Characterization of the Activity of the PI3K/mTOR ... · Small Molecule Therapeutics Characterization of the Activity of the PI3K/mTOR Inhibitor XL765 (SAR245409) in Tumor Models

Small Molecule Therapeutics

Characterization of the Activity of the PI3K/mTOR InhibitorXL765 (SAR245409) in Tumor Models with Diverse GeneticAlterations Affecting the PI3K Pathway

Peiwen Yu, A. Douglas Laird, Xiangnan Du, Jianming Wu, Kwang-Ai Won, Kyoko Yamaguchi, Pin Pin Hsu,Fawn Qian, Christopher T. Jaeger, Wentao Zhang, Chris A. Buhr, Paula Shen, Wendy Abulafia, Jason Chen,Jenny Young, Arthur Plonowski, F. Michael Yakes, Felix Chu, Michelle Lee, Frauke Bentzien, Sanh Tan Lam,Stephanie Dale, David J. Matthews, Peter Lamb, and Paul Foster

AbstractActivation of the PI3K (phosphoinositide 3-kinase) pathway is a frequent occurrence inhuman tumors and is

thought to promote growth, survival, and resistance to diverse therapies. Here, we report pharmacologic

characterization of the pyridopyrimidinone derivative XL765 (SAR245409), a potent and highly selective pan

inhibitor of class I PI3Ks (a, b, g , and d) with activity against mTOR. Broad kinase selectivity profiling of >130protein kinases revealed thatXL765 is highly selective for class I PI3Ks andmTORover other kinases. In cellular

assays, XL765 inhibits the formation of PIP3 in the membrane, and inhibits phosphorylation of AKT, p70S6K,

and S6 phosphorylation in multiple tumor cell lines with different genetic alterations affecting the PI3K

pathway. In a panel of tumor cell lines, XL765 inhibits proliferation with a wide range of potencies, with

evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL765

results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of

approximately 24 hours. Repeat dose administration of XL765 results in significant tumor growth inhibition in

multiple human xenograft models in nude mice that is associated with antiproliferative, antiangiogenic, and

proapoptotic effects. Mol Cancer Ther; 13(5); 1078–91. �2014 AACR.

IntroductionClass I PI3 kinases convert phosphatidylinositol 4,5-

bisphosphate (PIP2) tophosphatidylinositol 3,4,5-trispho-sphate (PIP3) in response to external cell stimuli (1, 2).Activation of class IA PI3Ks (phosphoinositide 3-kinases;PI3Ka, -b, and -d) ismediated by receptor tyrosine kinases(RTK). G-protein–coupled hormone receptors are impli-cated in activation of PI3Kb and class IB PI3K (PI3Kg ;ref. 3). Ras, another important mediator of extracellularstimuli, can also promote PI3K activation, and PI3K canmediate cellular transformation by Ras (4). Downstreameffectors of PI3K signaling, such as phosphoinositide-dependent kinase-1 (PDK1) and AKT, bind to PIP3 at thecell membrane and are subsequently activated by phos-phorylation (1). In turn, PDK1 and AKT activate growthpathways, inhibit apoptotic signaling, and regulate tran-sition through restriction points in the cell cycle viaphosphorylation of their respective substrates (1).

mTOR is the kinase component of two multisubunitcomplexes calledmTORC1 (includesmTOR/Raptor) andmTORC2 (includes mTOR/Rictor; refs. 5, 6). mTORC1 isactivated via PI3K pathway signaling and also via PI3K-independent mechanisms involving sensing of cellularamino acid levels, AMP levels, and hypoxia, and drivescellular growth by regulating protein translation anddegradation (5, 6). mTORC2 is activated via growth fac-tor–dependent signaling via mechanism(s) that are stillbeing elucidated, and regulates cell growth, proliferation,and survival via phosphorylation of theAKT kinase (5–7).

Dysregulation of PI3K pathway components, resultingin hyperactivated PI3K and/or mTORC1 signaling, isobserved in various cancers and correlates with tumorgrowth andsurvival (1). For example, the catalytic subunitof PI3Ka (p110a), encoded by the PIK3CA gene, is mutat-ed in 12% of human cancers (8). This is likely an under-estimate because many of these data are presumablygenerated by hotspot sequencing. In addition, the tumorsuppressor PTEN, which serves as a critical negativeregulator of PI3K signaling by converting PIP3 back toPIP2, is frequently deleted or downregulated in humantumors (1, 9). Moreover, the tumor suppressor gene LKB-1, which negatively regulates mTORC1, is mutated/inac-tivated in a variety of familial and sporadic tumors (10).PI3K pathway signaling is implicated in tumor cell inva-sion, migration, and dissemination (11). Genetic andpharmacologic approaches have demonstrated that PI3K

Authors' Affiliation: Exelixis, Inc., South San Francisco, California

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

Corresponding Author: A. Douglas Laird, Exelixis, Inc., 210 East GrandAvenue, South San Francisco, CA 94080. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0709

�2014 American Association for Cancer Research.

MolecularCancer

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signaling mediates VEGF production, neoangiogenesis,vascular permeability, and vessel integrity in preclinicaltumor models (12–14).Resistance to a variety of anticancer therapies, includ-

ing RTK inhibitors and genotoxic agents, has been attrib-uted to ongoing activation of the PI3K/PTEN pathway(15–17). PI3K pathway inhibitors have been shown tosensitize cancer cells to agents targetingHER2, MET, andEGFR as well as platinum drugs and taxanes (18–23).mTOR has been extensively explored as an oncology

target (24). Themacrolide antibiotic rapamycin is a potentinhibitor of the mTORC1 complex. Several analogs ofrapamycin have been tested clinically in various oncologyindications, and evidence of therapeutic benefit has beenobserved (24). For example, the U.S. Food and DrugAdministration has approved TORISEL (temsirolimus;Pfizer) for patients with advanced renal cell carcinoma(RCC) and Afinitor (everolimus; Novartis) for the treat-ment of patients with advanced RCC after failure oftreatment with sunitinib or sorafenib, and for advancedestrogen receptor–positive breast cancer after failure oftreatment with a nonsteroidal aromatase inhibitor. How-ever, the efficacy of these agentsmay be limited by the factthat rapamycin analogs do not inhibit mTORC2 (7). Inaddition, mTORC1 inhibition may enhance cell survivalby upregulating PI3K/AKT signaling via inhibition ofa mTORC1-dependent negative feedback loop actingthrough PI3K (24). Thus, selective inhibitors of PI3K andmTOR signaling have therapeutic potential as singleagents and in combination with other therapies for avariety of cancer indications and several such agents haveentered clinical testing in recent years (1, 24).XL765 (SAR245409) is a potent and selective inhibitor

of class I PI3Ks. In addition, XL765 also inhibits mTOR.In cellular assays, treatment with XL765 inhibits phos-phorylation of proteins downstream of PI3K andmTOR, including AKT and ribosomal protein S6 (S6RP),in multiple tumor cell lines with diverse molecularalterations affecting the PI3K pathway. In a broad panelof tumor cell lines XL765 inhibits proliferation with awide range of potencies, which seemed to be influencedby genetic background. Oral administration of XL765 inhuman xenograft tumor models in athymic nude miceresults in dose-dependent inhibition of PI3K pathwaycomponents with a duration of action of at least 24hours. As a single agent, XL765 shows significant tumorgrowth inhibition in multiple human xenograft modelsat well-tolerated doses. Taken together, these data sup-port the ongoing clinical investigation of XL765 for thetreatment of cancer.

Materials and MethodsIn vitro kinase inhibition assaysKinase activity for PI3K isoforms was measured as the

percentageofATPconsumedfollowing thekinase reactionusing luciferase–luciferin-coupled chemiluminescence aspreviously described (25), with ATP concentrations

approximately equal to the Km for each respective kinase.Kinase reactions were initiated by combining test com-pounds, ATP and kinase in a 20 mL volume. PI3Ka, PI3Kb,PI3Kg , and PI3Kd (Upstate Biotechnology) final enzymeconcentrations were 0.5, 8, 20, and 2 nmol/L, respectively.Asimilar assay formatwasused forDNAPK(DNAproteinkinase; purchased from Promega) and VPS34 [PIK3C3;prepared at Exelixis as an N-terminal tagged full-lengthhuman fusion protein, which was expressed in insect cellsusing Baculovirus Expression Vector Systems (BEVS)and affinity purified using glutathione sepharose]. VPS34assay buffer contained 20 mmol/L Tris-HCl, pH 7.5, 3.5mmol/L MnCl2, 100 mmol/L NaCl, 1 mmol/L DTT, and0.01% cholamidopropyldimethylammonio propanesulfo-nate (CHAPS). Of note, 0.5 mL dimethyl sulfoxide (DMSO)containing varying concentrations of the test compoundwasmixedwith 10mL enzyme solution (2� concentration).Kinase reactions were initiated by the addition of 10 mL ofliver phosphatidylinositol and ATP solution (2� concen-tration). Assay concentrations for VPS34, ATP, and phos-phatidylinositolwere40nmol/L, 1mmol/L, and5mmol/L,respectively.

Human tumor cell linesCell lines were obtained from the American Type Cul-

ture Collection (ATCC) in 2001 to 2005 and maintained inculture conditions at 37�C under 5% CO2. PC-3, MCF7,and A549 cells were maintained in Dulbecco’s ModifiedEagle Medium (DMEM; Cellgro 10-013-CV) containing10% FBS (heat inactivated; Cellgro; 35-016-CV), 1% non-essential amino acids (NEAA; Cellgro; 30-002-CI), and 1%penicillin–streptomycin (Cellgro). U87-MG and MDA-MB-468 cellsweremaintained in Eagle’sMinimumEssen-tial Medium (EMEM)-Alpha (Cellgro; 10-022-CV),DMEM/F-12 (Cellgro, 15-090-CV), respectively, supple-mentedwith 10% FBS, 2mmol/L L-glutamine, 1%NEAA,and 1% penicillin–streptomycin. LS174T cells were main-tained inMEM (GIBCO; 10370-021) containing 10% FBS, 2mmol/l L-glutamine, 1mmol/L sodiumpyruvate, and 1%penicillin–streptomycin. Ramos cells were maintained inRPMI-1640 (Cellgro; 10-040-CV) containing 10% FBS(Cellgro) and 1% penicillin–streptomycin (Cellgro).OVCAR-3 cellsweremaintained inRPMI-1640 containing20% FBS and 1% penicillin–streptomycin.

Immune-complex mTOR kinase—Westernimmunoblot analysis

mTORC1. HEK 293 (ATCC) cells were grown inDMEM (Cellgro) containing 10% FBS (Cellgro), 1%NEAA (Cellgro), and 1% penicillin–streptomycin (Cell-gro), and lysed in ice-cold lysis buffer containing 40mmol/L HEPES pH 7.5, 120 mmol/L NaCl, 1 mmol/LEDTA, 10 mmol/L Na pyrophosphate, 10 mmol/Lb-glycerophosphate, 50 mmol/L NaF, one tablet ofprotease inhibitors (Complete-Mini; EDTA-free; Roche),0.3% CHAPS, and 1.5 mmol/L Na3VO4. mTORC1 wasincubated with anti-mTOR antibody (N-19; Santa CruzBiotechnology; sc-1549) 1.5 hours to overnight. The

Pharmacodynamics and Antitumor Efficacy of XL765 (SAR245409)

www.aacrjournals.org Mol Cancer Ther; 13(5) May 2014 1079




resulting immune-complexes were immobilized onimmunoglobulin G (IgG) sepharose (GE Healthcare;17-0618-01), washed sequentially three times with lysisbuffer, once with wash buffer (50 mmol/L HEPES,pH 7.5, 40 mmol/L NaCl, and 2 mmol/L EDTA), andonce with kinase buffer (25 mmol/L HEPES, pH 7.5,50 mmol/L KCl, 20% glycerol, 10 mmol/L MgCl2,4 mmol/L MnCl2, 1 mmol/L DTT). The immune-com-plexes (equivalent to 1 � 106 cells) were preincubated at30�C with XL765 or 0.1% DMSO for 10 minutes, andthen subjected to kinase reaction for 30 minutes in a finalvolume of 20 mL (including 10 mL bed volume) contain-ing kinase buffer, 25 mmol/L ATP, and l mg 4EBP1(Exelixis). Kinase reactions were terminated by additionof 3.3 mL 4� sample buffer (Invitrogen; NP0007) con-taining 7% b-mercaptoethanol and analyzed byWesternimmunoblotting. Nitrocellulose membranes were incu-bated overnight at 4�C with 1/1,000 dilution of rabbitanti-mTOR (Upstate; 07-231) in 5% nonfat milk contain-ing TBST (TBS/0.1% Tween-20) or with 1/500 dilutionof rabbit anti-p4EBP1 [Cell Signaling Technology (CST),#9459] in 3% bovine serum albumin (BSA)/TBST, fol-lowed by incubation for 1 hours with a 1/5,000 dilutionof secondary immunopure peroxidase–conjugated goatanti-rabbit IgG (HþL; Pierce; 31462) in 5% nonfat milk/TBST. Phospho-4EBP1 and mTOR were detected withSuper Signal West Pico Substrate (Pierce; 34080). Thep4EBP1 blot was subsequently stripped and reprobedwith anti-4EBP1 antibody (1/1,000; CST#9452) with thetotal 4EBP1 signal detected as described above. Scanswere analyzed using ImageQuant software. The DMSOcontrol sample was used for normalization, and the IC50

value for XL765 was determined using XLfit4 software.mTORC2. HeLa (ATCC) cells were grown in suspen-

sion culture in EX-CELLHeLamedia (Sigma; 14591C) andlysed as described above with minor modifications. ThemTORC2 complexwas incubatedwith anti-RICTOR anti-body (Exelixis) for 2 hours and immune complexes(equivalent to 1� 107 cells) prepared as abovewithminormodifications. These were preincubated at 37�C with atest compound or 0.6% DMSO for 5 minutes, and thensubjected to a kinase reaction for 8 minutes in a finalvolume of 33 mL (including 5 mL bed volume) containingkinase buffer, 50 mmol/L ATP, and 0.75 mg AKT1 [full-length human AKT1 with amino-terminal Hi tag wasexpressed in Sf9 cells using standard procedures, puri-fied, then dephosphorylated with lambda protein phos-phatase (New England Biolabs; P0753L) before repurifi-cation and use as substrate]. Kinase reactions were sub-sequently terminated and resolved as described abovewith minor modifications, then transferred onto polyvi-nylidene difluoride (PVDF) membranes at 50 V for 20hours at 4�C. The membranes were blocked in 5% nonfatmilk in TBST for 1 hour and incubated overnight at 4�Cwith 1/1,000 dilution of rabbit anti-pAKT (S473; CST#4060) in 3% BSA/TBST. The membranes were washedthree times in TBST and incubated for 1 hour with a 1/10,000 dilution of secondary goat anti-rabbit horseradish

peroxidase (HRP) antibody (CST# 2125) in 5% nonfatmilk/TBST. The IC50 value for XL765 was determined asdescribed above.

PIP3 mass balance assayPC-3 (ATCC) and MCF7 (ATCC) cells were seeded at

2� 106 and 2.5� 106 cells, respectively, onto 10-cm dishesin culture medium and incubated at 37�C, 5% CO2 for 24hours. Growth medium was replaced with serum-freeDMEMand cellswere incubated for an additional 3 hours.Serial dilutions of test compounds in fresh serum-freemediumwere added to the cells in a final concentration of0.3%DMSO (vehicle) and incubated for 23minutes beforerecombinant human EGF stimulation (200 ng/mL; R&DSystems; 236-EG) for 2 minutes. After treatment, themedium was removed, and cellular material was precip-itated with ice-cold 10% trichloroacetic acid (TCA) andcollected by centrifugation. The pellet was washed with 3mL of 5% TCA/1 mmol/L EDTA. Neutral lipids wereextracted from the pellet with 3 mL of methanol:chloro-form (2:1), and then the acidic lipids were extracted with2.25 mL of methanol:chloroform:12 N HCl (80:40:1). Theorganic phase was separated from the aqueous phase bythe addition of 0.75mLof chloroform and 1.35mLof 0.1NHCl followed by centrifugation. The organic phase wasthen collected into a glass tube, dried under nitrogen gas,and resuspendedby sonication in awater bath in 120mLofthe PIP3 mass assay buffer (50 mmol/L HEPES, pH 7.4,150 mmol/L NaCl, and 1.5% sodium cholate).

Assays were conducted in 96-well plates (PerkinElmer;L2251692) by incubating 50 mL of the lipid extract with 50mL of the sensor complex in detection buffer (10 mmol/LTris-HCl, pH 7.2, 150 mmol/L NaCl, 7.5 mmol/L EDTA,0.1% Tween-20, and 1 mmol/L DTT) at ambient temper-ature in thedark for 2hours, andplateswere readusing anAlphaQuest reader (PerkinElmer). The sensor complexcontained 15mLof 100 nmol/LbiotinylatedPIP3 (Echelon;C-39B6), 15 mL of 100 nmol/L glutathione S-transferase(GST)–tagged GRP1 pleckstrin homology (Echelon; G-1200), and 20 mL of a mixture of donor and acceptorAlphaScreen beads (GST detection kit; PerkinElmer;6760603c). The PIP3 mass present was estimated by com-parison with standard curves constructed by addition ofknown amounts of diC8 PI(3,4,5)P3 standard (Echelon; P-3908) to the sensor complex.

pAKT and pS6 ELISApS6 ELISAwas performed as previously described (26)

with minor modifications. The pAKT ELISA assay wasperformed as follows: PC-3 (ATCC) cells were seeded at1.5� 105 cells per well in 6-well plates (NUNC; 140685) ingrowth medium then incubated at 37�C, 5% CO2 for 72hours, and the growthmediumwas replacedwith serum-free DMEM. Serial dilutions of the test compound in 0.3%DMSO (vehicle) were added to the cells and incubated for2 hours and 50 minutes. Cells were then stimulated with100 ng/mL EGF (R&D Systems; 236-EG) for 10 minutes.Cells were washed once with ice-cold PBS, harvested by

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Mol Cancer Ther; 13(5) May 2014 Molecular Cancer Therapeutics1080




briefly shaking in 100mLof TENN lysis buffer (20mmol/LTris-HCl, pH 7.5, 1mmol/LEDTApH8.0, 0.5%NP40, 150mmol/L NaCl) with protease and phosphatase inhibitors(1 mmol/L PMSF (phenylmethylsulfonylfluoride), 1mg/mL leupeptin, 1mg/mLaprotinin, 1mg/mLpepstatin,1 mmol/L EDTA, 1 mmol/L NaF, 20 mmol/L b-glycer-ophosphate, 1mmol/LNa-orthovanadate, and 5mmol/Lp-nitrophenyl phosphate), and transferred to 96-wellplates. Cells were lysed on ice for 20 minutes by pipettingup and down 10 times, and the supernatants were col-lected by centrifugation. ELISA assays for pAKTT308 andtotal AKT were performed with the AKT[pT308] ELISAKit (BioSource International; KHO0201) and AKT ELISAKit (BioSource International; KHO0101). IC50 values weredetermined on the basis of the ratio of pAKT to total AKTsignal in lysates fromcompound-treated cells, normalizedto lysates from DMSO-treated controls.

PI3K pathway profiling Western immunoblotanalysisPC-3, MCF7, A549, U87-MG, LS174T, MDA-MB-468,

andOVCAR-3 cellswere seeded at densities of 2� 106, 2�106, 1.2� 106, 2� 106, 2� 106, 1.2� 106, and 1� 106 cells,respectively, onto 10-cm dishes in their respective culturemediumand incubated at 37�C, 5%CO2 for 20 to 47 hours.The medium was replaced with test compounds dis-solved in the same media containing 0.3% DMSO, andthe cells were incubated for 3 hours. For growth factortreatment, the medium was replaced with test com-pounds dissolved in serum-free DMEM containing0.3% DMSO. After incubation for 3 hours, cells werestimulated with 100 ng/mL of EGF (R&D Systems; 236-EG) for 10 minutes. Cells were washed with ice-cold PBS,and directly lysed with cell lysis buffer (BioSource Inter-national; FNN0011) containing protease inhibitors (Com-plete-Mini; EDTA-free; Roche; 11836170001; aminoethyl-benzenesulfonyl fluoride; Sigma; A8456). Protein lysateswere analyzed by Western immunoblotting. PVDF mem-branes (Invitrogen) were incubated overnight at 4�Cwithprimary antibodies at the indicated concentrations in 3%BSA/TBST buffer, followed by incubation for 1 hour witha 1/10,000 dilution of secondary goat anti-rabbit HRPantibody (CST#7074) or a 1/3,000 dilution of secondarygoat anti-mouse HRP antibody (Amersham, NXA931) in5% nonfat milk/TBST. Signals were detected using ECL-plus (Amersham; RPN2132) and scanned using aTyphoon 9400 scanner (Molecular Devices). For totalprotein readouts, stripped membranes were incubatedwith the respective primary antibodies, with signal detec-tion as described above. Scans were analyzed using Ima-geQuant software. Phospho signals were normalized tothe corresponding total protein signals, the percentage ofinhibition relative to DMSO control was determined, andIC50 values were calculated using XLfit4 software. Thefollowing antibodies were used in Western Immunoblotanalysis: pAKT (T308; CST#4056; 1/500 dilution), pAKT(S473; BioSource International; 44-622G; 1/1,000), AKT(BioSource International; 44-607G; 1/1,000 dilution),

pp70S6K (T389; CST #9234; 1/1,000 dilution), p70S6K(Bethyl; A300-510A; 1/2,500 dilution), pS6 (S240/244;CST #2215; 1/2,000 dilution), S6 (CST #2217; 1/2,000dilution), pPRAS40 (T246; BioSource International; 44-1100G; 1/2,000 dilution), PRAS40 (BioSource Interna-tional AHO1031; 1/1,000 dilution), pGSK3b (S9; CST#9336; 1/1,000 dilution), GSK3b (CST #9315; 1/1,000dilution), p4EBP1 (T37/46; CST #9459; 1/1,000 dilution),4EBP1 (CST #9452; 1/1,000 dilution), cyclin D1 (EMDBiosciences; CC12; 1/1,000 dilution), pERK (Y204; SantaCruz Biotechnology; sc-7383, 1/1,000 dilution), and ERK(extracellular signal–regulated kinase; CST #9102; 1/1,000 dilution).

mTOR pathway assay in Ramos cellsRamos (ATCC) cells were seeded at a density of 0.3 �

106 cells/mL in growth medium. The next day, cells werecentrifuged, washed with serum-free medium supple-mented with 1% BSA (tissue culture tested; Sigma;A4919), resuspended in RPMI supplemented with 1%BSA, and incubated for 20 hours. The serum-starved cellswere centrifuged and resuspended in 5 mL of the savedmedia at 1 � 106 cells/mL. Cells were treated with testcompound administered at a finalDMSO concentration of0.3%. For the nutrient-depletion control, the serum-starved cells were washed once with PBS, and resus-pended in 5mL of PBSwith 0.3%DMSO.After incubationfor 2hours, cellswerewashedonce inPBS and lysed in 150mL of BioSource cell lysis buffer containing protease inhi-bitors (Complete-Mini; EDTA-free; Roche; AEBSF; Sig-ma). Lysates were analyzed byWestern immunoblotting.PVDF membranes were incubated overnight at 4�C with1/500 dilution of rabbit anti-pmTOR (CST#2974), with 1/1,000 dilution of rabbit anti-pp70S6K (CST#9234), or with1/1,000 dilution of rabbit anti–p4E-BP1 (CST#9459) in 3%BSA/TBST, followed by incubation for 1 hours with a 1/10,000 dilution of secondary goat anti-rabbit HRP anti-body (CST#7074) in 5% nonfat milk/TBST and signalsdetected and analyzed as described above. For detectionof total p70S6K and total 4E-BP1, stripped membraneswereprobedwith rabbit antitotal p70S6K (1/2,500, Bethyl;A300-510A) and with rabbit antitotal p4E-BP1 (1/1,000;CST#9452) with signals detected and analyzed asdescribed above.

Cell proliferation and cytotoxicity assaysCellular proliferation was assessed as previously

described (27) using the Cell Proliferation ELISA, Bromo-deoxyuridine (BrdUrd) Chemiluminescence Kit (Roche;Applied Science). Cytotoxicity was assessed using theATP Bioluminescence Assay as follows: PC-3, MCF7,A549, LS174T, MDA-MB-468, U87-MG, and OVCAR-3cells were plated at densities of 7 � 103, 1.5 � 104, 6 �103, 7 � 103, 7 � 103, 6 � 103, 1.5 � 104 cells per well,respectively, onto 96-well microtiter plates (Corning;3904) in culture medium, incubated at 37�C, 5% CO2 for18 hours, and then treated with a serial dilution of com-pound inmedium containing a final concentration of 0.3%






DMSO. Triplicate wells were used for each compoundconcentration. Control wells received 0.3% DMSO inmedia. Cultures were incubated at 37�C, 5% CO2 for anadditional 24 hours and cells were then assayed forviability using the ViaLight HS Kit (Cambrex; LT07-111).

Apoptosis assay (caspases 3/7 assay)PC-3,MCF7,A549, LS174T,MDAMB468,U87MG, and

OVCAR-3 cells were plated at densities of 5 � 103, 1.2 �104, 5� 103, 6� 103, 6� 103, 5� 103, and 1.2� 104 cells perwell, respectively, onto 96-wellmicrotiter plates (Corning;3904), in culture medium at 37�C, 5% CO2 for 18 hours,and then treated with a serial dilution of compound inmedium containing a final concentration of 0.3% DMSO.Triplicate wells were used for each compound concentra-tion. Positive control wells received 5 to 30,000 nmol/Ladriamycin (MCF7, A549, and LS174T) or 5 to 30,000nmol/L camptothecin (PC-3, U87-MG, OVCAR-3, andMDA-MB-468) and negative control wells received 0.3%DMSO inmedia. Backgroundwells contained no cells and0.3% DMSO in media. Following incubation at 37�C, 5%CO2 for an additional 48 hours, apoptosis was assessedusing the Apo-ONE Homogeneous Caspase-3/7 AssayKit (Promega; G7791). EC50 values were calculated on thebasis of the fluorescence of compound-treated wells com-pared with that of the corresponding positive control.

Anchorage-independent growth assay (soft agarassay)

Soft agar (60 mL/well of 0.75%; BD Biosciences) wasplated in a 96-well black plate (Nalge Nunc International)and allowed to solidify at 37�C for 20 minutes. A total of4.8� 103 PC-3 orMCF7 cells in 100 mL ofmedia containing0.375% agar, FBS (15% for PC-3 and 20% for MCF7), and1� concentrations of serial dilutions of XL765 were lay-ered over the base agar. After 10minutes, 60 mL of DMEM(GIBCO) containing FBS and 2-fold concentrated testcompounds were added over the cell layer. Followingequilibration of the media and the test compound, thefinal compound concentration was presumed to be 1� inall three layers. The cultureswere incubated for 14 days at37�C, 5% CO2. At day 7, 50 mL of fresh media containing10% FBS without compound was added to keep thecultures from drying out. At the completion of the incu-bation, themedia in the top layerwere removed, and40mLof media containing 50% Alamar Blue (BioSource Inter-national; DAL1025) were added to each well followed byincubation at 37�C, 5% CO2 for 4 hours and subsequentfluorescent detection.

Migration assayThe hepatocyte growth factor (HGF)–induced chemo-

taxis assay was performed as previously described (27).

PC-3,MCF7, andB16F10 cytotoxicity assays (AlamarBlue assay)

PC-3,MCF7, and B16F10 (ATCC) cells weremixedwitha series of diluted compounds in serum-free DMEM

(Gibco), DMEM containing 0.2% FBS, and serum-freeEBM-2 medium (Clonetics), respectively. Control wellsreceivedmediawith 0.25%DMSO alone. A total of 5� 103

cells were plated in each well of a 96-well plate andincubated at 37�C, 5%CO2 for 18 hours (PC-3) or 24 hours(B16F10 andHMVEC-L). At the end of the incubation, cellviability was determined using Alamar Blue solution(BioSource International).

Studies in tumor-bearing miceTumors were collected at the indicated time points and

tumor lysateswere prepared as previously described (25).Pooled lysates were analyzed by Western immunoblot-ting. PVDF membranes were incubated overnight at 4�Cwith primary antibodies to the respective phosphoepi-topes at the indicated concentrations in 3% BSA/TBSTbuffer. The membranes were then incubated for 1 hourwith a 1/10,000 dilution of secondary goat anti-rabbitHRP antibody (CST#7074) in 5% nonfat milk/TBST.Signals were detected using ECL-plus (Amersham;RPN2132) and scanned using Typhoon (MolecularDevices). To determine total protein levels, membraneswere stripped and incubated with the indicated primaryantibodies specific for the respective total proteins. Thesame procedure described above was followed to detectthe total protein signal. Scans were analyzed using ImageQuant software. The percentage of inhibition was deter-mined by normalizing the phosphoepitope signals to thetotal protein signals and then calculating thepercentage ofinhibition compared with vehicle control groups. Thefollowing antibodies were used in Western immunoblotanalysis of tumor extracts: pAKT (T308; CST#4056; 1/500dilution), pAKT (S473; CST #9271; 1/1,000), AKT (CST#9272; 1/1,500dilution), pp70S6K (T389;CST#9234; 1/500dilution), p70S6K (Bethyl; A300-510A; 1/2,000 dilution),pS6 (S240/244; CST #2215; 1/2,000 dilution), and S6 (CST#2217; 1/1,500 dilution).

In vivo efficacy studieswere performed in athymic nudemicepurchased fromTaconic andhousedaccording to theExelixis Institutional Animal Care and Use Committeeguidelines. Tumor cells were cultured in vitro in DMEM(Mediatech) supplemented with 10% FBS (20% for PC-3and OVCAR-3 cells), penicillin—streptomycin, and non-essential amino acids at 37�C in a humidified 5% CO2

atmosphere. On day 0, cells were harvested by brieftrypsinization, and 1 to 5 � 106 cells in 0.1 mL ice-coldHanks Balanced Salt Solution were implanted subcuta-neously (OVCAR-3) or intradermally (MCF7 and U-87MG) into the hind flank of female athymic nude mice. Inthe case of the MCF7 model, an estrogen pellet (IRA) wasimplanted subcutaneously at the nape of neck at the timeof tumor cell implantation. A total of 3 � 106 PC-3 cellswere similarly harvested and implanted subcutaneouslyinto the hind-flank of 5- to 8-week-old male nude mice.Tumor growth was monitored weekly with calipers untilstaging and dose initiation. During the dosing period,body and tumor weights were assessed as previouslydescribed (27). XL765 was formulated in sterile water/

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10 mmol/L HCl or water and administered at the indi-cated doses and regimens by oral gavage at a dose volumeof 10 mL/kg.

HistologyAfter euthanasia, tumors from animals administered

XL765 and/or other agents were excised and fixed in zincfixative (BD Pharmingen) for 24 to 48 hours before beingprocessed into paraffin blocks.Of note, 5-mm-thin sectionswere cut serially to represent the largest possible surfacefor each tumor and stained using standard immunohis-tochemical methods to detect Ki67 nuclear antigen (Lab-Vision) and CD31-positive tumor vessels (BD Pharmin-gen). CD31 was detected by biotinylated secondary anti-body followed by the avidin–biotin–peroxidase complex(BD Pharmingen). Ki67 was detected by Envisionþ anti-rabbit peroxidase complexed polymer (DAKO). Sectionswere counterstained with hematoxylin. The tumor meanvessel density and Ki67 index in tumor sections werequantified using the ACIS automatic cellular imagingsystem (Clarient Inc.). Themean number of tumor vesselsper mm2 was determined by analyzing eight to 15 fieldsacross the total tumor section. The percentage of Ki67-positive tumor cells was determined by sampling multi-ple representative fields of equal size across the totalviable tumor area of each section anddividing the numberofKi67-positive cells by the total number of cells identifiedper field. Apoptosis was assessed by TUNEL as previ-ously described (27). The results for each immunohisto-chemical readout were averaged for each tumor section,followed by averaging the results for each treatmentgroup (n ¼ 9–10). Statistical analyses were performedusing the standard two-tailed t test with Bonferroniadjustment for multiple comparisons against a singlecontrol group.

ResultsXL765 is a selective inhibitor of class I PI3Ks and ofmTOR in biochemical assaysXL765 (Fig. 1A) was identified following optimization

of a pyridopyrimidinone scaffold for in vivo PI3K/mTORpathway inhibition and drug-like properties. In assaysperformed using purified proteins in a luciferase-coupledchemiluminescence format, XL765 displayed potentinhibitory activity against class I PI3K isoforms p110a,p110b, p110d, and p120g , with IC50 values of 39, 110, 43,and 9 nmol/L, respectively (Table 1). The IC50 value forinhibition of PI3Ka by XL765 was determined at variousconcentrations of ATP, revealing XL765 to be an ATP-competitive inhibitor with an equilibrium inhibition con-stant (KI) value of 13 nmol/L.XL765 also inhibited mTOR (IC50 values of 160 and 910

nmol/L for mTORC1 and mTORC2, respectively) in animmune-complex kinase assay and the PI3K-relatedkinase DNAPK (IC50 value of 150 nmol/L). In contrast,XL765 had relatively weak inhibitory activity toward theclass III PI3K vacuolar sorting protein 34 (VPS34; IC50

value of �9.1 mmol/L). XL765 was also profiled against apanel of approximately130 protein kinases; no cross-reac-tivity was observed at concentrations below 1.5 mmol/L(Supplementary Table S1). All assays were performed atATP concentrations approximately equal to theMichaelisconstant (KM) values of the respective enzymes.

N

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pPRAS40T246

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103

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mo

l/L

Figure 1. A, chemical structure of XL765 (SAR245409). B, XL765 inhibitsPI3K pathway signaling in EGF-stimulated MCF7 cells. After incubationwith XL765 at the indicated concentrations, PI-103 (10 mmol/L), ZSTK474(10 mmol/L), or rapamycin (0.1 mmol/L), MCF7 cells were stimulated with100 ng/mL of EGF for 10minutes. The cells were then lysed and effects ofcompound on PI3K pathway signaling assessed by Westernimmunoblotting. C, XL765 inhibits the nutrient-dependent mTORsignaling pathway inRamos cells. Cells were starved in serum-freemediafor 20 hours, then treated with compounds at the indicatedconcentrations, DMSO, or serum- and nutrient-free PBS for 2 hours. Celllysates were prepared and analyzed by Western immunoblotting.






XL765 inhibits the PI3K pathway in multiple tumorcell models

MCF7 human mammary carcinoma cells and PC-3human prostate adenocarcinoma cells were selected forthe initial assessment of the effect of XL765 on signalingdownstream of PTEN/PI3K because they each have aprevalent genetic lesion that activates the PI3K pathway.MCF7 cells carry a heterozygous E545K-activating muta-tion in the p110a subunit of PI3K and PC-3 cells carry ahomozygous deletion of exons 3 to 9 of the PTEN tumorsuppressor gene. PIP3 is the product of a class I PI3Ksacting on the physiologic substrate PIP2. Hence, PIP3

levels serve as a direct assessment of PI3K activity. Con-sistent with its inhibitory activity against purified PI3Kproteins, XL765 inhibited EGF-induced PIP3 productionin PC-3 and MCF7 cells with IC50 values of 290 and 170nmol/L, respectively (Table 2). The ability of XL765 toinhibit phosphorylation of key signaling proteins down-stream of PI3K was examined by assessing its effects onEGF-stimulated phosphorylation of AKT and on nonsti-mulated phosphorylation of S6 in PC-3 cells by cell-basedELISA. XL765 inhibited these activitieswith IC50 values of250 and 120 nmol/L, respectively (Table 2).

The effects of XL765 on the PI3K signaling pathwaywere then examined by Western immunoblot analysis in

PC-3 andMCF7 cells (see Fig. 1B and Supplementary Fig.S1). The results were consistent in both cell lines. XL765inhibits AKT phosphorylation at both activation sites(T308 and S473) at concentrations consistent with theIC50 values determined by ELISA. The T308 phosphory-lation site on AKT is a substrate for PDK1 (1), whereas theS473 site is a substrate for mTORC2 (6). Inhibition of AKTsubstrate phosphorylation (PRAS40 andGSK3b) and inhi-bition of phosphorylation events downstream of mTOR(p70S6K, S6, and 4EBP1 phosphorylation) were also evi-dent. XL765 induces a decrease in the levels of cyclin D1protein, consistent with increased GSK3b activity as aresult of inhibition of AKT leading to GSK3b-mediatedphosphorylation and subsequent degradation of cyclinD1 (Fig. 1B). Overall, a similar range of compound con-centrations was required to inhibit PI3K proximal phos-phorylation events (AKT T308 phosphorylation) andphosphorylation events downstream of mTORC1 andmTORC2 (p70S6K phosphorylation and AKT S473phosphorylation).

The control compound ZSTK474 (an inhibitor of PI3K;Reference 28) at 10 mmol/L robustly decreased the levelsof all the phospho readouts assessed. The TORC1 inhib-itor rapamycin at 0.1 mmol/L did not inhibit the phos-phorylation of AKT or its direct substrates PRAS40 andGSK3b, but in fact seemed to stimulate phosphorylation ofAKT. This is consistent with relief of p70S6K-dependentnegative feedbackof PI3K (see Introduction).As expected,rapamycin significantly decreased p-p70S6K and pS6levels, consistent with its well-characterized ability toinhibit mTORC1. None of the compounds had significanteffects on ERK1/2 phosphorylation, consistent with bio-chemical profiling data. XL765 was further profiled inadditional cell lines bearing a variety of genetic lesionsthat activate/modulate the PI3K pathway. These wereOVCAR-3 (PIK3CA amplification), U87-MG (PTEN dele-tion), A549 (KRASmutation, loss-of-function mutation inthemTOR-directed tumor suppressor geneLKB-1),MDA-MB-468 (PTEN deletion), and LS174T (PIK3CA and KRASmutations) cells. XL765 demonstrated consistent activityin these cell lineswith nomarkeddifferences in sensitivitybeing evident (Supplementary Figs. S2 and S3).

XL765 inhibits PI3K-independent mTOR signalingTodirectly assess the impact of XL765 onmTOR in cells,

we used an approach that relies on the ability of nutri-tional signals to activate mTOR independent of PI3Kactivity. Regulation of mTOR signaling by nutrient avail-ability is predominant in certain transformed B cells (29),and we used the Burkitt lymphoma–derived cell lineRamos as a system to study the effect of XL765 on nutri-ent-dependent mTOR activity (Fig. 1C). Cells werestarved in serum-freemedia for 20 hours, and then treatedwith compounds, DMSO, or serum- and nutrient-free PBSfor 2 hours. Cell lysates were prepared and analyzed bygel electrophoresis and Western immunoblotting withanti-pmTOR, anti–p-p70S6K, and anti-p4EBP1 antibo-dies. Cells incubated in PBS show very low levels of

Table 1. The kinase inhibition profile of XL765

XL765

Family Kinase IC50 (nmol/L)

PI3K Class IA PI3Ka 39 � 10PI3Kb 110 � 30PI3Kd 43 � 3

Class IB PI3Kg 9 � 3Class III VPS34 9,060

PI3K-related mTORC1 160a

mTORC2 910a

DNAPK 150

NOTE: IC50 is the concentration required for 50% targetinhibition.aImmunoprecipitation kinase assay using cell lysates.

Table 2. Effects of XL765 on PIP3 productionand AKT and S6 phosphorylation

Cell linePIP3 IC50

(nmol/L)pAKT IC50

(nmol/L)apS6 IC50

(nmol/L)a

PC-3 290 250 120MCF7 170 ndb ndb

NOTE: See Materials and Methods for details.aIC50 values determined using ELISA assay.bnd, not determined.

Yu et al.





phosphorylation of p70S6K, 4EBP1, or the mTOR autop-hosphorylation site S2481, consistent with low mTORactivity. Incubation of cells in serum-free, nutrient con-taining media results in a robust upregulation of mTOR-dependent phosphorylation events. ZSTK474 and PI-103(30) at 10 mmol/L inhibit these phosphorylation events,consistent with their ability to directly inhibit mTORkinase activity in addition to PI3K activity. Rapamycintreatment at 0.1 mmol/L resulted in little if any decreasein mTOR autophosphorylation, but profoundly inhibitedp70S6K phosphorylation consistent with selective inhibi-tion of mTORC1. XL765 inhibited nutrient-dependentphosphorylation at all sites, with IC50 values of 160,340, and 3,000 nmol/L, for mTOR S2481, p70S6K, and4EBP1 phosphorylation, respectively. These results areconsistent with the mTOR kinase assay results presentedabove, and provide further evidence that XL765 is a directinhibitor of mTOR kinase activity.

Effects on proliferation in a panel of tumor cell linesIn MCF7 and PC-3 cells, XL765 inhibits proliferation

(monitored by BrdUrd incorporation) with IC50 values of1,070 and 1,840 nmol/L, respectively. When tested in abroad panel of tumor cell lines with diverse origins andgenetic backgrounds, XL765 was found to inhibit prolif-eration with a wide range of IC50 values (200 to >30,000nmol/L; Fig. 2; Supplementary Table S2). A breakdown of

sensitivity by genotype suggested that PIK3CA-mutantcell lines tended to be relatively sensitive to XL765, where-as RAS- or BRAF-mutant cell lines tended to be lesssensitive. Interestingly, severalRAS-mutant cell lineswererelatively insensitive to XL765 despite of their also har-boring PIK3CA mutations (Fig. 2; Supplementary TableS2). Cell lines with loss of PTEN showed a range ofsensitivities, with some (e.g., the prostate carcinoma linesZR75-1, LNCap, andPC-3) being sensitive andothers (e.g.,the glioblastoma cell lines U251, U373) being refractory.

Anchorage-independent growth in soft agar is consid-ered the most stringent assay for detecting malignanttransformation of cells. To further characterize the effectsof XL765 on tumor cell growth, an assay monitoring theanchorage-independent growth of PC-3 andMCF7 cells insoft agar over a 14-day period was used. XL765 inhibitscolony growth with an IC50 value of 270 nmol/L in PC-3cells and 230 nmol/L inMCF7 cells. These IC50 values aresignificantly lower than those required to inhibit growthof the cells in a monolayer, perhaps indicating anincreased reliance on PI3K pathway signaling for growthin three-dimensions.

To rule out direct cytotoxic effects of XL765 on tumorcells, its effects on cell viability were determined bybioluminescent measurement of cellular ATP. XL765did not reduce ATP levels in cells when incubatedfor 24 hours, indicating a lack of acute cytotoxicity

0

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Figure 2. Relative sensitivity of tumor cells to XL765 as a function of genetic status. a, Cell proliferation IC50 values are presented normalized to that for BT474(most sensitive cell line). See Materials and Methods and Supplementary Table S2 for details.






(Supplementary Table S2, footnote). Induction of cyto-plasmic caspases 3 and 7was examined as an indication ofapoptosis induction. XL765 did not affect the activity ofthese caspases at the doses and time point tested (Sup-plementary Table S2, footnote). In MCF7 cells, the anti-proliferative effects of XL765 were associated with aspecific block in the G1 phase of the cell cycle and anincrease of sub-G1 cell population (data not shown).Therefore, at least in MCF7 cells, XL765 exhibits antipro-liferative effects predominantly via blockade of the cellcycle rather than through cytotoxic or apoptotic effects.

XL765 inhibits tumor cell migrationOne of the hallmarks of aggressive tumor cells is the

ability tomigrate in response to chemotactic stimuli and toinvade surrounding tissue. HGF is one of the key stimu-lators of these behaviors, and cell lines expressing highlevels of the HGF receptor MET, are highly invasive andmetastatic in vivo. Because PI3K resides in the MET sig-naling pathway, the ability of XL765 to inhibit HGF-stimulated migration was tested in vitro. Murine B16melanoma cells express high levels ofMet,whichbecomeshighly phosphorylated when the cells are treated withHGF. In 10% serum, B16 cells plated in the top well of aTranswell chamber containing a barrierwith 0.8-mmporesshow very little ability to migrate to the lower chamberside. Addition of HGF to the lower Transwell chambergreatly increases migration through the barrier over a 24hours period.XL765 blocks this effectwith an IC50 value of601nmol/L (Supplementary Fig. S4). The cytotoxicity IC50

value of XL765 in B16 cells is 7,300 nmol/L, 12-fold higherthan the IC50 value for inhibition of migration. Therefore,inhibition of melanoma cell migration by XL765 is unlike-ly to be due to cytotoxicity.

XL765 inhibits the PI3K and mTOR pathways anddisplays robust antitumor activity in tumor-bearingmice

Lysates derived fromMCF7 xenograft tumors intrader-mally implanted into athymic nude mice contain highlevels of constitutively phosphorylatedAKT, p70S6K, andS6 proteins. The ability of XL765 to inhibit endogenousphosphorylation of AKT, p70S6K, and S6 was examinedfollowing a single oral dose of 10, 30, 100, or 300 mg/kg.The tumors were harvested 4, 24, or 48 hours after doseand homogenized in lysis buffer. Tumor lysates fromeachanimal (n ¼ 4) were then pooled for each group andanalyzed for levels of total and phosphorylated AKT,p70S6K, and S6 by Western immunoblotting (Fig. 3A).

Oral administration of XL765 causes a dose-dependentdecrease of phosphorylation of AKT, p70S6K, and S6 inthe tumors, reaching a maximum of 84% inhibition of S6phosphorylation at 30 mg/kg at 4 hours. The dose–response relationships (not shown) derived from the 4hours time point predict 50% inhibition of AKT, p70S6K,and S6 phosphorylation to occur at doses of 19 mg/kg(pAKTT308 and pAKTS473), 51 mg/kg (p-p70S6K), and 18mg/kg (pS6). Inhibition of AKT, p70S6K, and S6 phos-

phorylation in MCF7 tumors following a 30 mg/kg doseof XL765 was maximal at 4 hours, reaching 61% to 84%;however, the level of inhibition decreased to 0% to 42% by24 hours, and minimal or no inhibition was evident by 48hours (see Fig. 3A). Following a 100mg/kgdose of XL765,inhibition was also maximal at 4 hours (52%–75%). How-ever, in contrast with the 30 mg/kg dose, inhibition at 24hours (48%–71%)was almost comparablewith that seen at4 hours. Partial inhibition of some phosphoepitopes per-sisted through 48 hours (Fig. 3A).

Similarly, administration of XL765 caused a dose-dependent decrease of phosphorylation of AKT, p70S6K,and S6 in PC-3 tumors in vivo, reaching amaximumof 93%inhibitionofAKTphosphorylationat 300mg/kgat4hoursafter dose (Fig. 3B). The dose–response relationships (notshown) derived from the 4 hours time point predict 50%inhibition of AKT, p70S6K, and S6 phosphorylation tooccur at doses of 15 mg/kg (pAKTT308), 13 mg/kg(pAKTS473), 59 mg/kg (p-p70S6K), and 48 mg/kg (pS6).Consistent with the MCF7 data, for the 100 mg/kg doseinhibition (42%–60%) persisted through 24 hours afterdose. In both studies, bloodwas collected at the same timetumor tissue was harvested and plasma concentrations ofXL765 were assessed (Supplementary Table S3). On thebasis of these data, the plasma concentrations associatedwith inhibition of phosphorylation of AKT, p70S6K, andS6 by 50% in these tumor models ranged from approxi-mately 3 to 9 mmol/L.Hence, XL765 exhibited comparablepharmacodynamic activity in PIK3CA-mutant MCF7 andPTEN-deficient PC-3 xenograft tumor models.

Multiple tumor models were used to explore the effi-cacy and potency of repeat-dose oral administration ofXL765 with regard to tumor growth inhibition in vivo. Inaddition to the previously described MCF7 and PC-3models, the antitumor efficacy of XL765 was evaluatedin xenograftmodels, includingOVCAR-3 (humanovarianxenograft tumormodel exhibiting PIK3CA amplification),U-87 MG (human glioblastoma xenograft tumor modelharboring a deletion at codon 54 in the gene encodingPTEN, resulting in a frameshift), A549 [human non–smalllung cancer cell (NSCLC) xenograft tumor model harbor-ing a homozygous-activating mutation in KRAS and ahomozygous loss-of-function mutation in LKB1], andCalu-6 (humanNSCLC xenograft tumormodel harboringan activating mutation in KRAS).

XL765 administration results in significant antitumorefficacy in vivo in all of these models (Fig. 4) at doses thatproved well tolerated as assessed by daily monitoring ofmouse weights (Supplementary Fig. S5; no or minimalimpact on body weights compared with vehicle control).The most efficacious schedules were 30 mg/kg twice aday and 100 mg/kg every 2 days, which suggests thatsustained pathway inhibition is required for maximaleffect on tumor growth. These schedules generallyresulted in stasis of tumor growth, except in the PC-3model and the Calu-6 KRAS-mutant NSCLC model, inwhich tumors continued to grow although at a reducedrate. Immunohistochemical analyses of MCF7, PC-3, and

Yu et al.





A549 tumors collected at the end of the dosing periodrevealed significant, dose-dependent decreases in stain-ing for Ki67, a marker of cell proliferation. Moreover,XL765 administration was associated with increasedtumor cell apoptosis in MCF7 and A549 tumors andmodestly decreased tumor vascularization in MCF7,PC-3, and A549 tumors (Table 3). Thus, inhibition of PI3Kand mTOR by XL765 results in antiproliferative, proa-poptotic and antivascular effects in xenograft tumors.Plasma concentrations at the end of these efficacy studieswere similar to those seen following single-dose admin-istration. For example, in the MCF7 efficacy study,average plasma concentrations for the 30 mg/kg doseadministered once daily were 8.8, 5.3 mmol/L, and belowthe limit of detection at the 1, 4, and 24 hours time points,respectively (n ¼ 3/time point).The antitumor activity observed in the subcutaneousU-

87 MG glioblastoma xenograft model prompted us to

examine the pharmacodynamic activity of XL765 in themouse brain as ameasure ofwhether the compound couldeffectively cross the blood–brain barrier. Lysates frombrains of nontumor-bearing mice show significant PI3Kpathway activity as judged by levels of pAKT and pS6.Four hours following a single oral dose of 30 or 100mg/kgXL765, pAKT and pS6 levels are substantially reduced,demonstrating that XL765 can cross the blood–brain bar-rier and inhibit the PI3K pathway (Fig. 4).

DiscussionPrevious experience with highly selective inhibitors of

signal transduction pathways has revealed the existenceof unanticipated regulatory mechanisms that can act tolimit the efficacy of pathway inhibition. An example is theupregulation of AKT phosphorylation that occurs as theresult of relief of a negative feedback loop followinginhibition of mTORC1 by the rapamycin class of mTOR

MCF-7

PC-3

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Dose (mg/kg) 10 30 100 10 30 100 10 30 100484848 482424Time (h) 4 4 4 4 24 24

p-p70S6KT389

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Figure 3. Administration of XL765inhibits PI3K pathway signaling inMCF7 and PC-3 tumors. A singledose of XL765 or vehicle wasadministered by oral gavage toMCF7 (A) or PC-3 (B). Tumorswereresected at the indicated timesafter dose and the effects of XL765on phosphorylation of AKT,p70S6K, and S6were assessed byWestern immunoblotting.






inhibitors. Similarly, selective B-RAF inhibitors can trig-ger a "paradoxical" activation of C-RAF in the context ofactivated RAS as a result of allosteric effects on B-RAF/C-RAF dimers. Inhibition at a single node in a pathway also

allows for the development of resistance via pathwayactivation downstream of the point of intervention. Com-biningmultiple inhibitors that affect the samepathway, ordeveloping a single compound that inhibits multiple

Figure 4. XL765 administration results in tumor growth inhibition of established xenograft tumors. A, MCF7; B, PC-3; C, OVCAR-3; D, U87-MG; E, A549; or F,Calu-6 tumor cells were implanted and when tumors reached approximately100 mg in size administration of vehicle or XL765 was initiated at the indicateddoses and regimens (day 0/1, day of grouping; dosing was initiated on day 1). Data points, mean � SE for each treatment group (n ¼ 9–10). D, theinset in the U-87 MG tumor growth inhibition graph shows the effect of XL765 at the indicated oral dose on pAKT and pS6 levels in normal mouse brain at4 hours after dose. PO BID, orally twice a day; PO QD, orally every day; PO Q2D, orally every 2 days.

Yu et al.





members of a pathway is, therefore, an attractiveapproach to limit or circumvent these issues. For example,emerging data suggest that combinations of B-RAF andMEK inhibitors are superior to either agent alone for thetreatment of B-RAF–mutant metastatic melanoma.The PI3K/mTORpathway is one of themost frequently

activated signaling pathways in human cancer, in part,due to the mutation, amplification, or deletion of keypathway regulatory components. Activation of the path-way promotes tumor cell proliferation, survival, andresistance to anticancer therapies. As a result, extensiveefforts are being devoted to identifying and developingsmall-molecule inhibitors that affect different nodes of thepathway. Activation of the pathway is subject to regula-tion at multiple points and by a wide variety of signals,including growth factors, cellular energy levels, nutrition-al status, and oxygenation. We, therefore, elected to opti-mize a compound that would inhibit two key nodes in thepathway, PI3K and mTOR, with the aim of maximizingpathway blockade in the context of multiple genetic back-grounds andunder a variety of environmental conditions.XL765 (SAR245409) inhibits both class I PI3Ks aswell as

mTOR. Although XL765 seems to be more potent againstPI3Ka than against PI3Kb when assessed by biochemicalassays using purified kinases, in cellular assays XL765shows comparable activity versus PI3K pathway signal-ing in MCF7 breast (PIK3CA-mutant) and PC-3 prostate

(PTEN-deleted) tumor cells. PI3Kb is considered to be themajor driver of dysregulated PI3K pathway activity asso-ciated with PTEN deficiency (31).

Moreover, XL765 showed comparably robust and per-sistent pharmacodynamic activity against these cell lineswhen theywere grownas xenograft tumors inmice. Thesedata demonstrate that XL765 exhibits functionally com-parable activity against PI3Ka and PI3Kb in cultured cellsand xenograft tumors.

In biochemical assays XL765 was generally less potentagainstmTOR than against class I PI3K isoforms. In tumorcells, however, XL765 inhibits mTOR-dependent phos-phorylation events and PI3K-independent, nutrient-stim-ulated mTOR activity with a potency comparable withthat demonstrated for PI3K-dependent signaling, sug-gesting potential for concerted PI3K/mTOR inhibition incellular and in vivo models.

Our survey of the effects on XL765 onmultiple phospho-epitopes in the PI3K signaling pathway in six tumor celllines with differing genetic backgrounds revealed consis-tent inhibition downstream of both PI3K and mTOR. Wesaw no evidence for feedback upregulation within thepathwayorwithrespect toERKphosphorylation.However,it is important to note that our data are not exhaustivewith respect to either genotype or phosphorylation sitessurveyed, and are limited to a single time point. Theprofile of XL765 is clearly differentiated from that of

Table 3. Immunohistochemical analysis of proliferation, vascularization, andapoptosis inMCF7,PC-3, andA549 xenograft tumors

Ki67 Analysis CD31 Analysis Apoptotic index (TUNEL)

Group% of Positivecellsa

% ofReductionb MVD

% ofReductionb

% of Positivecells

Foldincreaseb

MCF7Vehicle, 10 mL/kg PO QD 43 � 4 na 50 � 7 nd 1.0 � 0.5 naXL765, 10 mg/kg PO QD 37 � 5 15 (ns) 28 � 4 44 3.8 � 2.5 4 (ns)XL765, 30 mg/kg PO QD 27 � 6 39 33 � 12 34 (ns) 5.3 � 1.6 6XL765, 30 mg/kg PO BID 23 � 3 48 23 � 7 54 3.5 � 1.8 4XL765, 100 mg/kg PO Q2D 7 � 8 84 41c 18 36 � 8 37

PC-3Vehicle, 10 mL/kg PO QD 21 � 2 na 34 � 4 na nd ndXL765, 30 mg/kg PO QD 13 � 4 37 28 � 6 17 (ns) nd ndXL765, 30 mg/kg PO BID 10 � 2 51 27 � 4 19 nd ndXL765, 100 mg/kg PO Q2D 18 � 2 16 26 � 3 22 nd nd

A549Vehicle, 10 mL/kg PO QD 32 � 5 na 37 � 8 na 1.0 � 0.3 naXL765, 30 mg/kg PO QD 21 � 3 33 35 � 10 6 (ns) 3.8 � 0.7 4XL765, 30 mg/kg PO BID 16 � 3 50 29 � 6 22 3.3 � 0.5 3XL765, 100 mg/kg PO Q2D 20 � 2 39 31 � 8 18 (ns) 7.1 � 0.8 7XL765, 100 mg/kg PO BIW 22 � 4 33 34 � 6 8 (ns) 6.1 � 1.7 6

Abbreviations: MVD, mean vessel density; na, not applicable; nd, not determined; POB.I.D, orally twice a day; POQD, orally everyday;PO Q2D; orally every 2 days.aValues are mean � SD.bValues are relative to vehicle control (in all cases P < 0.05 except where indicated as ns, not significant).cOn the basis of one evaluable tumor (insufficient viable tissue to score in 8 of 9 tumors).






rapamycin, which in all cell lines tested is a potent inhib-itor of mTORC1-dependent p70S6K and S6 phosphory-lation, but in some cell lines augmented PI3K activity asassessed by AKT phosphorylation, consistent with pre-vious reports.

XL765 exhibits awide range of antiproliferative activityagainst tumor cells grown as monolayers. In MCF7 cells,these effects were associated with a G1 arrest but not withacute cytotoxicity or induction of apoptosis. When sensi-tivity toXL765 is examined in relation to genotype, there isa trend suggesting enhanced sensitivity of cells exhibitingPIK3CA-activating mutations, consistent with similarobservations previously reported for the PI3K inhibitorsGDC-0941 and CH5132799 (32, 33). Likewise, the relativeinsensitivity of RAS-mutant cell lines, regardless ofPIK3CA status, to inhibition of proliferation by XL765 isconsistent with preclinical observations with other PI3Kpathway–targeting agents (23). However, we note that anumber of K-RAS–mutant lines such as the A549 NSCLCline were quite sensitive to XL765. These cells have aconcomitant deletion of the LKB1 gene, which may serveto sensitize them to inhibition of mTOR. Likewise, PTEN-deleted cell lines had awide range of sensitivities toXL765with some being very sensitive and some being refractory.The basis for these differences is not understood, butpresumably reflects varying degrees of dependence onPI3K pathway signaling for proliferation as a result ofalterations in other pathways that affect growth.

Inmultiple xenograft tumormodels, oral administrationofXL765 resulted in substantial tumorgrowth inhibition atwell-tolerated doses. The most efficacious schedules were30mg/kg twice a day or 100mg/kg every 2 days, suggest-ing that efficacy is associated with more continuous inhi-bition of the pathway. These models encompass multiplegenetic lesions activating the PI3K pathway, specifically aPIK3CA E545K mutation (MCF7), PIK3CA amplification(OVCAR-3), PTEN deletion (PC-3 and U-87 MG), KRASmutation (A549 and Calu-6), and LKB1 mutation (A549).The fact that efficacy was observed in all these modelssuggests that XL765may have broad utility in tumorswithactivation of the PI3K pathway. On the basis of the immu-nohistochemical/immunofluorescence analyses conduc-ted on tumor xenografts following repeat dosing of XL765,antitumor efficacy was associated with a combination ofantiproliferative and proapoptotic effects, with a modestimpact on tumor angiogenesis. These proapoptotic effectsin vivo, which are not evident on cultured tumor cells,likely reflect targeting of the tumor microenviroment inaddition to tumor cells themselves, which is consistentwith the antiangiogenic effects evident.

In the majority of the xenograft models, complete ornear complete inhibition of tumor growth (but not regres-sion) was observed, with the exception of the PC-3 andCalu-6 models, which were relatively resistant. Overall,our data are not extensive enough todeterminewhether invitro sensitivity is generally predictive of efficacy in xeno-graftmodels. It is alsonot yet clearwhether thepresence ofPIK3CA mutations or PTEN deficiency will be predictive

of greater clinical responsiveness to PI3K pathway inhi-bitors in general, although an analysis based on combin-ing the results ofmultiple early-stage trials suggested thatPIK3CA H1047R mutations are associated with response(34). Intensive molecular profiling of tumors in the ongo-ing XL765 clinical studies is being performed to furtherexplore this question.

Because we observed significant antitumor efficacy inthe U-87 MG glioblastoma model, we assessed the phar-macodynamic activity of XL765 in mouse brain. At thesame doses associated with efficacy in the subcutaneousxenograft model, XL765 effectively inhibited PI3K path-way signaling in the brain, supporting its potential utilityfor the treatment of central nervous systemmalignancies.Consistent with this observation, XL765 has demonstrat-ed significant efficacy in an orthotopic glioblastoma xeno-graft model, both as a single agent and in combinationwith temozolomide (35). In addition, recent data from aclinical trial in which XL765 was administered to glio-blastoma patients before surgical removal of recurringlesions showed significant inhibition of PI3K pathwaysignaling in glioblastoma tumor tissue following XL765dosing (36). XL765 is currently in phase I and II clinicalstudies as a single agent or in combination with othertargeted or cytotoxic agents in patients with solid tumors,lymphoma, and leukemia (NCT01390818, NCT01410513,and NCT01403636).

Disclosure of Potential Conflicts of InterestJ. Young has ownership interest (including patents) in Exelixis, Inc. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K.-A. Won, K. Yamaguchi, F. Qian, W. Zhang,C.A. Buhr, P. Shen, F.M. Yakes, P. Lamb, P. FosterDevelopment of methodology: X. Du, J. Wu, K.-A. Won, K. Yamaguchi,P.P. Hsu, F. Qian, C.T. Jaeger, W. Zhang, C.A. Buhr, P. Shen, W. Abulafia,J. Young, F. Chu, M. Lee, S.T. LamAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Yu, A.D. Laird, X. Du, J. Wu, K.-A. Won,K. Yamaguchi, F. Qian, C.T. Jaeger, W. Abulafia, J. Chen, J. Young,A. Plonowski, F.M. Yakes, S.T. LamAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): P. Yu, A.D. Laird, X. Du, K.-A. Won,K. Yamaguchi, P.P. Hsu, F. Qian, C.T. Jaeger, W. Zhang, W. Abulafia,J. Young, A. Plonowski, M. Lee, F. Bentzien, S.T. Lam, S. Dale, P. LambWriting, review, and/or revision of the manuscript: P. Yu, A.D. Laird,K.-A. Won, W. Zhang, J. Young, F. Bentzien, S.T. Lam, D.J. Matthews,P. Lamb, P. FosterAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A.D. Laird, K.-A. Won, W. Zhang,W. Abulafia, J. Young, D.J. MatthewsStudy supervision: P. Yu, A.D. Laird, K.-A. Won, P.P. Hsu, J. Young,A. Plonowski, F.M. Yakes, D.J. Matthews, P. Lamb, P. Foster

AcknowledgmentsThe authors thank Coumaran Egile for critical reading of the article.

Requests relating to provision of XL765 (SAR245409) should be directed toCoumaran Egile at sanofi ([email protected]).

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 August 27, 2013; revised February 21, 2014; accepted February24, 2014; published OnlineFirst March 14, 2014.

Yu et al.





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2014;13:1078-1091. Published OnlineFirst March 14, 2014.Mol Cancer Ther Peiwen Yu, A. Douglas Laird, Xiangnan Du, et al. Affecting the PI3K Pathway(SAR245409) in Tumor Models with Diverse Genetic Alterations Characterization of the Activity of the PI3K/mTOR Inhibitor XL765

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Characterization of the Activity of the PI3K/mTOR ... · Small Molecule Therapeutics Characterization of the Activity of the PI3K/mTOR Inhibitor XL765 (SAR245409) in Tumor Models