AT13148 Is a Novel, Oral Multi-AGC Kinase Inhibitor with ... · AT13148 Is a Novel, Oral Multi-AGC...

Cancer Therapy: Preclinical

AT13148 Is a Novel, Oral Multi-AGC Kinase Inhibitor withPotent Pharmacodynamic and Antitumor Activity

Timothy A. Yap1, Mike I. Walton1, Kyla M. Grimshaw2, Robert H. te Poele1, Paul D. Eve1, Melanie R. Valenti1,Alexis K. de Haven Brandon1, Vanessa Martins1, Anna Zetterlund1, Simon P. Heaton1, Kathrin Heinzmann1,Paul S. Jones3, Ruth E. Feltell2, Matthias Reule2, Steven J. Woodhead2, Thomas G. Davies2, John F. Lyons2,Florence I. Raynaud1, Suzanne A. Eccles1, Paul Workman1, Neil T. Thompson2, and Michelle D. Garrett1

AbstractPurpose:Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including

AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous

inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance

compared with a single pathway component.

Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel

clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression

microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148.

Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate

phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells

with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-

mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine

tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine

473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant

reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis

genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream

regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed.

Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent

pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT

inhibitors. AT13148will nowbe assessed in a first-in-human phase I trial.Clin Cancer Res; 18(14); 3912–23.

�2012 AACR.

IntroductionThe class I phosphoinositide 3-kinases (PI3K) are key

mediators of intracellular signaling between themembrane-bound receptor tyrosine kinases (RTKs) and downstreameffector molecules, which control many vital cellular func-tions, including survival, growth, proliferation, andmotility(1, 2). Downstream of these PI3Ks lies a network of serine/

threonine kinases, including several members of the AGCkinase family, such as AKT, also known as protein kinase B(PKB), phosphoinositide-dependent kinase 1 (PDK1),p70S6 kinase (p70S6K), p90 ribosomal S6 kinase (RSK),serum- and glucocorticoid-induced kinase (SGK), and Rhokinase (ROCK; refs. 3, 4). ThePI3K-AKTaxis of this signalingnetwork is hyperactivated in multiple cancers through dif-ferent mechanisms, including the deregulation of upstreamRTKs, for example, insulin-like growth factor-1 receptor(IGF-1R), and genetic alterations of PIK3CA, PTEN, or AKTgenes,AKT1,2, and3 (1, 2). Thus, pharmacologic inhibitionof this pathway is an area of great therapeutic interest (5).

Several drugs targeting the PI3K-AKT pathway are cur-rently in clinical development, including inhibitors of PI3K,AKT, andmTORC1/2 (5, 6). However, inhibiting PI3K-AKTsignaling at a single node has shown relatively limitedclinical efficacy to date. There are several possible explana-tions for this. First, AKT inhibition has been shown torelieve feedback suppression of RTK expression and activity,which may attenuate antitumor activity (7). Second, PI3Kderegulation may promote cancer through both AKT-

Authors' Affiliations: 1Cancer Research UK Cancer Therapeutics Unit,Division of Cancer Therapeutics, The Institute of Cancer Research, Sutton;2Astex Pharmaceuticals, Cambridge; and 3Cancer Research UK DrugDevelopment Office, Research Operations and Funding, London, UnitedKingdom

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author:Michelle D. Garrett, Cancer Research UK CancerTherapeutics Unit, Division of Cancer Therapeutics, The Institute of CancerResearch, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey SM25NG, United Kingdom. Phone: 44-20-8722-4352; Fax: 44-20-8722-4126;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-3313

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(14) July 15, 20123912

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Published OnlineFirst July 15, 2012; DOI: 10.1158/1078-0432.CCR-11-3313

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dependent and AKT-independent mechanisms, the latterinvolving the AGC kinases PDK1 and SGK (8). Third,inhibition of a single node such as PI3K or AKT may allowclinical resistance as reported for the selective BRAF inhib-itor vemurafenib (PLX4032, Roche; ref. 9). Furthermore,concurrent blockade of multiple components of the PI3Knetworkmay have greater therapeutic value than inhibitionof any single target (10). Thus, the simultaneous inhibitionof several essential nodes of the PI3K signaling networkmayprovide greater overall suppression of key pathways, withthe potential for improved therapeutic efficacy across abroader range of cancer types with less opportunity forresistance to develop.A possible strategy for such a targeted combinatorial

blockade is the development of a drug that inhibits AKTtogether with other key AGC kinases that form part of thePI3K signaling network. In this paper, we present the firstdetailed pharmacologic characterization of the novel, oralclinical drug candidate AT13148, a potent multi-AGCkinase inhibitor. AT13148 was identified using high-throughput X-ray crystallography and fragment-based leaddiscovery techniques (11–14). We show that this orallybioavailable drug candidate achieves active tumor expo-sures, induces robust pharmacodynamic biomarker mod-ulation and apoptosis in cancer cells with clinically relevantgenetic defects both in vitro and in vivo, and exhibits anti-tumor efficacy inmultiple human tumor xenograft models.Moreover, we show that AT13148-induced hyperphosphor-ylation of Ser473 AKT is not a therapeutically relevantreactivation step for this compound, in contrast to previoussuggestions for other ATP-competitive inhibitors of AKT(15). Finally, we describe gene expression microarray stud-

ies that characterize the underlying molecular mechanismsof action of AT13148, including compensatory feedbackloops that reveal differences comparedwith amore selectiveAKT inhibitor. On the basis of these studies, AT13148 isnow undergoing preclinical development before entry intophase I clinical trials.

Materials and MethodsCell culture and reagents

All human cancer cell lines were purchased from theAmerican Type Culture Collection and grown in theirrecommended culture medium, containing 10% FBS at37�C in an atmosphere of 5% CO2 and passaged for lessthan 6 months. AT13148 (16), CCT128930 (17), andLY294002 (Calbiochem, Merck Biosciences) were made upas 10 mmol/L stocks in dimethyl sulfoxide (DMSO).

In vitro kinase assaysAT13148was assayed against 40 kinases (NationalCentre

for Kinase Profiling, Dundee, UK) and the percentage inhi-bition at 10mmol/L of AT13148wasdetermined. IndividualIC50 values were measured for selected kinases using ATPconcentrations equivalent to the Km for each enzyme(Invitrogen).

Protein immunoblotting and immunoassayCells were harvested, lysates prepared, protein estima-

tions conducted, andWesternblots undertaken as described(18), using the following antibodies: pSer473 AKT, AKT,pSer9 GSK3b, GSK3b, pSer235/236 S6 ribosomal protein(S6RP), S6RP, pSer330 NDRG, NDRG, pSer157 VASP,VASP, pSer19 MLC2, MLC2, pThr24 FOX01/pThr32FOX03a, FOX01, PRAS40, cleaved PARP, IRS2, pThr1135Rictor, Rictor, cyclin E2, c-MYC (Cell Signaling Technolo-gy), PIK3IP1 (Abcam), pThr246 PRAS40 (Upstate), cleavedcaspase-3 (Epitomics), cyclin D1, p27, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Neomarkers).

Western blot analysis of MES-SA cells was conducted asabove, except that samples were lysed in 40 mmol/L Tris/HCl pH 7.5, 274mmol/L NaCl, 2% Triton-X-100, and 20%glycerol. Samples were resolved by SDS-PAGE, blotted ontonitrocellulose filters, blocked with Odyssey blocking buffer(LI-COR Biosciences) and incubated overnight with therespective antibodies (pSer9 GSK3b, GSK3b, pSer240/244S6RP, S6RP, pThr1462 tuberin, tuberin, and cleaved PARP(Cell Signaling Technology). Primary antibodies werelabeled with infrared IRDye-labeled secondary antibodies(LI-COR Biosciences) and proteins visualized using theOdyssey Infrared Imager (LI-COR Biosciences).

For studies using the Meso Scale Discovery (MSD) elec-trochemoluminescence platform, U87MG cell lysates wereprepared as described earlier and probed for pSer473 AKT,AKT, pSer9 GSK3b, GSK3b, pThr421/424 p70S6K, andp70S6K according to the manufacturer’s instructions (19).

Cell-cycle effects and Annexin V stainingFollowing drug or vehicle treatment, U87MG cells were

labeled with either bromodeoxyuridine or propidium

Translational RelevanceDeregulated phosphoinositide 3-kinase (PI3K)-AKT

pathway signaling through AGC kinases is implicatedin many cancers. The simultaneous inhibition of mul-tiple AGC kinases may increase antitumor activity andminimize clinical resistance compared with a singlekinase target. The clinical candidate AT13148 is a novel,oral, multi-AGC kinase inhibitor, which has potentpharmacodynamic and antitumor activity in humantumor xenografts with clinically relevant genetic defectsin vitro and in vivo, and shows a distinct mechanism ofaction from selective AKT inhibitors. AT13148 showedlinear pharmacokinetics, achieved therapeutically activedrug concentrations, and induced biomarker changesconsistent with AGC inhibition in human tumor xeno-grafts. Moreover, we show for the first time that induc-tion of AKT phosphorylation at serine 473 by AT13148,as reported for other ATP-competitive inhibitors of AKT,is not a therapeutically relevant reactivation step for thiscompound. These detailed preclinical and mechanisticdata will facilitate the forthcoming first-in-human phaseI trial of AT13148.

AT13148, a Potent, Oral AGC Kinase Inhibitor

www.aacrjournals.org Clin Cancer Res; 18(14) July 15, 2012 3913




iodide as described and analyzed by flow cytometry (18).The proportion of cells in each phase of the cell cycle wasdetermined using either the Cell Quest Pro Software pack-age (BD Biosciences) or WinMDI 2.8. Annexin V stainingwas carried out according to themanufacturer’s instructions(BD PharMingen)

In vivo studiesPharmacokinetic and pharmacodynamic analyses. All

procedures were in accordance with UK Home Office reg-ulations under the Animals (Scientific Procedures) Act1986, approved by The Institute of Cancer Research’s EthicsCommittee and in accordance with published guidelines(20). Mice were allowed access to food andwater ad libitum.

For pharmacokinetic analysis,male athymic BALB/cmicewere obtained from Harlan. AT13148 was formulated in10% DMSO, 1% Tween-20, and 89% saline and adminis-tered at 5 mg/kg i.v. or p.o. Duplicate samples of heparin-ized whole blood were collected by cardiac puncture at 1, 2,4, 6, 8, 16, 24, and 72 hours after dosing. Plasma and tissues(liver, kidney, spleen, and muscle were also taken) wereprepared and frozen at �20�C until analysis. AT13148 wasextracted from plasma and tissues using acetonitrile con-taining an internal standard and quantified using a liquidchromatography tandem mass spectrometry (LC-MS/MS)method and appropriate standard curves. Pharmacokineticparameters were determined using WinNonLin softwareversion 5.2.

To assess pharmacokinetic and pharmacodynamic rela-tionships, a single dose of AT13148 (30, 40, or 50 mg/kgp.o.) was given to groups of 3 female athymic (CrTac:Ncr-Fox1nu) mice bearing established subcutaneous xeno-grafts of MES-SA, BT474, or male athymic mice bearingPC3 xenografts. The drug was formulated in vehiclecontaining 10% DMSO 1% Tween-20 in 89% saline.Plasma and tumor samples (n ¼ 3) were obtained at2, 6, and 24 hours after compound or vehicle adminis-tration. Tumors were divided into 2 halves, snap frozen,stored at �20�C and each half used for pharmacokineticand pharmacodynamic analyses, respectively. For phar-macokinetic analysis, tissue samples were first homoge-nized in 5 volumes (w/v) of acetonitrile/water (50/50).AT13148 was extracted from plasma and tissue homo-genates and quantified as described earlier.

For pharmacodynamic studies, tumors were ground to apowder under liquid nitrogen, lysed and centrifuged toremove debris. Protein content was measured using BCAor Bradford reagent and samples evaluated by Westernblots or by MSD analysis as described earlier.

Efficacy studiesHuman MES-SA, BT474, and PC3 tumor cells were

injected s.c. into the right flank of female or male athymicmice. When tumors reached �100 mm3 mean volume,animals were randomized and treated with vehicle (10%DMSO, 1% Tween-20, and 89% saline) or AT13148 p.o.using the dosage schedules detailed in the figures. Bodyweight and tumor size were determined as previously

described (17) 3 times weekly. For the MES-SA study,%T/C represents mean tumor volume of treated animalsdivided by mean control tumor volume, expressed as apercentage on any particular day. For the BT474 and PC3studies, %T/C represents mean tumor weight of treatedanimals divided by mean control tumor weight at the endof the experiment, expressed as a percentage.

Statistical analysesStatistical significancewasdeterminedusing 1- or 2-tailed

t tests as appropriate with GraphPad Prism 5.0.Other methods including those for crystallography and

microarrays studies are detailed in the SupplementaryMaterial.

ResultsIdentification of a potent ATP-competitive inhibitor ofkey AGC kinases

AT13148 was discovered using fragment-based screeningcombined with structure-based design as previously describ-ed (refs. 13 and16; Fig. 1A). The structure of AT13148 boundin the ATP pocket of the PKA-AKT chimera was solved byX-ray crystallography (Fig. 1B). The use of this PKA-basedsurrogate provides a robust and validated structural system

Glycine loop

Asn 171Met 173

Tyr 122

A

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AT13148

CI

OH

NH2

NH

N

Figure 1. The structure and biologic activity of AT13148. A, chemicalstructure of AT13148. B, crystal structure of AT13148 bound torecombinant human PKA-AKT chimera.

Yap et al.

Clin Cancer Res; 18(14) July 15, 2012 Clinical Cancer Research3914




for understanding interactions between inhibitors and AKT(14). As expected, the structure shows that AT13148 fulfillsthe requirements of the canonical 3-point pharmacophoreneeded for potent binding to the ATP site of AKT, forminghydrogen-bonding interactions with the kinase hinge, elec-trostatic interactions with the ribose site, and hydrophobiccontacts with a lipophilic pocket in the glycine-rich loop.Screening of AT13148 against a panel of kinases at 10

mmol/L revealed >80% inhibition of the structurally relatedAGCkinases AKT, PKA, ROCK2, p70S6K,MSK, RSK1/2, andSGK (Supplementary Table S1). Further studies showed thatIC50 values for p70S6K, PKA, ROCKI, and ROCKII were allless than 10 nmol/L and those for AKT1, 2, and 3 were 38,402, and 50 nmol/L, respectively (Supplementary TableS2). For the related AGC kinases RSK1 and SGK3, theIC50 valueswere 85 and63nmol/L, respectively. In contrast,IC50 values for the non-AGC kinases CHK2 and Aurora Bwere both greater than 800 nmol/L. Therefore, AT13148 is a

potent inhibitor of the AGC kinases p70S6K, PKA, ROCKI,and ROCKII that also potently inhibits the related familymembers AKT1, AKT2, AKT3, RSK1, and SGK3 (Supple-mentary Table S2).

AT13148 inhibits the proliferation and AGC kinaseactivity of cancer cells

AT13148 potently inhibited proliferation with GI50values of 1.5 to 3.8 mmol/L across a selected panel of cancercell lines (Supplementary Table S3) representing commonhuman malignancies with deregulation of PI3K-AKT-mTOR or RAS-RAF pathways. The effect of 1-hour exposureto AT13148 on AKT and p70S6K signaling was initiallyexplored in PTEN-deficient U87MG glioblastoma cells (Fig.2A). Marked induction of pSer473 AKT occurred at allconcentrations. Nevertheless, phosphorylation of the 2AKTsubstrates GSK3b and PRAS40 was inhibited at AT13148concentrations >1 and 5 mmol/L AT13148, respectively.

CA

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Figure 2. The effect of AT13148 exposure on AGC kinase biomarker expression. PTEN-deficient U87MG human glioblastoma cells were incubated withAT13148 for 1 hour (A andB) at the concentrations indicated or (C) with 10mmol/L AT13148 for the times indicated. D, PTEN-deficientMES-SA human uterinesarcoma cells were incubated for 1 hour with AT13148 at the concentrations indicated. Immunoblotting was carried out for the proteins indicated. GAPDHwas used as a loading control. C, no treatment control; D, DMSO vehicle control; LY, positive control (PI3K inhibitor LY294002, 30 mmol/L).






Phosphorylation of the p70S6K substrate, Ser235/236S6RP, was also inhibited at concentrations >5mmol/L. Totalprotein levels remained generally constant throughout thisstudy, apart from PRAS40, which increased with AT13148treatment (Fig. 2A).

Immunoblotting after 1-hour treatment with AT13148in U87MG cells also showed clear inhibition of phos-phorylation on direct substrates of the AGC kinasesPKA, ROCK, and SGK, namely Ser157 VASP, Ser19 MLC2,and Ser330 NDRG1, respectively (Fig. 2B). Inhibition ofpSer157 VASP and pSer19 MLC2 was observed from0.5 mmol/L AT13148, whereas pSer330 NDRG1 wasinhibited from 5 mmol/L AT13148. Expression of totalprotein for all 3 AGC kinase substrates remained constantup to 20 mmol/L AT13148.

We next determined the effect of 10 mmol/L AT13148 onAKT andp70S6K signaling output over time inU87MGcells(Fig. 2C). Induction of pSer473 AKT was detected at theearliest time point assayed (0.5 hours) and sustainedthroughout the compound exposure time. Decreases inboth pSer9GSK3b andpThr246PRAS40were also observedfrom0.5 to 24 hours, while a decrease in pSer235/236 S6RPwas first detected at 1 hour. Total protein levels remainedgenerally constant throughout the time course.

Inhibition of AKT and p70S6K signaling was alsoobserved in PTEN-deficient MES-SA cells after treatmentwith AT13148 (Fig. 2D). A 1-hour drug exposure markedlyinhibited phosphorylation of the AKT substrates GSK3band tuberin, and thedownstreamp70S6K substrate S6RP, atAT13148 concentrations of 3 mmol/L or above, with min-imal effects on total protein levels. Taken together, thesebiomarker data clearly show that AT13148 can inhibit anumber of AGC kinases, including AKT, in human tumorcell lines in vitro.

Phosphorylation of AKT substrates remainssuppressed, despite pSer473 AKT induction byAT13148

ATP competitive inhibitors of AKT induce phosphoryla-tion on Ser473 of this kinase and this may have therapeuticimplications (15). Specifically, this phosphorylated formof AKT has been shown to be hyperactive in vitro whendissociated from the inhibitor, thus potentially leading toactivation of AKT targets in cells, and in turn promotingoncogenesis. To address this, we monitored both pSer473AKT and phosphorylation of targets downstream of AKTin U87MG cells in vitro after exposure to AT13148 at 1and 10 mmol/L for 1 and 24 hours, followed by theremoval of compound for 0, 4, 8, or 24 hours (Supple-mentary Fig. S1A and S1B). Analysis of total and phos-pho-protein signals using the quantitative MSD electro-luminescence immunoassay revealed that the pSer473AKT signal was strongly induced in U87MG cells at bothconcentrations and all time points, and was sustainedeven after 24 hours of compound removal. In contrast,both the pSer9 GSK3b and pThr421/424 S6K signals wereinhibited under the same conditions and only showedpartial recovery up to 24 hours after compound removal.

From these studies it can be concluded that althoughAT13148 induces phosphorylation on Ser473 AKT, com-pound removal does not cause downstream phosphory-lation signals to recover rapidly or to rebound to greaterthan pretreatment levels.

Pharmacokinetics and pharmacodynamics of AT13148To determine if therapeutically active concentrations of

AT13148 can be achieved in vivo, the pharmacokineticprofile of this drug candidate was investigated in BALB/cmice. After administration of 5mg/kg i.v., AT13148 showeda low plasma clearance of 1.68 L/h/kg, which is less thanhalf liver blood flow, and a large volume of distribution of9.05 L/kg with a terminal half-life of 2.83 hours (Fig. 3A;Supplementary Table S4). Oral drug administration of5 mg/kg of AT13148 resulted in complete bioavailability.Increasing oral doses from 5 to 50 mg/kg showed linearpharmacokinetics, with plasma AUC0–¥ increasing in pro-portion with dose (Fig. 3B).

Figure 3C shows the concentrations of AT13148 achievedin athymic mouse plasma and HER2-positive PIK3CA-mutant BT474 human breast cancer xenografts after theadministration of 2 daily doses of 40mg/kg p.o. Consistentwith the large volume of distribution described, tumorAT13148 concentrations greatly exceeded plasma concen-trations at 2, 6, and 24 hours with tumor:plasma ratios of8.5, 7.0, and 13.6, respectively. Moreover, tumor AT13148concentrations were at least 9 times greater than the in vitroGI50 value of 1.8 mmol/L for this cell line, maintained over24 hours.

Pharmacodynamic biomarker changes measured in thesame BT474 xenografts by MSD immunoassay are shownin Fig. 3D. There was an increase in pS473 AKT at 2, 6, and24 hours after AT13148 treatment, consistent with in vitroobservations (Fig. 2C). Importantly, phosphorylation ofthe AKT substrate GSK3b and the downstream targetp70S6K was significantly decreased, consistent with sus-tained inhibition of AKT activity by AT13148 in vivo at thisdose. Comparable studies in PTEN-deficient PC3 humanprostate tumor xenografts also showed high AT13148tumor:plasma ratios and significant decreases in the phos-phorylation of AKT biomarkers (Supplementary Fig. S2Aand S2B). Clear inhibition of phosphorylation of the AKTsubstratesGSK3b, tuberin, and the p70S6K target S6RPwerealso observed in PTEN-deficient MES-SA human uterinetumor xenografts after treatment with 40 and 50 mg/kgp.o. of AT13148 over 24 hours (Fig. 4A; SupplementaryFig. S3). Importantly, induction of cleaved PARP wasobserved at these doses of AT13148 over 24 hours, indi-cating that AT13148 induces apoptosis in solid tumors.

Taken together, these data suggest that AT13148marked-ly inhibits the activity of both AKT and p70S6KAGCkinasesin human tumor xenografts with differentially activatedPI3K pathways after oral administration.

In vivo antitumor activity of AT13148Following the demonstration of clear in vitro activity and

promising in vivo pharmacokinetic and pharmacodynamic

Yap et al.





properties indicating target modulation, the antitumoractivity of AT13148 was assessed in multiple human tumorxenograft models. Figure 4B shows that AT13148 markedlyinhibited the growth of established MES-SA human uterinesarcoma tumor xenografts at the same doses of 40 and 50mg/kg that also resulted in pharmacodynamic changesindicative of target engagement (Fig. 4A; SupplementaryFig. S3); the percentage treated/control (%T/C) was 41%and 54% at 40 and 50 mg/kg p.o. of AT13148, respectively,when measured on day 11 of treatment.Further antitumor studies were undertaken in estab-

lished BT474 (Fig. 4C) and PC3 human tumor xenograftmodels (Supplementary Fig. S2C). AT13148 inhibited thegrowth of both these models giving T/C values of 35.8%on day 26 and 65.4% on day 27, for BT474 and PC3respectively. These results are consistent with boththe pharmacokinetic and pharmacodynamic data (Figs.

3A–D and Fig. 4A; Supplementary Figs. S2A, S2B, and S3),which indicate that for this oral route of administration,plasma drug concentrations were at or just above the invitro GI50 values, whereas tumor drug concentrationsgreatly exceeded GI50 values for at least 24 hours forthese tumor cells. AT13148 also showed growth inhibi-tion in the mutant KRAS A549 lung adenocarcinomaxenograft model (Supplementary Fig. S4). Tumor growthwas significantly inhibited in the MES-SA, BT474, andPC3 tumor xenografts (P < 0.01), with marked growthinhibition in A549 (P ¼ 0.0788) and minor animalweight loss in all studies (Supplementary Figs. S2D,S4B, S5A, and S5B). Our results therefore clearly showthat orally administered AT13148 induces sustained inhi-bition of the AGC kinases AKT and p70S6K and exhibitsmarked antitumor effects in 4 genetically relevant humantumor xenograft models.

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Figure 3. Characterization of the pharmacokinetic properties and pharmacodynamic effects of AT13148 in vivo. A, plasma and tissue pharmacokinetics ofAT13148 inmice following i.v. andoral (p.o.) administration. Values aremean�SE for 3 to 5miceper timepoint. B, relationship betweendose andexposure forAT13148 after single-dose oral administration in mice. Data from 2 independent experiments (open and closed circles). C, concentrations of AT13148measured in plasma (open bar) and HER2-positive, PIK3CA-mutant BT474 breast cancer xenografts (closed bar) taken 2, 6, and 24 hours after thesecond dose of AT13148 given p.o. at 40 mg/kg on 2 consecutive days of treatment (i.e., 1 cycle). Dotted line represents 96-hour GI50 value (1.6 mmol/L) inBT474 cells in vitro. Bars showmean�SE for 3 determinations. D, quantification of pharmacodynamic biomarker changes (ratio of phospho vs. total protein)measured using the MSD electrochemoluminescent platform for the tumors shown in C. Dashed line represents this ratio in vehicle-treated controlsas 1.0. Values are mean � SE for 3 determinations. Statistics: �, P < 0.05; ��, P < 0.01; ��, P < 0.001 significantly different from control. Cont, control.






Gene expression microarray analysisThe molecular and cellular responses of the multi-AGC

kinase inhibitor AT13148 drug candidate were exploredby gene expression microarray and compared withthose of the previously described AKT-selective inhibitorCCT128930 (17) after 6-hour treatment of U87MG cells at0.1 mmol/L, 1�GI50, and 3�GI50 concentrations (Figs. 5A–D and 6A–C). The expression levels of 563 genes weresignificantly altered after AT13148 treatment, whereas theexpression levels of 898 genes were significantly changedwith CCT128930 (Fig. 5A and B; Supplementary Table S5Aand S5B). Importantly, there was an overlap of 147 genesthat exhibited significantly altered expression in responseto both AT13148 and CCT128930 (Fig. 5B).

The effect of each compound on AKT pathway signalingwas also simultaneously confirmedbyWestern blot analysis(Fig. 5C). Effects on AKT signaling were reflected by per-turbations in the gene expression network (Fig. 6A–C).Specifically, inhibition of AKT has been reported to blockphosphorylation of the forkhead transcription factorsFOXO3a and FOXO1,which then translocate to the nucleusand activate transcription of downstream targets. The neg-ative cell-cycle regulators p27KIP1 and cyclinG2, encoded byCDKN1B andCCNG2, respectively (21, 22) are both FOXOtargets and their gene expression levels were upregulated inresponse to both compounds, although the level of induc-tion appeared stronger with CCT128930 (Fig. 6A; Supple-mentary Table S5A and S5B). The upregulation in expres-

sionwas also confirmed for p27KIP1 at the protein level (Fig.5C). Expression of the FOXO1 gene is itself transcriptionallyregulated by FOXO3a (23, 24), and FOXO1 expression wasincreased upon treatment with both compounds by geneexpressionmicroarray and confirmed at the protein level byWestern blot analysis (Figs. 5C and 6B; SupplementaryTable S5A and S5B).

Interestingly, the gene expression network analysis alsorevealed evidence of the induction of both negative andpositive feedback mechanisms (Fig. 6C). Both PIK3CAand IRS2 can be transcriptionally regulated by FOXO3aand induction of both has been reported after treatmentof cells with PI3K inhibitors (25–27). This could poten-tially lead to reactivation of the PI3K/AKT pathway. Weobserved increased expression of both PIK3CA and IRS2genes after treatment with AT13148 and CCT128930 (Fig.6C and Supplementary Table S5A and S5B). We alsonoted a concentration-dependent band shift downwardof IRS2 on the Western blots for CCT128930, which wasless marked for AT13148 (Fig. 5C). We confirmed usinglambda phosphatase that this band shift is because ofhypophosphorylation of IRS2 (Fig. 5D). AKT RNA inter-ference (RNAi) studies confirmed that this band shift wasAKT-dependent (Fig. 5D). The more specific AKT inhib-itor CCT128930 induced a greater mobility shift andmarked accumulation of IRS2 protein, compared withthat observed for AT13148 or AKT RNAi, suggesting agreater blockade on targets that impact on IRS2. However,

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Figure 4. Pharmacodynamicbiomarker and antitumor activity ofAT13148 in human tumorxenografts. A, effect of a singledose of 50mg/kgAT13148 given p.o. on pharmacodynamicbiomarkers in PTEN-deficientMES-SA human uterine sarcomaxenografts. Protein expressionwas assessed by immunoblottingusing GAPDH as a loading control.B, antitumor activity of AT13148 at40 or 50 mg/kg p.o. on anintermittent schedule [every 3 daysor (daily on 2 consecutive days)every 5 days] in established MES-SA human uterine sarcomaxenografts. C, antitumor activity ofAT13148 at 40 mg/kg p.o. on 2consecutive days with 3 days restbetween treatments in HER2-positive, PIK3CA-mutant BT474human breast cancer xenografts.Values are mean � SE for 5 to 8mice per time point. Statistics: ��,P< 0.01 significantly different fromcontrol.

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this loss of phosphorylation on IRS2 does not lead todownstream reactivation of the PI3K-AKT pathway, as canbe seen by the continued suppression of phosphorylationof AKT substrates (Fig. 5C). Expression of the tumorsuppressor PIK3IP1, a potential transcriptional target ofFOXO1 and CREB1 (28), was also induced at the genetranscription level following treatment with both com-pounds (Fig. 6C; Supplementary Table S5A and S5B).This induction was confirmed at the protein level (Fig.5C). PIK3IP1 has been shown to decrease PI3K p110aactivity both in vitro and in vivo (29, 30).Gene ontology (GO) analysis (31, 32) for the biologic

processes of the 147 common genes that showed signifi-cantly altered expression in response to both AT13148 andCCT128930 revealed thatmost genes affected were involvedin regulation of the cell cycle, and apoptosis (Figs. 5B and 6Aand B; Supplementary Table S6A–S6C). GO analysis for thegenes showing altered expression with the specific AKTinhibitor CCT128930 revealed that "cell cycle" was themostcommon term in the top 20most statistically significant (by

P value)GO categories, which is consistent with gene expres-sion changes previously observed with PI3K inhibitors(refs. 33, 34; Supplementary Table S6B). The effects ofCCT128930 on the expression of cell-cycle genes were gen-erally greater than with AT13148 (Fig. 6A). For example, thedownregulation of positive cell-cycle regulators such asCDC25A, CDC6, and CCNE1 was more pronounced withCCT128930 than AT13148. However, in contrast to thedownregulation of CCND1 in response to CCT128930, amodest but significant and reproducible increase wasobserved with AT13148, which was confirmed by TaqMan(data not shown). The downregulation of cyclin D1 byCCT128930was also confirmed at the protein level, whereasAT13148 had no effect on the level of cyclinD1 protein (Fig.5C; Supplementary Fig. S6A and S6B). CCT128930 alsocaused a decrease in the expression of the proapoptoticmarker c-MYC, both at the gene and protein level, whereasAT13148did not (Fig. 5B; Supplementary Fig. S6A and S6B).

In contrast to CCT128930, the GO analysis for AT13148identified "cell death," "programmed cell death," and

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"apoptosis" as the most common terms in the 20 GOcategories with the most statistically significant P values.(Supplementary Table S6C). These data are consistent withthe potent induction of apoptosis seen after treatment withAT13148 both in vitro and in vivo. Furthermore, a directcomparison of the cellular effects of AT13148 (GI50 ¼ 3.3mmol/L, Supplementary Table S3) versus CCT128930 (GI50¼ 6.3 mmol/L; ref. 17) identified a marked increased inPARP and caspase-3 cleavage with AT13148 versusCCT128930 at approximately equipotent doses of 10 and20 mmol/L, respectively (Supplementary Fig. S6A and S6B).This was further emphasized by the fact that AT13148showed a significantly greater degree of Annexin V stainingthanCCT128930 at equipotent concentrations, again indic-ative of a higher level of apoptosis with AT13148 (Supple-mentary Fig. S6C). Moreover, AT13148 inhibited the phos-phorylation of the AKT substrates GSK3b, PRAS40, FOXO1,FOXO3a at similar concentrations to CCT128930 (Fig. 5C;Supplementary Fig. S6A and S6B), but was 10-fold morepotent against the ROCK substrate MCL2 (SupplementaryFig. S6A and S6B), suggesting that inhibition of this AGCkinasemay contribute to the increased apoptotic cell death.Furthermore, there was a clear difference in the cell-cycleeffects of the 2 compounds, with CCT128930 causing apredominant G1/S arrest with loss of S-phase at increasingconcentrations, in contrast to a predominant G2/M arrestseen with AT13148 (Supplementary Figs. S6D, S6E, andS7).Summarizing this gene network analysis, both the AGC

kinase inhibitor AT13148 and the more AKT-selectiveCCT128930 show molecular effects in cancer cells consis-tent with blockade of AKT signaling, leading to changes ingene expression that include induction of upstream regu-lators.However, the 2 agents clearly alsohavedistinct effectsin cancer cells. Although CCT128930 primarily modulatesgenes in the network regulating cell cycle and causes a G1

phase arrest, AT13148 has a predominant effect on apo-ptosis genes and causes a greater apoptotic phenotype, witha secondary effect on cell cycle at the G2–M phase.

DiscussionPI3K signaling is commonly deregulated in cancer and

the oncogenic signal is transmitted predominantly throughAGC kinases, such as AKT, p70S6K, PDK1, SGK, and ROCK(3).We report for the first time the detailed biologic activityof a novel, potent, oral clinical drug candidate AT13148,which is a multi-AGC kinase inhibitor discovered usingfragment-based screening combined with structure-baseddesign. We have showed that AT13148 is a potent inhibitorof selected AGC kinases including AKT, p70S6K, PKA, SGK,and ROCK (Supplementary Table S2).In this study, we have used the pharmacologic audit trail

that we originally conceptualized and subsequently advo-cated (35, 36) to guide the biomarker-driven drug discoveryand development of AT13148. Thus, we incorporateddetailed pharmacokinetic and pharmacodynamic studiesto confirm adequate drug exposure with concomitant target

and pathway blockade. We have showed that AT13148 hasantiproliferative activity in a range of in vitro models har-boring different genetic abnormalities, including pathogen-ic PTEN, KRAS, PIK3CA, and HER2 aberrations (Supple-mentary Table S3). Interestingly, the GI50 values obtainedfrom these tumor cell lines were broadly similar despite thedifferent oncogenic alterations and therefore we intend toexpand these observations to amuch larger panel of humancancer cell lines. These results may be in part because of thesimultaneous blockade of different AGC kinases, reducingopportunities for the disruption of negative feedback loopsand cross-talk with other key signaling pathways, and thusattenuating the potential for intrinsic resistance. Further-more, in our in vitro studies with PTEN-deficient U87MGhuman glioblastoma and similarly PTEN-deficient MES-SAuterine sarcoma tumor cell lines, we have shown thatAT13148 causes substantial blockade of AKT, p70S6K, PKA,ROCK, and SGK substrate phosphorylation in both a con-centration- and time-dependent manner, confirming thatAT13148 can simultaneously inhibit multiple AGC kinasesin these cancer cells (Fig. 2A–D).

Having showed promising pathway modulation andantiproliferative effects on cancer cells in vitro, our subse-quent pharmacokinetic studies showed that drug exposureis related linearly to the administered oral dose of AT13148(Fig. 3A and B). Furthermore, oral administration ofAT13148 gave high tumor:plasma concentrations for atleast 24 hours in mice bearing HER2-positive, PIK3CA-mutant BT474 breast or PTEN-deficient PC3 human pros-tate cancer xenografts (Fig. 3C; Supplementary Fig. S2A).These exposures greatly exceeded the in vitro antiprolifera-tive GI50 values that would be predicted to produce phar-macodynamic biomarker modulation, pathway blockade,and antitumor efficacy. Indeed, inhibition of signalingoutput was confirmed by the marked inhibition of phos-phorylation on both AKT and p70S6K substrates for up to24 hours in both of these cancermodels, as well as in PTEN-deficient MES-SA uterine tumor xenografts (Figs. 3Dand 4A; Supplementary Figs. S2B and S3). Subsequently,oral efficacy studieswithAT13148 showedantitumor effectsin all 3 clinically relevant human tumor xenografts (Fig. 4Band C; Supplementary Fig. S2C). These data indicate thatAT13148 exhibits promising single-agent antitumor activityafter oral administration and support its clinical evaluation.Apart from a monotherapy drug development strategy,AT13148 may also be considered for rational combinationregimens, especially with other targeted therapies thatinduce the activation of compensatory pathways, for exam-ple AKT phosphorylation after mTORC1 inhibitionobserved with everolimus (37). In keeping with other AKTinhibitors currently in the clinic, toxicologic studies withAT13148 revealed some early hyperglycemia, but the effectswere largely equivocal (data not shown). As might beexpected from its target kinase profile, AT13148 alsoshowed vascular smooth muscle contraction, hypotension,and tachycardia (data not shown), but these perturbationsreturned to normal after repeat dosing, suggesting an adap-tive response. Gene expression microarray analysis of






normal tissuesmight help to identify those genes associatedwith drug toxicity.

We have shown that robust inhibition of AGC kinaseactivity occurs in cancer cells both in vitro and in vivo, despitethe observed induction of phosphorylation on Ser473 ofAKT by AT13148, which binds into the ATP pocket of thiskinase. This type of induction has been seenwith other ATP-competitive inhibitors of AKT and shown to be because ofdirect inhibitor binding, rather than a regulatory pathwayfeedback mechanism (15). Furthermore, it has beenshowed that this phosphorylated formof AKT is hyperactivesuggesting that in vivo treatment with an ATP competitiveinhibitor of AKT may promote tumor cell growth (15).However, our studies have shown that despite the inductionof pSer473 AKT, the removal of AT13148 from cancer cellsin vitro does not lead to increased phosphorylation of AKTsubstrates (Supplementary Fig. S1A and S1B). Furthermore,our in vivo pharmacodynamic and efficacy studies withAT13148 (Figs. 3D and 4A–C; Supplementary Figs. S2–S4), using the doses and schedules presented in this paper,indicate that AKT is not hyperactivated and does not pro-mote tumor cell growth but rather, signaling output andtumor growth are inhibited. It is not possible to conclude atthis point whether the observed inhibition of AKT signalingoutput is because of the fact that AT13148 inhibits multipleAGC kinases, or is associated with the pharmacologic prop-erties of this inhibitor. However, our data provide evidenceboth in vitro and in vivo that the AKT pathway is inhibitedrather than activated with the AGC kinase inhibitorAT13148 and that such an inhibitor approach is a viabletherapeutic anticancer strategy.

Our gene expressionmicroarray studies inPTEN-deficientU87MG human glioblastoma cells identified an overlap of147 genes that exhibited significantly altered expression inresponse to both AT13148 and CCT128930 (Fig. 5B). Thissuggests a component of sharedmechanismof actionon thegene network that correlated with inhibition of the IGF-PI3K-AKT-mTORpathway (Figs. 5C and 6A–C). Of interest,increased expression of upstream positive regulators wasobserved especially of IRS2, which also seems to be at leastpartly regulated by FOXO1 and FOXO3a. The increasedexpression of genes encoding IRS2 and PI3K p110a couldpotentially lead to reactivation of the pathway, as has beenshown previously for IRS2 with the pan-class I PI3K inhib-itor, GDC-0941 (34). However, although we observedincreased Ser473 phosphorylation on AKT with bothCCT128930 and AT13148 (Fig. 5C), downstream targetswere still dephosphorylated and the pathway remainedinactive.

Both compounds altered the expressionof genes involvedin cell-cycle regulation and apoptosis (Figs. 5C and 6A andB; Supplementary Fig. S6). The enrichment for cell-cyclegenes is very similar to that seen with the dual pan-class IPI3K/mTOR inhibitor PI-103 (33), suggesting that thesegene expression changes are pathway related. However,effects were greater for CCT128930 than AT13148, consis-tent with the former being amore AKT-selective compound.Downregulation of positive cell-cycle regulators, such as

cyclin E and CDC6, and upregulation of negative cell-cycleregulators including p27KIP1 in response to treatment withboth AT13148 and CCT128930, correlated with a substan-tial decrease in S-phase cells. However, althoughCCT128930, like other inhibitors of PI3K/AKT signaling,gave apredominantG1 arrest, AT13148didnot. Thismaybeexplained by the fact that in contrast to CCT128930,AT13148 did not cause decreased expression of the majorG1 regulator, cyclin D1 at either the gene expression orprotein level (Fig. 5C; Supplementary Fig. S6A and S6B).Consequently, therewas an equal distribution of cells eitherside of S phase, expressed as an increase in the G2/M phase(Supplementary Figs. S6D, S6E, and S7). Conversely, themost enriched population of genes showing altered expres-sion with AT13148 but not CCT128930 are those involvedin the control of apoptosis. This molecular phenotype isrecapitulated at the cellular level where a much greaterinduction of apoptosis is observedwithAT13148 comparedwith CCT128930 at the same concentrations (Supplemen-tary Fig. S6A–S6C). We hypothesize that these differencesare a result of the targeting of several key AGC kinases byAT13148, in contrast to the more AKT-specific effects ofCCT128930.

In conclusion,we have disclosed here for the first time thedetailed mechanism of action and therapeutic potential ofthe novel, potent, multi-AGC kinase inhibitor, and oraldrug candidate AT13148. We report the preclinical phar-macologic audit trail for AT13148 that supports its clinicaldevelopment, including the pharmacokinetic—pharmaco-dynamic–antitumor activity relationship in clinically rele-vant human tumor xenografts. In addition, our detailedgene expression microarray analysis has revealed thatAT13148 shows a distinct gene expression profile thatcorrelates with a marked apoptotic rather than cytostaticphenotype, emphasizing the functional differencesbetween its properties as a multi-AGC kinase inhibitor incontrast to a more AKT-selective inhibitor. In view of thepotential mechanistic advantages detailed above, and thepotent antitumor activity observed at well-tolerated dosesagainst established human tumor xenografts with clinicallyrelevant genetic drivers, the clinical use of such an AGCkinase inhibitor strategy will now be assessed in a first-in-human phase I trial of AT13148.

Disclosure of Potential Conflicts of InterestT.A. Yap,M.I.Walton, R.H. te Poele, P.D. Eve,M.R. Valenti, A.K. de Haven

Brandon, V. Martins, A. Zetterlund, S.P. Heaton, K. Heinzmann, F.I. Ray-naud, S.A. Eccles, P. Workman, and M.D. Garrett are current or formeremployees of The Institute of Cancer Research, which has a commercialinterest in the development of AKT inhibitors, including AT13148, andoperates a rewards for inventors scheme. K.M.Grimshaw, R. Feltell,M. Reule,S.J. Woodhead, T.G. Davies, J.F. Lyons, and N.T. Thompson are current orformer employees of Astex Therapeutics, which also has a commercialinterest in the development of AKT inhibitors including AT13148. BothAstex Therapeutics and The Institute of Cancer Research have been involvedin a commercial collaboration with Cancer Research Technology Limited(CRT) to discover and develop inhibitors of AKT and intellectual propertyarising from this programhas been licensed to AstraZeneca. P.Workman hasa commercial research grant from Yamanouchi (now Astellas), PiramedPharma, and Astex Pharmaceuticals; ownership interest (including patents)from Piramed Pharma (acquired by Roche) and Chroma Therapeutics; andis a consultant/advisory board member for Piramed Pharma, ChromaTherapeutics, Novartis, Wilex, and Nextech Ventures.

Yap et al.





Grant SupportGrant support was provided to T.A. Yap, M.I. Walton, P.D. Eve, M.R.

Valenti, A.K. de Haven Brandon, V. Martins, A. Zetterlund, F.I. Raynaud, S.A.Eccles, P.Workman, andM.D. Garrett by Cancer ResearchUK (CR-UK) grantC309/A8274; to S.P. Heaton by CR-UK grant C51/A6883; to R.H. te Poele byCR-UK grant C51/A7401; to K. Heinzmann by Marie Curie Early StageFunding; and to P. Workman by CR-UK grant number C309/A8992. P.Workman is a Cancer Research Life Fellow. Additional support was providedto S.A. Eccles andM.D.Garrett by The Institute of Cancer Research. This workwas carried out as part of a funded research collaboration with Astex

Therapeutics. The authors acknowledge NHS funding to the NIHR Biomed-ical Research Centre.

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.

ReceivedDecember 23, 2011; revisedMay 1, 2012; acceptedMay 15, 2012;published OnlineFirst July 10, 2012.

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2012;18:3912-3923. Published OnlineFirst July 15, 2012.Clin Cancer Res Timothy A. Yap, Mike I. Walton, Kyla M. Grimshaw, et al. Pharmacodynamic and Antitumor ActivityAT13148 Is a Novel, Oral Multi-AGC Kinase Inhibitor with Potent

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