OSI-027 (20 mmol/L) and rapamycin (20 mmol/L) onpAKT (S473) in 12 and 24 cancer cell lines,respectively

Percentage of cell lines showingpAKT (S473)

Stimulation No change Inhibition

Rapamycin(20 mmol/L, 24 h)

15/24 (62) 6/24 (25) 3/24 (13)

OSI-027(20 mmol/L, 24 h)

0/12 (0) 2/12 (17) 10/12 (83)






treated animals for 14 days (Fig. 4C), whereas adminis-tration of OSI-027 on a twice daily schedule of 25 mg/kgfor 14 days resulted in robust 90% median TGI (data notsown). Pharmacodynamic analysis showed that althoughthe lower dose of 25 mg/kg showed significant inhibition(>79%) of 4E-BP1 phosphorylation, it only lasted forabout 8 hours with inhibition of p4E-BP1 completelyreversed by 16 hours postsingle dose of OSI-027 at 25mg/kg, corresponding to plasma concentrations of lessthan 0.13 mmol/L (Supplementary Fig. S8). These datasuggest that sustained inhibition of tumor p4E-BP1 by

more than 50% seems to be necessary for substantial TGIin this model.

Tumor samples from the same animals were also usedto quantify the in vivo effects of OSI-027 on phosphoryla-tion of the mTORC2 substrate AKT (S473). After a single25 mg/kg dose, greater than 60% inhibition of tumorpAKT was observed for up to 4 hours with a rebound ofpAKT content as drug plasma levels decreased (Supple-mentary Fig. S8). A single dose of 65 mg/kg resulted inmarked inhibition (>50%) of pAKT for up to 16 hourswith a rebound of pAKT content at 24 hours (Fig. 4B).

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Figure 2. Antiproliferative effects of OSI-027 and rapamycin. A, rapamycin-insensitive cell lines (IC50 > 20 mmol/L) were treated with OSI-027 for 72 hoursin a dose-dependentmanner. Relative cell viabilities weremeasured by usingCell TiterGlo and plotted as%DMSO control using prism. B, rapamycin-sensitive(IC50 < 0.075 mmol/L) cell lines were treated with OSI-027 for 72 hours and dose–response curve is plotted as % DMSO control using prism. C, Rh1,Rh1/mTORrr, Rh30, Rh30/mTORrr, and Rh30/Rapa10K cells were treated with OXA-01 and rapamycin for 72 hours and cell viability was measured by usingCell TiterGlo reagent.

Bhagwat et al.





In vivo antitumor activity of OSI-027 and rapamycinin colorectal tumorsThe effects of OSI-027 were initially evaluated in the

human colorectal xenograft models GEO and COLO 205,selected in part due to insensitivity to rapamycin in vitro(data not shown). In the COLO 205 tumormodel, oral OSI-027 treatment at 65 mg/kg once daily for 12 days resulted

in 100% median TGI with 37% regression, whereas rapa-mycin treatment (20 mg/kg i.p., d1-5, d8-12) resulted in79% median TGI (Fig. 4D). Statistical analysis showedsignificant difference between the efficacy of rapamycinand OSI-027 treatments (P < 0.001). At the end of dosing,tumor samples were removed at 8 and 24 hours andanalyzed for phospho-S6, phospho-4E-BP1, and phos-pho-AKT. OSI-027 treatment resulted in significant inhibi-tion of both mTORC1 and mTORC2 effectors at 8 hourswith recovery by 24 hours (Fig. 4E and SupplementaryFig. S9). The correspondingmedian plasma concentrationsat 8 and 24 hours were 22 and 0.77 mmol/L, respectively.On theother hand, rapamycin treatment showedsustainedinhibition of only the mTORC1 effector pS6, but had littleto no effect on mTORC2 signaling, as assessed by tumorp4E-BP1 (T37/46) and pAKT (Ser473; Fig. 4E and Supple-mentary Fig. S9). These data show that an mTORC1/mTORC2-specific inhibitor such as OSI-027 can achievesuperior inhibitory effects on pAKT and p4E-BP1 in vivo ascompared with an mTORC1-selective inhibitor such asrapamycin, and this inhibition of tumor pAKT and p4E-BP1 correlated with improved antitumor activity.

Similarly, improved tumor growth inhibitory activityof OSI-027 was observed in the GEO xenograft model as

Figure 3. Proapoptotic effect ofOSI-027, OXA-01, and rapamycin.A, induction of caspase-3/7 wasmeasured in BT-474 and IGR-OV1cancer cell lines by treatment withdrugs for 48 hours by usingCaspaseGlo reagent. Data arefrom 2 separate experiments andpresented as mean � SD. B, IGR-OV1 cells treated with OSI-027 (20mmol/L), rapamycin (20 mmol/L),and DMSO for 48 hours andstained with propidium iodide todetermine cell death (sub-G1

fraction) by FACS analysis.

–2 –1 0 1 20

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Table 2. Summary table showing differentiationof OXA-01 from rapamycin in Rh1, Rh1/mTORrr, Rh30, Rh30/mTORrr, and Rh30/Rapa10K cell lines

Proliferation IC50, mmol/L

Rapamycin OXA-01

Rh1 0.0005 0.5Rh1/mTORrr 1.5 1.7Rh30 0.0005 0.4Rh30/mTORrr 4.4 0.4Rh30/Rapa10K >10 1.0






compared with the efficacy of rapamycin (Fig. 4F).Oral OSI-027 administration at 65 mg/kg once dailyfor 14 days resulted in 95% median TGI, whereas rapa-mycin treatment (20 mg/kg i.p., d1-5, d8-12) resulted in75% median TGI (Fig. 4F). Statistical analysis showedsignificant difference between the rapamycin and OSI-027 treatment groups (P < 0.05).

Evaluation of in vivo efficacy of OSI-027 in mTORpathway–dependent xenograft models

The antitumor efficacy of OSI-027 was evaluated inPIK3CA mutant SKOV-3 and KRAS mutant OVCAR-5human ovarian cancer xenograft models. In the SKOV-3model,OSI-027 at 50mg/kg oncedaily for 14days resultedin significant TGI (100%median TGI)with 15% regression.

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Figure 4. Pharmacodynamic effects and efficacy of OSI-027 in tumor xenograft models. Correlation of mean plasma drug concentrations (red line) andpercent p4E-BP1 (T37/46; (blue line; A) and pAKT (Ser473; green line; B) content over time (n ¼ 4/time point) following 65 mg/kg oral single dose of OSI-027in MDA-MB-231 xenograft model. C, dose-dependent efficacy of OSI-027 in MDA-MB-231 model. Efficacy of OSI-027 (oral dose) and rapamycin(i.p. dose) in COLO 205 (D) and GEO (F) models. OSI-027 treatment was significantly more effective than rapamycin treatment (P < 0.001 in COLO 205and P < 0.05 in GEO). E, pharmacodynamic effects of OSI-027 and rapamycin in COLO 205 tumors. COLO 205 tumors were collected at 8 and 24 hoursafter 12 days of dosing (n ¼ 2) and immunoblotted for p4E-BP1 (T37/46), pS6 (S235/236), and pAKT (S473). The bands were quantified and plottedas a percentage of vehicle-treated controls.

Bhagwat et al.





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Figure 5. OSI-027 inhibits tumor growth in mTOR-activated tumor models. A, efficacy of OSI-027 in PIK3CA mutant SKOV-3 ovarian xenograft model.SKOV-3 tumors were treated with OSI-027 at 50 mg/kg once daily and 25 mg/kg twice daily for 14 days by oral gavage. Alternatively, steady-stateconcentrations of OSI-027 were delivered by using 14-day mini pumps implanted s.c. in tumor-bearing animals. Steady-state concentrations of 0.5 and2 mmol/L were achieved by using a dose of 0.12 mg/kg/h and 0.49 mg/kg/h, respectively. B, actual plasma concentrations achieved by using mini-pumps ondays 5 and 13 in tumor-bearing animals (n ¼ 4). C, efficacy of OSI-027 in KRAS mutant OVCAR-5 ovarian xenograft model. OVCAR-5 tumors were treatedwith OSI-027 at 50 mg/kg once daily and 25 mg/kg twice daily for 14 days by oral gavage and at 6 mmol/L steady-state concentrations by using mini-pumps.D, pharmacodynamic effects of OSI-027 delivered by using mini-pumps. Phosphorylation of 4E-BP1 (T37/46) was evaluated in satellite OVCAR-5 tumor-bearing animals at various steady-state concentrations on day 11. E, efficacy of intermittent dosing of OSI-027 in PTEN-null IGR-OV1 ovarian xenograftmodel. IGR-OV1 tumors were treated with OSI-027 at 50 mg/kg once daily for 14 days, 150 mg/kg once every 3 days � 5 doses, or 300 mg/kg once every7 days � 2 doses. F, Ki67 nuclear staining determined by immunohistochemistry in SKOV-3 xenografts collected 8 hours after 3 daily doses of OSI-027 at65 mg/kg. BID, twice daily; QD, once daily; Q3D, once every 3 days; Q7D, once every 7 days.






In addition, twice daily dosing at 25 mg/kg twice dailycorresponding to a total daily dose of 50mg/kg resulted incomparable TGI (100% median TGI with 12% regression;Fig. 5A). In contrast, treatment of OVCAR-5 (KRASmutant) tumors with 50 mg/kg once daily or 25 mg/kgtwice daily was only marginally efficacious showing 47%and 51%median TGI, respectively (Fig. 5C). Furthermore,to better understand the relationship between TGIand drug pharmacokinetics, continuous dosing of OSI-027 delivered via 14-day osmotic pumps was evaluated.In the OSI-027–sensitive tumor model, SKOV-3 mainte-nance of a steady-state concentration of approximately0.5 mmol/L for 14 days was sufficient to achieve maximalefficacy equivalent to 50 mg/kg once daily dosing (100%medianTGIwith20%regression;Fig. 5A). Figure 5Bshowsthe actual plasma drug concentration on days 5 and 13 forestimated 0.5 and 2 mmol/L levels delivered via osmoticpump. Interestingly, in the OVCAR-5 model, which is lesssensitive to OSI-027, a higher steady-state plasma concen-tration of 6 mmol/L was necessary to achieve efficacyequivalent to 50 mg/kg once daily dosing (Fig. 5C).Steady-state concentrations of 0.5 and 2 mmol/L wereinactive in this model (data not shown). Pharmacodyna-mic evaluation in additional OVCAR-5 tumor-bearingmice on day 11 of treatment showed that tumor phos-pho-4E-BP1 inhibition in tumor samples was achievedonly at 6 mmol/L steady state (Fig. 5D), and this pharma-codynamic inhibition correlated with efficacy (Fig. 5C).We further evaluated the effects of intermittent dosingof OSI-027 in the PTEN null IGR-OV1 ovarian carcinomaxenograftmodel.OSI-027 administration at 50mg/kgoncedaily for 14 days resulted in 100% TGI (Fig. 5E). OSI-027administration at 150 mg/kg once every 3 days� 5 doses,corresponding to the same cumulative dose of 50 mg/kgper day, resulted in equivalent TGI (100% median TGIwith 17% regression). Similarly, a high intermittent doseof 300 mg/kg administered once weekly resulted in com-parable efficacy (100% TGI with 45% regression). Similarresults were observed with the SKOV-3 model (data not

shown). Pharmacokinetic analysis at the 300 mg/kg doseshowed that more than 20 mmol/L OSI-027 plasma con-centrations were maintained for at least 96 hours, with adecline to 0.58 mmol/L by 120 hours.

Finally, we determined the level of proliferation inSKOV-3 tumors by staining tumor samples for Ki67.OSI-027 was administered at 65 mg/kg once daily for3 days and tumor sampleswere collected 8 hours post lastdose. OSI-027 treatment resulted in 51.8% � 4.2% inhibi-tion of Ki67 staining compared with vehicle-treated con-trol tumors (Fig. 5F), confirming the antiproliferativeeffects of OSI-027.

Broad-spectrum antitumor activity of OSI-027The antitumor efficacy of OSI-027 was further evalu-

ated in a wide variety of human cancer xenograft models.OSI-027 administered orally at a 50 or 65 mg/kg dailydose showed significant inhibition of tumor growth andinduced tumor regression in several models of breast,colon, lung, prostate, lymphoma, and head and neckcancer (Table 3). In all studies, OSI-027 was well toleratedwith less than 10% body weight loss observed during thetreatment period.

Discussion

Clinical studies with rapamycin and rapalogs havevalidated mTOR as an anticancer target. However, theeffectiveness of rapalogs may be limited by lack ofinhibition of mTORC2 signaling functions and activa-tion of AKT via release of the S6K–IRS-1 negative feed-back loop. These considerations led to intensive effortsto develop mTOR-selective kinase inhibitors that com-pletely suppress both mTORC1 and mTORC2 activitiesin cancer cells. In this article, we provide biochemical,cellular, and in vivo evidence that OSI-027 is a potentand selective dual inhibitor of mTORC1 and mTORC2and is mechanistically and functionally distinct fromrapamycin.

Table 3. Broad-spectrum antitumor activity of OSI-027

Cell line Tumortype

PTEN/PIK3CA/KRAS/BRAFmutation status

OSI-027 dose,daily

Tumor growthinhibition (%)

Maximumregression (%)

MCF-7 Breast PIK3CA mt 65 mg/kg 87 9MDA-MB-468 Breast PTEN null 50 mg/kg 99 26HCT-116 Colon PIK3CA mt, KRAS mt 65 mg/kg 100 18NCI-H292 Lung wt 50 mg/kg 61 8NCI-H460 Lung PIK3CA mt, KRAS mt 50 mg/kg 84 0DU145 Prostate wt 65 mg/kg 58 0RL Lymphoma wt 65 mg/kg 100 27FADU HNSCC wt 50 mg/kg 86 0

NOTE: OSI-027 was administered orally once daily at indicated dose for 14 days. The percentages of TGI and regression werecalculated as described in Materials and Methods. n ¼ 8 animals per group in all studies.Abbreviation: HNSCC, head and neck squamous cell carcinomas.

Bhagwat et al.





As predicted, OSI-027 potently inhibited phosphoryla-tion of AKT on Ser 473 in the majority of cell lines tested.Surprisingly, phosphorylation of AKT on Thr 308, aPDK1 phosphorylation site was also inhibited by OSI-027 in several tumor cell lines we have investigated andsimilar inhibition of pAKT (T308) has been reported withmTORC1/mTORC2-selective inhibitors such as AZD-8055, Ku-0063794, PP242, and INK128 (15, 21–22, 31).But our observation differs from results with the selectivemTORC1/mTORC2 inhibitor WYE-132 or genetic abla-tion of mTORC2, which did not inhibit AKT phosphor-ylation on T308. It is unclear whether this distinction isassociated with differences in selectivity profile or differ-ences in the cell lines used. More detailed comparisons ofthese mTORC1/mTORC2-selective inhibitors are clearlyneeded to understand underlying difference in theirmechanisms of action.OSI-027 profoundly inhibits proliferation of both rapa-

mycin-sensitive and -insensitive cancer cell lineswith IC50

values in low micromolar range. Inhibition of 4E-BP1(T37/46)phosphorylation byOSI-027 correlateswith inhi-bition of cell proliferation in rapamycin-insensitiveMDA-MB-231 and rapamycin-sensitive BT-474 breast cancer celllines (data not shown). The molecular mechanism forrapamycin sensitivity and insensitivity in these cell linesis not yetwell understoodandneeds further investigation.Treatment with OSI-027, but not rapamycin, results inprofound inhibition of 4E-BP1 (T37/46) phosphorylation,and it seems to translate into robust antiproliferativeeffects in the majority of cell lines tested. OSI-027 andother selective mTORC1/2 inhibitors have been shown toinhibit rapamycin-insensitive functions of mTORC1 (15,21–26). Surprisingly, OSI-027 induced substantial celldeath in several cell lines with PI3K/AKT pathway acti-vation.Wehypothesized that this cell death is likelydue toinduction of autophagy and/or apoptosis. The inhibitionof phosphorylation of 4E-BP1 at T37/46 has been asso-ciated with a greater inhibition of cap-dependent transla-tion and cellular proliferationwith concomitant inductionof autophagy (7). In most of the cell lines studied in vitro,rapamycin and rapalogs induce a partial growth inhibi-tion and limited autophagy (32). mTORC1 has beenshown to control autophagy by direct and indirectmechanisms (32, 33). Recently, OSI-027, but not rapamy-cin, has been shown to profoundly stimulate autophagy inK562 leukemic cells and RCC cell lines (34, 35). Alterna-tively, we have measured caspase-3/7 activation uponOSI-027 or rapamycin treatment todeterminewhether celldeath is mediated by apoptosis. Rapamycin failed toinduce caspase-3/7 in the majority of cell lines, whereasOSI-027 induced apoptosis in some cell lines with PI3K/AKT pathway activation. Indeed, quantitation of the sub-G1 fraction by flow cytometry indicates that OSI-027induced apoptotic cell death in IGR-OV1 cells. However,the observation of caspase-3/7 induction in PTEN-defi-cient IGR-OV1 and U-87 MG, but not PTEN-deficientMDA-MB-468, indicates that there is not a simple correla-tion between PTEN status and apoptosis induction. These

collective results suggest that OSI-027 can cause cell deathby inducing both autophagy and apoptosis in a cell type–specific manner.

OSI-027 was well tolerated in vivo and induced dose-dependent growth inhibition and/or regression in multi-ple tumorxenograftsmodels. Efficacywasassociatedwitha dose-dependent pharmacodynamic inhibition of bothp4E-BP1 and pAKT in tumors. These studies highlight thecritical importance of mTOR as a driver of tumor prolif-eration in vivo. OSI-027 was effective against tumors fromdiverse genetic backgrounds showing significant TGI andregression in tumors harboring PTEN deficiency (IGR-OV1, MDA-MB-468), mutant PIK3CA (MCF-7, SKOV-3),and mutant KRAS (MDA-MB-231, H460). Steady-statemodeling of OSI-027 drug concentration by using osmoticpumps in PIK3CA mutant (SKOV-3) and PIK3CA wild-type (OVCAR-5) xenograft models indicates that PIK3CAmutant models are more sensitive to OSI-027 treatment.Interestingly, in a PTEN-null IGR-OV1 model, a dose of300 mg/kg once weekly was well tolerated and as effica-cious as once daily dosing at 50 mg/kg. This efficacy wasattributed to sustained plasma drug concentrationsgreater than 20 mmol/L for at least 4 days after dosing.Taken together, thesedatasuggest that insensitive tumors,maintenance of trough drug concentrations of 0.5 mmol/Lachieved through daily or intermittent oral dosing areneeded for therapeutic efficacy. The efficacy of OSI-027and rapamycin was compared in GEO and COLO 205colorectal cancer xenografts dosing both agents at MTD,with rapamycin dosed i.p to compensate for its poor oralbioavailability. In these studies, OSI-027 induced signifi-cantly greater TGI than rapamycin. Interestingly, both ofthese cell lines have an active IGF-II/IGF-1R autocrineloop, thereby activating the PI3K/AKT pathway. Thelimited efficacy of rapamycin compared with OSI-027 inthese models is likely to be due to the fact that OSI-027inhibits bothmTORC1 andmTORC2,whereas rapamycinonly inhibits mTORC1. In all (>10) in vivo xenograftstested, the efficacy of OSI-027 was not restricted to PTENdeficiency, PIK3CA mutation, or PTEN mutation status.

In conclusion, specific and global targeting of mTOR incancer cells by dual mTORC1 and mTORC2 inhibitorshas further validated an essential role of mTOR in cellproliferation, survival, and tumor growth. OSI-027 ismechanistically and functionally distinct from rapalogsand inhibits growth of numerous models of breast, colon,ovary, lung, and prostate cancer. The preclinical phar-macology, broad-spectrum efficacy, and feasibility ofmultiple dosing schedules have supported the use ofOSI-027 as an anticancer agent and justified its entry intophase I clinical development as a promising new antic-ancer therapy for potential treatment of solid tumors andhematologic malignancies.

Disclosure of Potential Conflicts of Interest

Employment by OSI Pharmaceuticals for: S.V. Bhagwat, P.C. Gokhale,A.P. Crew, A. Cooke, Y. Yao, C. Mantis, J. Kahler, J. Workman, M. Bittner,






N.W. Gibson, L.D. Arnold, R. Wild, D.M. Epstein, and J.A. Pachter atthe time this work was conducted. Inventorship on patents for A.P. Crewand L.D. Arnold.

Acknowledgments

The authors thank Xin Chen, Hanqing Dong, and Paula A.R. Tavares-Greco for synthesizing OXA-01 and OSI-027, Anna Chan for assistance

with mechanistic and proliferation assays, David Young and Gina Sen-nello for Ki-67 and CD31 staining of the tumors.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 9, 2010; revised June 2, 2011; accepted June 8, 2011;published OnlineFirst June 14, 2011.

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Bhagwat et al.





2011;10:1394-1406. Published OnlineFirst June 14, 2011.Mol Cancer Ther Shripad V. Bhagwat, Prafulla C. Gokhale, Andrew P. Crew, et al. Inhibitor of mTORC1 and mTORC2: Distinct from RapamycinPreclinical Characterization of OSI-027, a Potent and Selective

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Preclinical Characterization of OSI-027, a Potent and ...mct.aacrjournals.org/content/molcanther/10/8/1394.full.pdffurther support the important role of mTOR as a driver of tumor growth