therapeutics antitumor efficacy profile of pki-402, a dual...

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Published OnlineFirst April 6, 2010; DOI: 10.1158/1535-7163.MCT-09-0954

Research Article Molecular
Cancer
Therapeutics

Antitumor Efficacy Profile of PKI-402, a DualPhosphatidylinositol 3-Kinase/Mammalian Target ofRapamycin Inhibitor

Robert Mallon1, Irwin Hollander1, Larry Feldberg1, Judy Lucas1, Veronica Soloveva3, Aranapakam Venkatesan2,Christoph Dehnhardt2, Efren Delos Santos2, Zecheng Chen2, Osvaldo dos Santos2,Semiramis Ayral-Kaloustian2, and Jay Gibbons1

Abstract

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PKI-402 is a selective, reversible, ATP-competitive, equipotent inhibitor of class I phosphatidylinositol3-kinases (PI3K), including PI3K-α mutants, and mammalian target of rapamycin (mTOR; IC50 versusPI3K-α = 2 nmol/L). PKI-402 inhibited growth of human tumor cell lines derived from breast, brain (glioma),pancreas, and non–small cell lung cancer tissue and suppressed phosphorylation of PI3K and mTOR effectorproteins (e.g., Akt at T308) at concentrations that matched those that inhibited cell growth. In MDA-MB-361[breast: Her2+ and PIK3CA mutant (E545K)], 30 nmol/L PKI-402 induced cleaved poly(ADP-ribose) polymer-ase (PARP), a marker for apoptosis. In vivo, PKI-402 inhibited tumor growth in MDA-MB-361, glioma(U87MG), and lung (A549) xenograft models. In MDA-MB-361, PKI-402 at 100 mg/kg (daily for 5 days,one round) reduced initial tumor volume of 260 mm3 to 129 mm3 and prevented tumor regrowth for 70 days.In MDA-MB-361 tumors, PKI-402 (100 mg/kg, single dose) suppressed Akt phosphorylation (at T308) andinduced cleaved PARP. Suppression of phosphorylated Akt (p-Akt) was complete at 8 hours and still evidentat 24 hours. Cleaved PARP was evident at 8 and 24 hours. In normal tissue (heart and lung), PKI-402(100 mg/kg) had minimal effect on p-Akt, with no detectable cleaved PARP. Preferential accumulation ofPKI-402 in tumor tissue was observed. Complete, sustained suppression of Akt phosphorylation may causetumor regression in MDA-MB-361 and other xenograft models. We are testing whether dual PI3K/mTORinhibitors can durably suppress p-Akt, induce cleaved PARP, and cause tumor regression in a diverse setof human tumor xenograft models. Mol Cancer Ther; 9(4); 976–84. ©2010 AACR.

Introduction

Phosphatidylinositol 3-kinase (PI3K)-α plays a key rolein the biology of human cancer. PI3K-α is a lipid kinasethat is a central component in the PI3K/Akt/mammaliantarget of rapamycin (mTOR) signaling pathway. Thispathway regulates cell proliferation, growth, survival,and apoptosis (1, 2). The deregulated activation ofPI3K-α and its downstream effectors, including Akt andmTOR, has been linked to tumor initiation and mainte-nance. PI3K/Akt/mTOR pathway activation can becaused by loss of PTEN (the phosphatase that regulatesPI3K signaling), overexpression or activation of somereceptor tyrosine kinases [e.g., epidermal growth factor

ffiliations: 1Oncology, 2Discovery Medicinal Chemistry, andSciences, Wyeth Research, Pearl River, New York

plementary material for this article is available at Molecularrapeutics Online (http://mct.aacrjournals.org/).

ding Author: Robert Mallon, Wyeth Research, 401 NorthRoad, Pearl River, NY 10965. Phone: 973-747-5906; Fax:31. E-mail: [email protected]

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receptor (EGFR) and HER-2], interaction with activatedRas, overexpression of the PI3K-α gene (PIKC3A), ormutations in PIKC3A that cause elevated PI3K-α kinaseactivity (1–3). Aberrantly elevated PI3K/Akt/mTORpathway signaling has been implicated in poor prognosisand survival in patients with various lymphatic tumors,as well as breast, prostate, lung, glioblastoma, melanoma,colon, and ovarian cancers (1–4). Additionally, PI3K/Akt/mTOR pathway activation contributes to resistanceof cancer cells to both targeted anticancer therapies andconventional cytotoxic agents (5, 6). An effective inhibitorof the PI3K/Akt/mTOR pathway could both preventcancer cell proliferation and induce programmed celldeath (apoptosis; refs. 1, 2, 5). Therefore, the quest oftheWyeth PI3K inhibitor discovery project was to identifypotent small-molecule inhibitors of the PI3K/Akt/mTORsignaling pathway, which we hypothesized would exertantitumor activity in a broad array of preclinical tumormodels.PKI-402 is an example of small molecules we have

identified as potent inhibitors of the PI3K/mTOR signal-ing pathway. PKI-402 is a reversible, ATP-competitive,and equipotent inhibitor of class I PI3Ks, including theE545K and H1047R PI3K-α mutants, and mTOR (IC50

2010 American Association for Cancer Research.

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PKI-402, a Dual PI3K/mTOR Inhibitor


versus PI3K-α = 2 nmol/L). Selectivity of PKI-402was established in a screen against 236 diverse humankinases. PKI-402 caused in vitro growth inhibition ofhuman tumor cell lines derived from a diverse set ofhuman tumor tissues, including breast, brain (glioma),pancreas, and non–small cell lung cancer (NSCLC)tissues. In vitro, PKI-402 suppressed phosphorylation ofPI3K and mTOR effector proteins, particularly phosphor-ylated Akt (p-Akt) at T308, at concentrations that closelymatched those that inhibited tumor cell growth. In MDA-MB-361, a breast tumor line with mutant PI3K-α (E545K)and elevated levels of Her2 receptor, PKI-402 inducedcleaved poly(ADP-ribose) polymerase (PARP), a markerfor apoptosis. In vivo, PKI-402 displayed antitumor activ-ity (i.v. route) in breast [MDA-MB-361: Her2+ and PIK3-CA (E545K)], glioma (U87MG and PTEN), and NSCLC(A549; K-Ras and STK11) xenograft models.A key question about PI3K/mTOR signaling inhibitors

is: can they cause tumor regression in preclinical models?Data we present here show that PKI-402 caused regres-sion in the MDA-MB-361 xenograft model. PKI-402 effectwas most pronounced at 100 mg/kg (daily for 5 days,one round), which reduced initial tumor volume andprevented tumor regrowth for 70 days. In MDA-MB-361 tumor tissue, PKI-402 at 100 mg/kg (single dose)fully suppressed p-Akt at both the T308 and the S473sites at 8 hours and induced cleaved PARP. At 24 hours,p-Akt suppression was still evident, as was cleavedPARP. Biomarker analysis of heart and lung tissueshowed minimal effect on p-Akt and no induction ofcleaved PARP by PKI-402 at 100 mg/kg. Preferentialaccumulation of PKI-402 in tumor tissue was observed.Data presented here indicate that sustained suppressionof p-Akt correlates with tumor regression in the MDA-MB-361 xenograft model.

Materials and Methods

Enzyme assaysEnzyme assays were done in fluorescent polarization

(FP) format, adapted from the Echelon K-1100 PI3K FPassay kit protocol (7). Human class I PI3Ks and PI3K-αmutants (E545K and H1047R) were produced in Sf9or purchased from Upstate Biotech. GST-GRP1 (murine)was produced in Escherichia coli and isolated by GST-Sepharose. Assay buffers were react ion buffer[20 mmol/L HEPES (pH 7.1), 2 mmol/L MgCl2, 0.05%CHAPS, and 0.01% β-mercaptoethanol] and stop/detec-tion buffer [100 mmol/L HEPES (pH 7.5), 4 mmol/LEDTA, 0.05% CHAPS]. FP reaction was run for 30 minat room temperature in 20 μL of reaction buffer contain-ing 20 μmol/L phosphatidylinositol 4,5-bisphosphate(PIP2), 25 μmol/L ATP, and <4% DMSO (compoundsolvent). FP reaction was stopped with 20 μL of stop/detection buffer (10 nmol/L probe and 40 nmol/L GST-GRP), and after 2 h, data were collected using an Envi-sion plate reader (Perkin-Elmer).

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Selectivity of PKI-402 was evaluated in the Invitrogen236 human kinase panel at [ATP] = Km for each enzyme.

Cell culture, growth inhibition, andtranslocation assaysCell lines obtained from the American Type Culture

Collection were MDA-MB-361, MDA-MB-468, T47D,MCF7, BT474, HT29, HCT116, DLD1, U87MG, H157,NCI-H460, A549, NCI-H1975, NCI-H1650, NCI-H2170, KB, 786-0, A498, MIA-PaCa-2, and PC3. Muta-tional status (http://www.sanger.ac.uk/genetics/CGP/cosmic/) of various oncogenes is listed in Table 1.U2OS cells engineered to monitor FOXO1–green fluores-cent protein (GFP) cellular translocation were from Ther-mo Scientific. All cell lines were propagated at 37°C in5% CO2 incubators in supplier-recommended growthmedium.Cell growth inhibition was determined using the

CellTiter 96 AQueous proliferation assay from Promega.Manufacturer's protocol was used, with adjustmentsfor cell line growth characteristics. Data were collected

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Table 1. PKI-402 IC50 values in human tumorcell line growth inhibition assays

Cell line

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IC50

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Breast
MDA-MB-361 PIK3CA 0.006 0.001 MDA-MB-468 PTEN, RB, p53 0.009 0.001 T47D p53, PIK3CA 0.013 0.002 BT474 PIK3CA, p53 0.026 0.006 MCF7 PIK3CA 0.101 0.014
Colon
HCT116 K-Ras, PIK3CA 0.033 0.002 HT29 B-Raf, PIK3CA, p53 0.136 0.041 DLD1 K-Ras, PIK3CA 0.227 0.009
Lung (NSCLC)
NCI-H157 K-Ras, STK11, p53 0.036 0.004 NCI-H460 K-Ras, PIK3CA, STK11 0.089 0.003 A549 K-Ras, STK11 0.116 0.031 NCI-H2170 p53 0.127 0.027 NCI-H1975 EGFR, PIK3CA, p53 0.210 0.015 NCI-H1650 EGFR, p53 0.349 0.061
Pancreas
MIA-PaCa-2 K-Ras, p53 0.051 0.003
Brain (glioma)
U87MG PTEN 0.077 0.017
Prostate
PC3 PTEN, p53 0.016 0.002
Epidermoid
KB 0.027 0.003
Renal
A498 VHL 0.268 0.002 786-0 PTEN, VHL, p53 0.334 0.060
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after 72 h using a Wallac Victor2 V (Perkin-Elmer)1420 multilabel HTS counter. FOXO-GFP translocationin U2OS cells was quantified after 60-min PKI-402 expo-sure using a Cellomics ArrayScan VTI Reader.

Cell lysis and western blottingBriefly, 3 × 105 cells were seeded onto six-well microtiter

plates (Nunc) 24 h before being exposed to PKI-402 incomplete growth medium. Cells were exposed tocompound for 4 h (unless indicated otherwise in figurelegends). Subsequently, cell growth medium wasremoved and cells were washed twice with cold (4°C)PBS. Cell lysis buffer (0.2 mL) was added with mixing toinsure cell lysis. Cell lysis buffer was 20 mmol/LTris-HCl(pH 7.5), 150 mmol/L NaCl, 1 mmol/L Na2EDTA,1 mmol/L EGTA, 1% NP40, 2.5 mmol/L sodium pyro-phosphate, 1 mmol/L β-glycerophosphate, 1 mmol/LNa3VO4, and 1 μg/mL leupeptin. Cell lysates were spunat 14,000 rpm for 30 s. Supernatant (60 μL) was combinedwith 30 μL of 3× protein gel loading buffer [187.5 mmol/LTris-HCl (pH 6.8), 6% (w/v) SDS, 15% glycerol, 0.03%(w/v) bromphenol blue, and 125 mmol/L DTT].Samples were boiled (5 min) and subjected to SDS-PAGE, transferred to nitrocellulose, and probed withvarious antibodies. Antibodies from Cell Signaling Tech-nology were anti-Akt, anti–p-Akt at T308, anti–p-Aktat S473, anti-4EBP1, anti–phospho-4EBP1 (p-4EBP1) atT37/46, anti–p70 S6 kinase (p70S6K), anti–phospho-p70S6K (p-p70S6K) at T389, anti-PARP, anti–cleavedPARP at D214, anti–phospho-endothelial nitric oxidesynthase (ENOS) at S1177, anti–phospho-glycogensynthase kinase 3α/β (GSK3α/β) at S21/9, anti–phospho-PRAS40 at T246, and anti–phospho-p44/42mitogen-activated protein kinase (MAPK; extracellularsignal-regulated kinase 1/2; T202/Y204). Conjugate(horseradish peroxidase) secondary antibody enabledchemiluminescent detection. Inhibition of protein phos-phorylation was quantified from Western blots analyzedon the Bio-Rad Fluor-S MultiImager using Quantity OneAnalysis software.

Caspase activation assayThe Caspase-Glo 3/7(Promega) luminescent assay was

used to measure caspase-3/7 activity in MDA-MB-361and HCT116 cell lines. Cells were exposed to PKI-402 for4 or 24 h. Assays were done in 96-well format (∼4,000 cellsper well). Caspase-3/7 activity was quantitated using aWallac Victor2 V 1420 multilabel HTS counter.

Establishment of xenograft tumors, efficacy studies,and biomarker analysisEstablishment of tumors, group randomization, tumor,

and body weight recording during efficacy studieswere described elsewhere (8). PKI-402 or vehicle wasadministered by i.v. route. All in vivo studies usingnude mice were conducted under an approved Ins-titutional Animal Care and Use Committee protocol.Tumor weight was calculated by the formula tumor

Mol Cancer Ther; 9(4) April 2010


weight (mg) = (d2 × D/2), where d and D are the shortestand longest diameters of the tumor, respectively,measured in millimeters. Significant (statistically, Stu-dent's t test) reduction in the tumor growth of treatedgroups compared with controls (vehicle) was defined asP < 0.05.Pharmacodynamic (biomarker) measurements were

done on tumor-bearing female nude mice administeredPKI-402. Tumor or normal tissue samples were collectedfrom euthanized animals, homogenized, washed twicewith cold (4°C) PBS, and then treated with cell lysis buffer(described above). Cell lysateswere processed and probedwith the various antibodies as described above.

Results

Enzyme assaysPKI-402 (Fig. 1) is a pan class I PI3K inhibitor with a

2 nmol/L IC50 against PI3K-α. IC50 determinations forPKI-402 against class I PI3Ks (α, β, δ, and γ), the twomost common mutant forms of PI3K-α (E545K andH1047R; ref. 9), and the PI3K-related kinase family mem-ber mTOR are shown in Fig. 1.PKI-402 is an ATP-competitive triazolopyrimidine scaf-

fold compound (Supplementary Data). Mutant forms ofPI3K-α reported to have elevated lipid kinase activity(10) were inhibited by PKI-402 at concentrations equiva-lent to the IC50 for wild-type PI3K-α. PI3K-β, PI3K-δ, andPI3K-γ isoforms were inhibited at concentrations only 3-to 8-fold higher than that of PI3K-α. mTOR kinase inhi-bition (IC50, 3 nmol/L) indicated that PKI-402 was anequipotent class I PI3K/mTOR inhibitor.Characterization of PKI-402 against a panel of 236 hu-

man protein kinases (Invitrogen) confirmed a highly se-lective profile (Supplementary Data). In this kinase panel,only C-Raf and B-Raf (wild-type and V600E mutant)were inhibited by PKI-402. Subsequent IC50 values of7 μmol/L were determined for all Raf enzymes. TheIC50s for PKI-402 against all other kinases in the Invitro-gen panel were >10 μmol/L.

Cell growth inhibition assayTable 1 lists IC50 data for human tumor cell growth

inhibition by PKI-402. Cell lines from an array of tumortissues, including breast, colon, lung, epidermoid, renal,pancreas, brain, and prostate, were tested. PKI-402 was apotent cell growth inhibitor in most human tumor celllines, with IC50 values of <250 nmol/L (17 of 20). In allcases where examined, PKI-402 was found to suppressphosphorylation of downstream effectors of PI3K signal-ing (e.g., Akt) at concentrations that closely matchedgrowth inhibition IC50.Higher IC50 values against A498, 786-0, and NCI-

H1650 (IC50 range, 268–349 nmol/L) were observed,but when we evaluated phosphorylation of the PI3K ef-fector Akt (T308) in A498 and 785-0, we found that it oc-curred at concentrations that closely matched growthinhibition IC50s (Supplementary Data).

Molecular Cancer Therapeutics





PKI-402 in vitro profile in biomarker, caspaseactivation, and FOXO1-GFP translocation assaysPKI-402 effect on phosphorylation of PI3K and mTOR

effector proteins and activation of caspase-3/7 in MDA-MB-361 [Her2+ and PIK3CA (E545K)]. The effect of PKI-402 on a representative group of PI3K/mTOR effectorproteins in MDA-MB-361 is shown in Fig. 2A. This wasdone to link PKI-402 enzyme inhibition to cellular anti-proliferative effects. PKI-402 lipid kinase activity in cellswas not directly measured. Class I PI3Ks convert PIP2 toPIP3 (phosphatidylinositol 3,4,5-trisphosphate) at the in-ner cell membrane (1, 2). PIP3 is bound by the pleckstrinhomology domains of both PDK1 and Akt serine/threo-nine kinases, resulting in their close proximity at the in-ner cell membrane where PDK1 phosphorylates (at T308)and activates Akt. Suppression of cellular PIP3 by PKI-402 was indirectly shown by potent (IC50, <10 nmol/L)suppression of Akt phosphorylation at T308 (Fig. 2A).Full activation of Akt kinase occurs when the mTOR-con-taining TORC2 protein complex phosphorylates Akt atS473. Figure 2A shows that potent (IC50, <30 nmol/L)suppression of Akt phosphorylation at S473 was alsocaused by PKI-402. PKI-402 did not affect the overallAkt content in MDA-MB-361 cells at any concentrationtested, indicating that p-Akt suppression was not an ar-tifact of compound cytotoxicity.TORC1 as well as TORC2 mTOR complexes were in-

hibited by PKI-402. Examples of PKI-402 effect onTORC1 were suppression of 4EBP1 and p70S6 kinasephosphorylation. Both p70S6K and 4EBP1 phosphoryla-tion was inhibited at IC50s of <10 nmol/L (Fig. 2A).PKI-402 suppression of Akt phosphorylation caused

consequent effects on Akt effectors such as PRAS40,ENOS, and GSK3. PRAS40 regulates mTOR activity,ENOS generates nitrous oxide in blood vessels and is in-volved in regulation of vascular function and angiogen-esis, and GSK3 is a serine/threonine protein kinase that



regulates cell cycle progression and glucose metabolism(2, 11, 12). Akt phosphorylation of PRAS40 at T246 wassuppressed at an IC50 of <30 nmol/L (Fig. 2A). Akt phos-phorylation of ENOS at S1177 and GSK3α/GSK3β at S9/S21 was suppressed by PKI-402 at IC50s of <10 nmol/L(Fig. 2A).An effect of PKI-402 particularly evident in MDA-MB-

361 cells was induction of cleaved PARP, an indicator ofcells undergoing apoptosis (13, 14). Complete PKI-402suppression of Akt phosphorylation in MDA-MB-361correlated with detectable cleaved PARP at 0.1 μmol/LPKI-402 (Fig. 2A). Cleaved PARP was detected inMDA-MB-361 within 4 hours after exposure to PKI-402.We also measured PKI-402 activation of caspase-3

(Caspase-3/7 assay, Promega) in MDA-MB-361 becausecaspase-3 is a critical mediator of apoptosis, being eitherpartially or totally responsible for the proteolytic cleavageof many key proteins including the nuclear enzymePARP (14). Figure 2B shows that PKI-402 caused a dose-dependent increase in caspase-3/7 activity at 4 hours.MDA-MB-361 cells overexpress the Her2 receptor; there-fore, we tested the ability of HKI-272 (irreversible Her2kinase inhibitor; ref. 15) or PD0325901 [MAP/extracellularsignal-regulated kinase kinase (MEK) inhibitor; ref. 16] toeither induce caspase-3/7 activity alone or enhance PKI-402–induced caspase activity. Neither PD0325901 norHKI-272 caused caspase-3/7 activation at concentrationstested (Fig. 2B). When PKI-402 was combined with0.1 μmol/L (shown) or 1.0 μmol/L (data not shown) of ei-ther PD0325901 or HKI-272, caspase-3/7 activation inMDA-MB-361 cells was unaffected. These data indicatethe significant addiction of MDA-MB-361 to PI3K/mTORsignaling tomaintain cell viability. Less than 10% ofMDA-MB-361 cells exposed to PKI-402 at 0.3 μmol/L (or higher)for 24 hours remained viable (Supplementary Data).PKI-402 effect on FOXO1(FKHR)-GFP translocation

in U2OS. Forkhead proteins (e.g., FOXO1) comprise a

Figure 1. Structure of PKI-402 and IC50 (μmol/L)inhibition data for class I PI3Ks, the mostfrequently occurring mutant forms (E545K andH1047R) of PI3K-α, and mTOR.

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highly conserved family of transcription factors that con-trol genes encoding proteins regulating insulin produc-tion, apoptosis, and cell cycle (17). FOXO1 activity isregulated by Akt-mediated phosphorylation. Akt-phos-phorylated FOXO1 is sequestered in the cytosol by 14-3-3 protein, and unphosphorylated FOXO1 locates tothe cell nucleus. Figure 2B shows that PKI-402, whichsuppressed Akt phosphorylation (T308 and S473;Fig. 2A), caused translocation of FOXO1(FKHR)-GFP tocell nuclei in U2OS cells engineered (Thermo Scientific)to monitor compound-mediated FOXO1-GFP nucleartranslocation. The IC50 value for PKI-402 effect onFOXO1-GFP nuclear translocation was 21 nmol/L(Fig. 2C). Figure 2D shows FOXO1-GFP (green) cyto-plasmic distribution in untreated U2OS cells (left), withnuclear translocation evident (at right) after 1-hour expo-sure to PKI-402.



PKI-402 induction of cleaved PARP and activation ofcaspase-3/7 in HCT116 cells. To determine whether thePKI-402 effect on cell survival observed in MDA-MB-361 breast tumor cells was unique, or also evident in oth-er tumor cells, we evaluated the sensitivity of the colontumor line HCT116 (K-Ras and PIK3CA) to this com-pound. In vitro growth inhibition IC50 of HCT116 byPKI-402 was 0.033 μmol/L. Suppression of p-Akt-T308was observed at ≥0.1 μmol/L after 4-hour exposure toPKI-402. However, no cleaved PARP was evident after4-hour exposure to PKI-402 up to 3 μmol/L. Longer ex-posure (24 hours) of HCT116 to PKI-402 again showedsuppression of p-Akt-T308 (IC50, <0.1 μmol/L; Fig. 3A,top), with only a trace cleaved PARP band at 3.0 μmol/L.Because HCT116 cells have both PIK3CA and K-Ras

mutations, we tested whether a MEK inhibitor,PD0325901, could influence PKI-402 effect on cleaved

Figure 2. In vitro profile of PKI-402. MDA-MB-361 [Her2+/PIK3CA (E545K)] cells were exposed to PKI-402 (0.003–0.3 μmol/L) for 4 h. A, PKI-402suppression of p-Akt (both T308 and S473) and suppression of phosphorylation of Akt effectors [GSK3 (at S9/21), PRAS40 (at T246), and ENOS (at S1177)]and mTOR effectors [p70S6K (at T389) and p-4EBP1 (at T37/46)]. PKI-402 induction of cleaved PARP (cPARP) at 0.1 and 0.3 μmol/L. B, caspase-3/7activity in MDA-MB-361 after a 4-h exposure to PKI-402, HKI-272 (Her2 inhibitor), or PD0325901 (MEK inhibitor) at 0.03 to 3.0 μmol/L. (◊), maximalcaspase-3/7 activity was caused by 1.0 to 3.0 μmol/L PKI-402. Neither HKI-272 (□) nor PD0325901 (Δ) induced caspase-3/7 activity. C and D, PKI-402combined with 0.1 μmol/L of either HKI-272 (×) or PD0325901 (+) did not increase caspase-3/7 activity. U2OS (FOXO1-GFP) cells were exposed toPKI-402 (0.003–10.0 μmol/L) for 1 h. C, nuclear to cytoplasmic ratio of FOXO1-GFP in U2OS cells after 60-min incubation with PKI-402 (IC50, 21 nmol/L).D, translocation of FOXO1-GFP (green) in U2OS from the cytoplasm (left) to the nucleus (right) after 60-min exposure to PKI-402. Nuclei (blue) werestained with Hoechst 33342.






PARP induction. PD0325901 IC50 for growth inhibition ofHCT116 was 0.230 μmol/L, and IC50 for phosphorylatedMAPK (p-MAPK) (T202/Y204) suppression was ∼0.050μmol/L (24 hours). Cleaved PARP was not detected inHCT116 after PD0325901 exposure at 24 hours (up to3.0 μmol/L; data not shown). When HCT116 cells wereexposed to 0.1 μmol/L PD0325901 and PKI-402 (from0.03 to 3.0 μmol/L) for 24 hours, a substantial increasein cleaved PARP was observed. Cleaved PARP signalwas detectable at 0.1 μmol/L, with a dose-dependent in-crease up to 3.0 μmol/L (Fig. 3A, bottom).The effect of PKI-402 alone and in combination with

PD0325901 on caspase-3/7 activity was measured at24 hours (Fig. 3B). PKI-402, or PD0325901, alone weaklyinduced caspase-3/7 activity at the highest compoundconcentration (3.0 μmol/L). In contrast, PD0325901(0.1 μmol/L) combined with PKI-402 increased caspase-



3/7 activity at PKI-402 concentrations from 0.03 to3.0 μmol/L (5-fold increase at 3.0 μmol/L). This effectwill be investigated in future in vivo efficacy studies withHCT116.

In vivo efficacy and biomarker profile of PKI-402 inMDA-MB-361 (breast) tumor xenograftsPKI-402 at 25, 50, and 100 mg/kg caused regression of

MDA-MB-361 tumors (Fig. 4A). PKI-402 effect was mostpronounced at 100 mg/kg (daily for 5 days, one round),which reduced an initial tumor volume of 260 mm3 to129 mm3 and prevented tumor regrowth for 70 days.PKI-402 given at 100 mg/kg daily for 5 days was welltolerated; however, continuous exposure at this levelfor longer than 7 days did cause weight loss. Tumor re-growth occurred between days 16 and 20 when PKI-402was administered at 25 and 50 mg/kg (daily for 5 days,two rounds; both regimens well tolerated). However,when PKI-402 was readministered at 100mg/kg, large tu-mors were significantly (P < 0.02) reduced in volume. Inthe 25 mg/kg treatment group, PKI-402 readministered at100 mg/kg (daily for 5 days, one round) on day 37 caused∼650 mm3 tumors to shrink to ∼200 mm3, a ∼69% reduc-tion in tumor volume. In the 50 mg/kg treatment group,PKI-402 readministered on day 45 (100 mg/kg, daily for3 days, two rounds) caused ∼600 mm3 tumors to ulti-mately shrink to ∼200 mm3, a ∼58% reduction in tumorvolume. In Fig. 4B, photographic evidence of PKI-402 ef-fect on MDA-MB-361 tumors treated at 100 mg/kg (dailyfor 5 days, one round) is shown.Biomarker evaluation was done on MDA-MB-361

tumor tissue from mice treated with 25, 50, and100 mg/kg (single dose) of PKI-402. Figure 4C showsthe complete suppression of p-Akt (at T308 and S473)and p-p70S6K at 8 hours after administration of PKI-402at 100 mg/kg. Distinct PKI-402 effect on this set ofPI3K/mTOR effector proteins was also evident at the 50and 25 mg/kg dosing levels. Cleaved PARP was evidentwhen PKI-402 was given at 50 and 100 mg/kg. Table 2lists serum and tumor tissue concentrations after 50 or100 mg/kg single-dose administration of PKI-402. At50 mg/kg PKI-402, preferential accumulation in tumortissue relative to serum was evident at 4 hours and per-sisted up to 8 hours. Tumor/serum ratio (6.2) peaked at6 hours. Tumor/serum ratio for PKI-402 at 100 mg/kgwas 8.9 at 24 hours.The effect of PKI-402 on PI3K/mTOR effector protein

phosphorylation and cleaved PARP in both tumor andnormal (e.g., heart and lung) tissue was assessed. InMDA-MB-361 tumor tissue, PKI-402 (100 mg/kg, singledose) effect on p-Akt (both T308 and S473), p-p70S6K,and p-pS6 was reduced (compared with 8 hours) but stillevident at 24 hours (Fig. 4D, left). Cleaved PARP signal,however, was equal if not greater than that observed at8 hours (Fig. 4C). In both heart (Fig. 4D, right) and lungtissue (data not shown) from nude mice bearing MDA-MB-361 tumors, PKI-402 (single dose, 100 mg/kg)

Figure 3. PKI-402 suppression of p-Akt-T308, induction of cleavedPARP, and activation of caspase-3/7 alone, and with 0.1 μmol/LPD0325901 (MEK) in HCT116 (K-Ras and PIK3CA) at 24 h. A, top,cleaved PARP, p-Akt-T308, p-MAPK (T202/Y204), and actin afterPKI-402 (0.03–3.0 μmol/L) alone for 24 h [IC50s, <0.03 μmol/L(p-Akt-T308) and ≥3.0 μmol/L (cleaved PARP)]. No PKI-402 effect wasevident on p-MAPK (T202/Y204) or actin. Bottom, effect of combinedPKI-402 and 0.1 μmol/L PD0325901. The p-Akt-T308 IC50 was <0.03μmol/L, cleaved PARP was now evident at 0.1 μmol/L, and p-MAPK(T202/Y204) was fully suppressed. B, induction of caspase-3/7 activity byPKI-402 (□), PD0325901 (Δ), and combined (◊) PKI-402 and PD0325901(at 0.1 μmol/L) in HCT116 exposed to compounds for 24 h.




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reduced p-Akt (T308 and S473) at 24 hours, but there wasno detectable cleaved PARP.

In vivo efficacy of PKI-402 in A549 (NSCLC) andU87MG (glioma) tumor xenograftsPKI-402 was tested for antitumor efficacy in the A549

(NSCLC) and U87MG (glioma) xenograft models. A549cells contain mutant K-Ras and STK11. Because these



mutations influence both PI3K and mTOR activity, wetested PKI-402 in vivo growth-inhibitory effects on A549tumors. Figure 5A shows that PKI-402 significantly (P <0.02) inhibited the growth of A549 tumors in nude miceat 25 and 50 mg/kg (daily for 5 days, for three rounds).PKI-402 inhibition of U87MG tumor growth showed

efficacy in a PTEN-negative glioblastoma multiforme tu-mor line. PKI-402 at 100 mg/kg (daily for 5 days, one

Figure 4. In vivo efficacy against MDA-MB-361 xenografts and in vivo biomarkers in tumor and heart tissue. A, PKI-402 given at 100 mg/kg (Δ) daily for 5 d forone round or at 25 (▴) or 50 (▪) mg/kg daily for 5 d for two rounds. The 100 mg/kg treatment caused tumor regression with no regrowth until day ∼70.Initial tumor regression in both 50 and 25 mg/kg groups was followed by tumor regrowth at days 16 to 20. Readministration of PKI-402 (↓, at 100 mg/kg, dailyfor 3 d) either once (25 mg/kg group, on day 37) or twice (50 mg/kg group, on day 45 and then again on day 53) caused shrinkage (>50%) of large tumors.Points, mean tumor volume from 10 mice; bars, SEM. B, a control tumor and a tumor from the 100 mg/kg treatment group at day 14. C, cleaved PARP,p-Akt (T308 and S473), p-p70S6K (T389), p-S6 (S235/236), and actin status in tumor xenograft tissue. PKI-402 was given in single-dose format at 100, 50, and25 mg/kg. Tumor tissue was harvested at 8 h after compound administration. Suppression of protein phosphorylation and cleaved PARP induction wasgreatest at the 100 mg/kg dose. D, comparison PKI-402 effect on MDA-MB-361 tumor and heart tissues in MDA-MB-361 tumor-bearing nude mice. PKI-402was given at 100 mg/kg (single dose) and tissue was harvested at 24 h. Note the lack of cleaved PARP in the heart tissue.

Table 2. Concentration (ng/mL) of PKI-402 in serum and tumor following a single dose

Pharmacokinetic summary
ng/mL
Compound
Route Dose Formulation
2010 Am

1 h

erican As

4 h

Molec

sociation f

6 h

ular Can

or Cancer

8 h

cer Thera

Research

24 h

PKI-402
i.v. 50 mg/kg Saline/lactic acid Serum 6,228 1,215 527 549 Tumor 8,319 6,576 3,265 2,219
PKI-402
i.v. 100 mg/kg Saline/lactic acid Serum 814 132 Tumor 2,813 1,184
NOTE: PKI-402 accumulation in tumor tissue was observed at 4 to 8 h (50 mg/kg) and at 8 to 24 h (100 mg/kg).

peutics

.




round) caused significant (P < 0.01) reduction in tumorgrowth relative to untreated controls (Fig. 5B). Rapidgrowth of U87MG tumors in this model was evidentby the ∼5-fold increase in control tumor size overthe 7-day study period. PKI-402 clearly attenuatedthis growth, permitting only a ∼2.3-fold increase intumor size.

Discussion

We identified PKI-402 in enzyme assays that measuredactivity of class I PI3Ks and mTOR. PKI-402 was a potentinhibitor of these enzymes, including the most fre-quently occurring mutant forms of PI3K-α (8). Analysisof PKI-402 against a panel of 236 human kinases showeda highly selective profile. In vitro, PKI-402 inhibition ofcell growth and suppression of Akt phosphorylationcorrelated with induction of apoptosis in MDA-MB-361(breast) human tumor cells. The proapoptotic effects ofPKI-402 alone or in combination (i.e., PD0325901) onother cell lines (HCT116; Fig. 3) suggest that PKI-402could have a broad range of activity and possibly morethan a cytostatic effect on xenograft tumor models.A key question about PI3K/mTOR signaling inhibitors

is: can they cause tumor regression in preclinical models?Based on current data disclosed about other PI3K/mTORinhibitors (e.g., BEZ235, PI-103, and SF1126; refs. 18, 19),PKI-402 shows a distinctive single-agent profile in that itcan potently induce cell death in vitro and cause tumorregression in vivo (MDA-MB-361 model).Indeed, at 25, 50, and 100 mg/kg, PKI-402 caused re-

gression of MDA-MB-361 tumors (Fig. 4A). PKI-402 effectwas most pronounced at 100 mg/kg (daily for 5 days, oneround), which reduced an initial tumor volume (260 mm3

to 129 mm3) and prevented tumor regrowth for 70 days.Tumor regrowth occurred between days 16 and 20 whenPKI-402 was administered at 25 and 50 mg/kg (daily for5 days, two rounds). However, these enlarged tumorsremained sensitive to PKI-402. For example, when PKI-402 was readministered at 100 mg/kg (daily for 3 days,one round) at day 37 (25 mg/kg group), large tumors(∼600 mm3) were reduced in volume by 69%.Biomarker analysis done with MDA-MB-361 tumor tis-

sue from mice given PKI-402 at 100 mg/kg (single dose)showed sustained suppression of Akt phosphorylation atboth the T308 and the S473 sites and induction of cleavedPARP. Suppression of p-Akt was complete at 8 hours an-d still evident at 24 hours, whereas cleaved PARP wasevident at both 8 and 24 hours. Biomarker analysis ofheart and lung tissue from MDA-MB-361 tumor-bearingmice given PKI-402 at 100 mg/kg showed minimal effecton p-Akt and no induction of cleaved PARP (Fig. 4D).Preferential accumulation of PKI-402 in tumor tissuewas observed (Table 2). It may be that complete andsustained suppression of p-Akt is needed to causetumor regression in the MDA-MB-361 and other tumorxenograft models. We are doing detailed pharma-cokinetic, pharmacodynamic, and antitumor efficacy



studies to confirm this hypothesis. If this link is estab-lished, it could become a key criterion for advancingcompounds to clinical development.Finally, we established PKI-402 antitumor efficacy in

additional in vivo models. These were A549 (NSCLC;K-Ras/STK11) and U87MG (glioma, PTEN). Clinical out-come for NSCLC is especially bleak (20). PKI-402 efficacyagainst A549 tumors (Fig. 5) supports the concept thatinhibition of the K-Ras effector PI3K is an effective anti-tumor strategy (1, 5). In cells lacking functional STK11,

Figure 5. PKI-402 in vivo efficacy against A549 (NSCLC) and U87MG(glioma) xenografts. A, PKI-402 antitumor activity against A549xenografts at 10 mg/kg ( ), 25 mg/kg ( ), and 50 (▪) mg/kg (21-d study).B, PKI-402 antitumor activity against U87MG xenografts at 100 mg/kg(▴) in a 7-d study. Points, mean tumor volume from 10 mice; bars, SEM.




Mallon et al.

984


such as A549, defective AMPK regulation of mTOR, andin turn deregulation of mTOR-controlled cell metabo-lism, suggests possible sensitivity to PKI-402 mTOR-inhibitory effects. PKI-402 significantly inhibited thegrowth of A549 tumors in nude mice at both 25 and50 mg/kg (daily for 5 days, three rounds).As with NSCLC, patients with PTEN-negative glio-

blastoma multiforme tumors generally have a poor prog-nosis (21). In clinical settings, such tumors are resistant toEGFR inhibitors, radiotherapy, and most alkylatingagents (22). PKI-402 (100 mg/kg, daily for 5 days) inhib-ited U87MG tumor growth, with significant reduction intumor growth relative to untreated controls. Despite thehigh dosing level needed for efficacy, these data suggestthat compounds such as PKI-402 may be useful either asa single agent or in combination with cytostatic or cyto-



toxic (e.g., temozolomide; ref. 23) drugs in treatment ofglioblastoma multiforme.

Disclosure of Potential Conflicts of Interest

All authors are employees of Wyeth Research. No other potential con-flicts of interest were disclosed.

Acknowledgments

We thank Ker Yu and Lourdes Toral-Barza for evaluating PKI-402 intheir mTOR kinase assay.

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 10/13/2009; revised 12/30/2009; accepted 01/14/2010;published OnlineFirst 04/06/2010.

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2010;9:976-984. Published OnlineFirst April 6, 2010.Mol Cancer Ther Robert Mallon, Irwin Hollander, Larry Feldberg, et al. Rapamycin InhibitorPhosphatidylinositol 3-Kinase/Mammalian Target of Antitumor Efficacy Profile of PKI-402, a Dual

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